Patent application title: TRANSGENIC ALGAE ENGINEERED FOR HIGHER PERFORMANCE
Inventors:
Pat J. Unkefer (Los Alamos, NM, US)
Pat J. Unkefer (Los Alamos, NM, US)
Penelope S. Anderson (Los Alamos, NM, US)
Penelope S. Anderson (Los Alamos, NM, US)
Thomas J. Knight (Raymond, ME, US)
Thomas J. Knight (Raymond, ME, US)
IPC8 Class: AC12N112FI
USPC Class:
435471
Class name: Chemistry: molecular biology and microbiology process of mutation, cell fusion, or genetic modification introduction of a polynucleotide molecule into or rearrangement of nucleic acid within a microorganism (e.g., bacteria, protozoa, bacteriophage, etc.)
Publication date: 2016-05-19
Patent application number: 20160137972
Abstract:
The present disclosure relates to transgenic algae having increased
growth characteristics, and methods of increasing growth characteristics
of algae. In particular, the disclosure relates to transgenic algae
comprising a glutamine phenylpyruvate transaminase transgene and to
transgenic algae comprising a glutamine phenylpyruvate transaminase
transgene and a glutamine synthetaseClaims:
1. A transgenic alga comprising a plant-derived or algal-derived
glutamine phenylpyruvate transaminase (GPT) transgene and a glutamine
synthetase transgene, wherein each of said GPT transgene and said GS
transgene is operably linked to a promoter.
2. The transgenic alga according to claim 1, wherein the alga is a green alga.
3. The transgenic alga according to claim 2, wherein the green alga is a Chlorella species.
4. The transgenic alga according to claim 1, wherein the GPT transgene is a plant-derived GPT.
5. The transgenic alga according to claim 1, wherein the GPT is an algal-derived GPT.
6. The transgenic alga of claim 4, wherein the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO 24, SEQ ID NO: 30, SEQ ID NO:31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 44, and SEQ ID NO: 47, an amino acid sequence that is at least 75% identical to SEQ ID NO: 2, an amino acid sequence that is at least 75% identical to SEQ ID NO: 9, an amino acid sequence that is at least 75% identical to SEQ ID NO: 15, an amino acid sequence that is at least 75% identical to SEQ ID NO: 19, an amino acid sequence that is at least 75% identical to SEQ ID NO: 21, an amino acid sequence that is at least 75% identical to SEQ ID NO 24, an amino acid sequence that is at least 75% identical to SEQ ID NO: 30, an amino acid sequence that is at least 75% identical to SEQ ID N0:31, an amino acid sequence that is at least 75% identical to SEQ ID NO: 32, an amino acid sequence that is at least 75% identical to SEQ ID NO: 33, an amino acid sequence that is at least 75% identical to SEQ ID NO: 34, an amino acid sequence that is at least 75% identical to SEQ ID NO: 35, an amino acid sequence that is at least 75% identical to SEQ ID NO: ID NO: 36, an amino acid sequence that is at least 75% identical to SEQ ID NO: 44, and an amino acid sequence that is at least 75% identical to SEQ ID NO: 47.
7. The transgenic alga according to claim 5, wherein the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 48, and an amino acid sequence that is at least 75% identical to SEQ ID NO: 48.
8. The transgenic alga according to claim 1, wherein the GS transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 39, an amino acid sequence that is at least 75% identical to SEQ ID NO: 4, an amino acid sequence that is at least 75% identical to SEQ ID NO: 7, and an amino acid sequence that is at least 75% identical to SEQ ID NO: 39.
9. The transgenic alga according to claim 1, wherein the GPT and GS transgenes are incorporated into the genome of the alga.
10. A progeny of any generation of the transgenic alga of claim 9, wherein said progeny comprises said GPT transgene and said GS transgene.
11. The transgenic alga of claim 9, which displays a faster growth rate, increased chlorophyll production, increased ribulose bisphosphate carboxylase (RUBISCO) level, increased nitrogen use efficiency, increased biomass yield, increased GPT activity, increased GS activity, increased 2-oxoglutaramate levels, and/or increased tolerance to salt or saline conditions when compared to a control wild-type or untransformed alga.
12. A method for increasing growth characteristics of an alga relative to an wild type or progenitor alga of the same species, comprising: (a) introducing a plant-derived or algal-derived GPT transgene into the alga; (b) introducing a GS trans gene into the alga or a progeny of the alga; (c) expressing the GPT transgene and the GS transgene in the alga or the progeny of the alga; and, (d) selecting an alga having an increased growth characteristic relative to an alga of the same species that does not comprise the GPT transgene or the GS transgene.
13. The method according to claim 12, wherein the increased growth characteristic is selected from the group consisting of: faster growth rate, increased chlorophyll production, increased ribulose bisphosphate carboxylase (RUBISCO) level, increased nitrogen use efficiency, increased biomass yield, increased GPT activity, increased GS activity, increased 2-oxoglutaramate levels, and/or increased tolerance to salt or saline conditions.
14. A transgenic alga comprising a plant-derived or algal-derived GPT transgene, wherein said GPT transgene is operably linked to a promoter.
15. The transgenic alga according to claim 14, wherein the alga is a green algae.
16. The transgenic alga according to claim 15, wherein the green alga is a Chlorella species.
17. The transgenic alga according to claim 14, wherein the GPT transgene is a plant-derived GPT.
18. The transgenic alga according to claim 14, wherein the GPT is an algal-derived GPT.
19. The transgenic alga of claim 17, wherein the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO 24, SEQ ID NO: 30, SEQ ID NO:31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 44, and SEQ ID NO: 47, an amino acid sequence that is at least 75% identical to SEQ ID NO: 2, an amino acid sequence that is at least 75% identical to SEQ ID NO: 9, an amino acid sequence that is at least 75% identical to SEQ ID NO: 15, an amino acid sequence that is at least 75% identical to SEQ ID NO: 19, an amino acid sequence that is at least 75% identical to SEQ ID NO: 21, an amino acid sequence that is at least 75% identical to SEQ ID NO 24, an amino acid sequence that is at least 75% identical to SEQ ID NO: 30, an amino acid sequence that is at least 75% identical to SEQ ID N0:31, an amino acid sequence that is at least 75% identical to SEQ ID NO: 32, an amino acid sequence that is at least 75% identical to SEQ ID NO: 33, an amino acid sequence that is at least 75% identical to SEQ ID NO: 34, an amino acid sequence that is at least 75% identical to SEQ ID NO: 35, an amino acid sequence that is at least 75% identical to SEQ ID NO: 36, an amino acid sequence that is at least 75% identical to SEQ ID NO: 44, and an amino acid sequence that is at least 75% identical to SEQ ID NO: 47.
20. The transgenic alga according to claim 18, wherein the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 48, and an amino acid sequence that is at least 75% identical to SEQ ID NO: 48.
21. The transgenic alga according to claim 14, wherein the GPT transgene is incorporated into the genome of the alga.
22. A progeny of any generation of the transgenic alga of claim 21, wherein said progeny comprises said GPT transgene.
23. The transgenic alga of claim 21, which displays a faster growth rate, increased chlorophyll production, increased ribulose bisphosphate carboxylase (RUBISCO) level, increased nitrogen use efficiency, increased biomass yield, increased GPT activity, increased GS activity, increased 2-oxoglutaramate levels, and/or increased tolerance to salt or saline conditions when compared to a control wild-type or untransformed alga.
24. A method for increasing growth characteristics of an alga relative to an wild type or progenitor alga of the same species, comprising: (a) introducing a plant-derived or algal-derived GPT transgene into the alga; (b) expressing the GPT transgene in the algae or the progeny of the alga; and, (c) selecting an alga having an increased growth characteristic relative to an alga of the same species that does not comprise the GPT transgene.
25. The method according to claim 24, wherein the increased growth characteristic is selected from the group consisting of: faster growth rate, increased chlorophyll production, increased ribulose bisphosphate carboxylase (RUBISCO) level, increased nitrogen use efficiency, increased biomass yield, increased GPT activity, increased GS activity, increased 2-oxoglutaramate levels, and/or increased tolerance to salt or saline conditions.
Description:
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S. application Ser. No. 13/037,149, filed Feb. 28, 2011, which claims the benefit of U.S. Provisional Application No. 61/308,974, filed Feb. 28, 2010, which is hereby incorporated by reference in its entirety.
REFERENCE TO A "SEQUENCE LISTING," A TABLE, OR A COMPUTER PROGRAM LISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE
[0003] The Sequence Listing written in file 87485-921786-SEQLIST.TXT, created on Mar. 2, 2015, 1430,105 bytes, machine format IBM-PC, MS-Windows operating system, is hereby incorporated by reference in its entirety for all purposes.
FIELD
[0004] The present disclosure relates to transgenic algae having increased growth characteristics, and methods of increasing growth characteristics of algae. The disclosure also relates to recombinant polynucleotides for the generation of transgenic algae having increased growth characteristics.
BACKGROUND OF THE INVENTION
[0005] The global biodiesel market demand is estimated to reach 37 billion gallons by 2016, growing at an average annual growth rate of 42%. Europe will be the major market for the next decade or so, closely followed by the US market. To meet this increased market demand, additional oil sources, especially non-edible oils, need to be explored (Li et al., 2008 Appl. Microbiol. Biotechnol. 80:749-756). Microalgae seems to be the only source of renewable biodiesel that has the potential to displace petroleum-derived transportation fuels without the controversial argument "Food or Fuel" and to help the nation reach the 2003 Biofuels Directive target of achieving greenhouse gas savings (Christi, 2007, Biotechnol. Adv. 25:294-306; Christi, 2008, Trends Biotechnol. 26:126-131; Cockerill and Martin, 2008, Biotechnol. Biofuels 1:9).
[0006] The most advanced biotechnology being applied to algal growth has been the creation of the antennae mutants that have less light harvesting machinery in the cell, which allows a greater fraction of the light to pass through an individual cell. This light then strikes other cells deeper in the culture. This is viewed as advantageous because some of the light energy striking a normal cell is in excess and is lost as fluorescence. These mutants do not suffer this loss of excess energy; it is available to other deeper cells in the culture. Thus the overall culture accumulates biomass faster. These mutants then grow using their normal rates of metabolism. In addition, some are attempting to engineer herbicide resistance genes into the production strains to allow competing algae in a production bioreactor to be controlled with the herbicide.
[0007] Numerous algal biofuels companies populate the landscape; it is reasonable to expect at least 20 of them will be producing algal oil at large scale within a year. Microalgal biodiesel is technically feasible (Gouveia et al. 2009 J. Ind. Microbiol. Biotechnol 36:269-274). However technoeconomic analyses show that for microalgal biofuels to be economically competitive with petrodiesel, the production, harvesting and extraction steps must be optimized and costs reduced. The production step must be increased substantially to increase the overall total biomass production. The degree to which the production rate can be improved within the constraints of the fixed costs of the production reactor, will dictate how much other costs must be reduced to achieve profitability or even the bottom line. The technology described herein can be expected to address that need.
[0008] In plants, the organic compound 2-oxoglutaramate is a powerful signal metabolite which regulates the function of a large number of genes involved in the photosynthesis apparatus, carbon fixation and nitrogen metabolism. A number of transaminase and hydrolyase enzymes known to be involved in the synthesis of 2-hydroxy-5-oxoproline in animals have been identified in animal liver and kidney tissues (Cooper and Meister, 1977, CRC Critical Reviews in Biochemistry, pages 281-303; Meister, 1952, J. Biochem. 197:304). In algae, the biochemical synthesis of 2-hydroxy-5-oxoproline has not been established. Moreover, the function of 2-hydroxy-5-oxoproline in algae is unknown.
[0009] Unkefer et al., U.S. Pat. No. 6,593,275, disclose a dramatic increase in the growth rate of algae when treated with 2-hydroxy-5-oxoproline. Continuously culturing the algae in the presence of this compound or mixtures of this compound with other prolines will enrich sub-strains of the algae that respond well to the prolines.
BRIEF SUMMARY OF THE INVENTION
[0010] The present disclosure relates to transgenic algae having increased growth characteristics. In one embodiment, the invention relates to transgenic algae having enhanced (faster) growth rates. Applicants have recently identified the enzyme glutamine phenylpyruvate transaminase (GPT) as a catalyst of 2-hydroxy-5-oxoproline (2-oxoglutaramate) synthesis in plants, and here disclose that transgenic algae engineered to over-express plant-derived GPT and glutamine synthetase (GS1) genes grow faster and produce higher amounts of chlorophyll compared to wild type algae.
[0011] In one embodiment, the disclosure provides the generation of GPT+GS1 Chlorella. The double-transgenic Chlorella demonstrated substantially faster growth rates that the untransformed Chlorella grown under identical conditions for the same amount of time. Methods for the generation of the transgenic algae of the invention are provided.
[0012] In one embodiment, the disclosure provides a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter.
[0013] In one embodiment, the disclosure provides a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, and wherein the algae is a green algae.
[0014] In one embodiment, the disclosure provides a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, and wherein the algae is selected from a Chlorella or Chlamydomonas species.
[0015] In one embodiment, the disclosure provides a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, and wherein the GPT transgene is a plant-derived GPT.
[0016] In one embodiment, the disclosure provides a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, and wherein the GPT is an algal-derived GPT.
[0017] In one embodiment, the disclosure provides a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, wherein the GPT transgene is a plant-derived GPT, and wherein the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO 24, SEQ ID NO: 30, SEQ ID NO:31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 44, SEQ ID NO: 47, an amino acid sequence that is at least 75% identical to SEQ ID NO: 2, an amino acid sequence that is at least 75% identical to SEQ ID NO: 9, an amino acid sequence that is at least 75% identical to SEQ ID NO: 15, an amino acid sequence that is at least 75% identical to SEQ ID NO: 19, an amino acid sequence that is at least 75% identical to SEQ ID NO: 21, an amino acid sequence that is at least 75% identical to SEQ ID NO 24, an amino acid sequence that is at least 75% identical to SEQ ID NO: 30, an amino acid sequence that is at least 75% identical to SEQ ID NO:31, an amino acid sequence that is at least 75% identical to SEQ ID NO: 32, an amino acid sequence that is at least 75% identical to SEQ ID NO: 33, an amino acid sequence that is at least 75% identical to SEQ ID NO: 34, an amino acid sequence that is at least 75% identical to SEQ ID NO: 35, an amino acid sequence that is at least 75% identical to SEQ ID NO: 36, an amino acid sequence that is at least 75% identical to SEQ ID NO: 44, and an amino acid sequence that is at least 75% identical to SEQ ID NO: 47.
[0018] In one embodiment, the disclosure provides a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, wherein the GPT is an algal-derived GPT, and wherein the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 48, and an amino acid sequence that is at least 75% identical to SEQ ID NO: 48.
[0019] In one embodiment, the disclosure provides a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, and wherein the GS transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 39, an amino acid sequence that is at least 75% identical to SEQ ID NO: 4, an amino acid sequence that is at least 75% identical to SEQ ID NO: 7, and an amino acid sequence that is at least 75% identical to SEQ ID NO: 39.
[0020] In one embodiment, the disclosure provides a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, and wherein the GPT and GS transgenes are incorporated into the genome of the algae.
[0021] In one embodiment, the disclosure provides a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, wherein the algae is a green algae, and wherein the GPT and GS transgenes are incorporated into the genome of the algae.
[0022] In one embodiment, the disclosure provides a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, wherein the algae is selected from a Chlorella or Chlamydomonas species, and wherein the GPT and GS transgenes are incorporated into the genome of the algae.
[0023] In one embodiment, the disclosure provides a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, wherein the GPT transgene is a plant-derived GPT, and wherein the GPT and GS transgenes are incorporated into the genome of the algae.
[0024] In one embodiment, the disclosure provides a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, wherein the GPT is an algal-derived GPT, and wherein the GPT and GS transgenes are incorporated into the genome of the algae.
[0025] In one embodiment, the disclosure provides a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, wherein the GPT transgene is a plant-derived GPT, wherein the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO 24, SEQ ID NO: 30, SEQ ID NO:31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 44, SEQ ID NO: 47, an amino acid sequence that is at least 75% identical to SEQ ID NO: 2, an amino acid sequence that is at least 75% identical to SEQ ID NO: 9, an amino acid sequence that is at least 75% identical to SEQ ID NO: 15, an amino acid sequence that is at least 75% identical to SEQ ID NO: 19, an amino acid sequence that is at least 75% identical to SEQ ID NO: 21, an amino acid sequence that is at least 75% identical to SEQ ID NO 24, an amino acid sequence that is at least 75% identical to SEQ ID NO: 30, an amino acid sequence that is at least 75% identical to SEQ ID NO:31, an amino acid sequence that is at least 75% identical to SEQ ID NO: 32, an amino acid sequence that is at least 75% identical to SEQ ID NO: 33, an amino acid sequence that is at least 75% identical to SEQ ID NO: 34, an amino acid sequence that is at least 75% identical to SEQ ID NO: 35, an amino acid sequence that is at least 75% identical to SEQ ID NO: 36, an amino acid sequence that is at least 75% identical to SEQ ID NO: 44, and an amino acid sequence that is at least 75% identical to SEQ ID NO: 47, and wherein the GPT and GS transgenes are incorporated into the genome of the algae.
[0026] In one embodiment, the disclosure provides a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, wherein the GPT is an algal-derived GPT, wherein the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 48, and an amino acid sequence that is at least 75% identical to SEQ ID NO: 48, and wherein the GPT and GS transgenes are incorporated into the genome of the algae.
[0027] In one embodiment, the disclosure provides a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, wherein the GS transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 39, an amino acid sequence that is at least 75% identical to SEQ ID NO: 4, an amino acid sequence that is at least 75% identical to SEQ ID NO: 7, and an amino acid sequence that is at least 75% identical to SEQ ID NO: 39, and wherein the GPT and GS transgenes are incorporated into the genome of the algae.
[0028] In one embodiment, the disclosure provides the progeny of any generation of a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, wherein the GPT and GS transgenes are incorporated into the genome of the algae, and wherein said progeny comprises said GPT transgene and said GS transgene.
[0029] In one embodiment, the disclosure provides the progeny of any generation of a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, wherein the algae is a green algae, wherein the GPT and GS transgenes are incorporated into the genome of the algae, and wherein said progeny comprises said GPT transgene and said GS transgene.
[0030] In one embodiment, the disclosure provides the progeny of any generation of a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, wherein the algae is selected from a Chlorella or Chlamydomonas species, wherein the GPT and GS transgenes are incorporated into the genome of the algae, and wherein said progeny comprises said GPT transgene and said GS transgene.
[0031] In one embodiment, the disclosure provides the progeny of any generation of a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, wherein the GPT transgene is a plant-derived GPT, wherein the GPT and GS transgenes are incorporated into the genome of the algae, and wherein said progeny comprises said GPT transgene and said GS transgene.
[0032] In one embodiment, the disclosure provides the progeny of any generation of a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, wherein the GPT is an algal-derived GPT, wherein the GPT and GS transgenes are incorporated into the genome of the algae, and wherein said progeny comprises said GPT transgene and said GS transgene.
[0033] In one embodiment, the disclosure provides the progeny of any generation of a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, wherein the GPT transgene is a plant-derived GPT, wherein the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO 24, SEQ ID NO: 30, SEQ ID NO:31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 44, SEQ ID NO: 47, an amino acid sequence that is at least 75% identical to SEQ ID NO: 2, an amino acid sequence that is at least 75% identical to SEQ ID NO: 9, an amino acid sequence that is at least 75% identical to SEQ ID NO: 15, an amino acid sequence that is at least 75% identical to SEQ ID NO: 19, an amino acid sequence that is at least 75% identical to SEQ ID NO: 21, an amino acid sequence that is at least 75% identical to SEQ ID NO 24, an amino acid sequence that is at least 75% identical to SEQ ID NO: 30, an amino acid sequence that is at least 75% identical to SEQ ID NO:31, an amino acid sequence that is at least 75% identical to SEQ ID NO: 32, an amino acid sequence that is at least 75% identical to SEQ ID NO: 33, an amino acid sequence that is at least 75% identical to SEQ ID NO: 34, an amino acid sequence that is at least 75% identical to SEQ ID NO: 35, an amino acid sequence that is at least 75% identical to SEQ ID NO: 36, an amino acid sequence that is at least 75% identical to SEQ ID NO: 44, and an amino acid sequence that is at least 75% identical to SEQ ID NO: 47, wherein the GPT and GS transgenes are incorporated into the genome of the algae, and wherein said progeny comprises said GPT transgene and said GS transgene.
[0034] In one embodiment, the disclosure provides the progeny of any generation of a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, wherein the GPT is an algal-derived GPT, wherein the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 48, and an amino acid sequence that is at least 75% identical to SEQ ID NO: 48, wherein the GPT and GS transgenes are incorporated into the genome of the algae, and wherein said progeny comprises said GPT transgene and said GS transgene.
[0035] In one embodiment, the disclosure provides the progeny of any generation of a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, wherein the GS transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 39, an amino acid sequence that is at least 75% identical to SEQ ID NO: 4, an amino acid sequence that is at least 75% identical to SEQ ID NO: 7, and an amino acid sequence that is at least 75% identical to SEQ ID NO: 39, wherein the GPT and GS transgenes are incorporated into the genome of the algae, and wherein said progeny comprises said GPT transgene and said GS transgene.
[0036] In one embodiment, the disclosure provides a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, wherein the GPT and GS transgenes are incorporated into the genome of the algae, and wherein the transgenic algae displays a faster growth rate, increased chlorophyll production, increased ribulose bisphosphate carboxylase (RUBISCO) level, increased nitrogen use efficiency, increased biomass yield, increased GPT activity, increased GS activity, increased 2-oxoglutaramate levels, and/or increased tolerance to salt or saline conditions when compared to an analogous wild-type or untransformed algae.
[0037] In one embodiment, the disclosure provides a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, wherein the algae is a green algae, wherein the GPT and GS transgenes are incorporated into the genome of the algae, and wherein the transgenic algae displays a faster growth rate, increased chlorophyll production, increased ribulose bisphosphate carboxylase (RUBISCO) level, increased nitrogen use efficiency, increased biomass yield, increased GPT activity, increased GS activity, increased 2-oxoglutaramate levels, and/or increased tolerance to salt or saline conditions when compared to an analogous wild-type or untransformed algae.
[0038] In one embodiment, the disclosure provides a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, wherein the algae is selected from a Chlorella or Chlamydomonas species, wherein the GPT and GS transgenes are incorporated into the genome of the algae, and wherein the transgenic algae displays a faster growth rate, increased chlorophyll production, increased ribulose bisphosphate carboxylase (RUBISCO) level, increased nitrogen use efficiency, increased biomass yield, increased GPT activity, increased GS activity, increased 2-oxoglutaramate levels, and/or increased tolerance to salt or saline conditions when compared to an analogous wild-type or untransformed algae.
[0039] In one embodiment, the disclosure provides a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, wherein the GPT transgene is a plant-derived GPT, wherein the GPT and GS transgenes are incorporated into the genome of the algae, and wherein the transgenic algae displays a faster growth rate, increased chlorophyll production, increased ribulose bisphosphate carboxylase (RUBISCO) level, increased nitrogen use efficiency, increased biomass yield, increased GPT activity, increased GS activity, increased 2-oxoglutaramate levels, and/or increased tolerance to salt or saline conditions when compared to an analogous wild-type or untransformed algae.
[0040] In one embodiment, the disclosure provides a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, wherein the GPT is an algal-derived GPT, wherein the GPT and GS transgenes are incorporated into the genome of the algae, and wherein the transgenic algae displays a faster growth rate, increased chlorophyll production, increased ribulose bisphosphate carboxylase (RUBISCO) level, increased nitrogen use efficiency, increased biomass yield, increased GPT activity, increased GS activity, increased 2-oxoglutaramate levels, and/or increased tolerance to salt or saline conditions when compared to an analogous wild-type or untransformed algae.
[0041] In one embodiment, the disclosure provides a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, wherein the GPT transgene is a plant-derived GPT, wherein the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO 24, SEQ ID NO: 30, SEQ ID NO:31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 44, SEQ ID NO: 47, an amino acid sequence that is at least 75% identical to SEQ ID NO: 2, an amino acid sequence that is at least 75% identical to SEQ ID NO: 9, an amino acid sequence that is at least 75% identical to SEQ ID NO: 15, an amino acid sequence that is at least 75% identical to SEQ ID NO: 19, an amino acid sequence that is at least 75% identical to SEQ ID NO: 21, an amino acid sequence that is at least 75% identical to SEQ ID NO 24, an amino acid sequence that is at least 75% identical to SEQ ID NO: 30, an amino acid sequence that is at least 75% identical to SEQ ID NO:31, an amino acid sequence that is at least 75% identical to SEQ ID NO: 32, an amino acid sequence that is at least 75% identical to SEQ ID NO: 33, an amino acid sequence that is at least 75% identical to SEQ ID NO: 34, an amino acid sequence that is at least 75% identical to SEQ ID NO: 35, an amino acid sequence that is at least 75% identical to SEQ ID NO: 36, an amino acid sequence that is at least 75% identical to SEQ ID NO: 44, and an amino acid sequence that is at least 75% identical to SEQ ID NO: 47, wherein the GPT and GS transgenes are incorporated into the genome of the algae, and wherein the transgenic algae displays a faster growth rate, increased chlorophyll production, increased ribulose bisphosphate carboxylase (RUBISCO) level, increased nitrogen use efficiency, increased biomass yield, increased GPT activity, increased GS activity, increased 2-oxoglutaramate levels, and/or increased tolerance to salt or saline conditions when compared to an analogous wild-type or untransformed algae.
[0042] In one embodiment, the disclosure provides a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, wherein the GPT is an algal-derived GPT, wherein the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 48, and an amino acid sequence that is at least 75% identical to SEQ ID NO: 48, wherein the GPT and GS transgenes are incorporated into the genome of the algae, and wherein the transgenic algae displays a faster growth rate, increased chlorophyll production, increased ribulose bisphosphate carboxylase (RUBISCO) level, increased nitrogen use efficiency, increased biomass yield, increased GPT activity, increased GS activity, increased 2-oxoglutaramate levels, and/or increased tolerance to salt or saline conditions when compared to an analogous wild-type or untransformed algae.
[0043] In one embodiment, the disclosure provides a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, wherein the GS transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 39, an amino acid sequence that is at least 75% identical to SEQ ID NO: 4, an amino acid sequence that is at least 75% identical to SEQ ID NO: 7, and an amino acid sequence that is at least 75% identical to SEQ ID NO: 39, wherein the GPT and GS transgenes are incorporated into the genome of the algae, and wherein the transgenic algae displays a faster growth rate, increased chlorophyll production, increased ribulose bisphosphate carboxylase (RUBISCO) level, increased nitrogen use efficiency, increased biomass yield, increased GPT activity, increased GS activity, increased 2-oxoglutaramate levels, and/or increased tolerance to salt or saline conditions when compared to an analogous wild-type or untransformed algae.
[0044] In one embodiment, the disclosure provides a method for increasing growth characteristics of an algae relative to a wild type or progenitor algae of the same species, the method including: (a) introducing a GPT transgene into the algae; (b) introducing a GS transgene into the algae or a progeny of the algae; (c) expressing the GPT transgene and the GS transgene in the algae or the progeny of the algae; and, (d) selecting an algae having an increased growth characteristic relative to an algae of the same species that does not contain a GPT transgene or a GS transgene.
[0045] In one embodiment, the disclosure provides a method for increasing growth characteristics of an algae relative to a wild type or progenitor algae of the same species, the method including: (a) introducing a GPT transgene into the algae; (b) introducing a GS transgene into the algae or a progeny of the algae; (c) expressing the GPT transgene and the GS transgene in the algae or the progeny of the algae; and, (d) selecting an algae having an increased growth characteristic relative to an algae of the same species that does not contain a GPT transgene or a GS transgene, and wherein the increased growth characteristic is selected from the group consisting of: faster growth rate, increased chlorophyll production, increased ribulose bisphosphate carboxylase (RUBISCO) level, increased nitrogen use efficiency, increased biomass yield, increased GPT activity, increased GS activity, increased 2-oxoglutaramate levels, and/or increased tolerance to salt or saline conditions.
[0046] In one embodiment, the disclosure provides a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter.
[0047] In one embodiment, the disclosure provides a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, and wherein the algae is a green algae.
[0048] In one embodiment, the disclosure provides a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, and wherein the algae is selected from a Chlorella or Chlamydomonas species.
[0049] In one embodiment, the disclosure provides a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, and wherein the GPT transgene is a plant-derived GPT.
[0050] In one embodiment, the disclosure provides a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, and wherein the GPT is an algal-derived GPT.
[0051] In one embodiment, the disclosure provides a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, wherein the GPT transgene is a plant-derived GPT, and wherein the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO 24, SEQ ID NO: 30, SEQ ID NO:31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 44, SEQ ID NO: 47, an amino acid sequence that is at least 75% identical to SEQ ID NO: 2, an amino acid sequence that is at least 75% identical to SEQ ID NO: 9, an amino acid sequence that is at least 75% identical to SEQ ID NO: 15, an amino acid sequence that is at least 75% identical to SEQ ID NO: 19, an amino acid sequence that is at least 75% identical to SEQ ID NO: 21, an amino acid sequence that is at least 75% identical to SEQ ID NO 24, an amino acid sequence that is at least 75% identical to SEQ ID NO: 30, an amino acid sequence that is at least 75% identical to SEQ ID NO:31, an amino acid sequence that is at least 75% identical to SEQ ID NO: 32, an amino acid sequence that is at least 75% identical to SEQ ID NO: 33, an amino acid sequence that is at least 75% identical to SEQ ID NO: 34, an amino acid sequence that is at least 75% identical to SEQ ID NO: 35, an amino acid sequence that is at least 75% identical to SEQ ID NO: 36, an amino acid sequence that is at least 75% identical to SEQ ID NO: 44, and an amino acid sequence that is at least 75% identical to SEQ ID NO: 47.
[0052] In one embodiment, the disclosure provides a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, and wherein the GPT is an algal-derived GPT, and wherein the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 48, and an amino acid sequence that is at least 75% identical to SEQ ID NO: 48.
[0053] In one embodiment, the disclosure provides a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, and wherein the GPT transgene is incorporated into the genome of the algae.
[0054] In one embodiment, the disclosure provides a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, wherein the algae is a green algae, and wherein the GPT transgene is incorporated into the genome of the algae.
[0055] In one embodiment, the disclosure provides a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, wherein the algae is selected from a Chlorella or Chlamydomonas species, and wherein the GPT transgene is incorporated into the genome of the algae.
[0056] In one embodiment, the disclosure provides a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, wherein the GPT transgene is a plant-derived GPT, and wherein the GPT transgene is incorporated into the genome of the algae.
[0057] In one embodiment, the disclosure provides a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, wherein the GPT is an algal-derived GPT, and wherein the GPT transgene is incorporated into the genome of the algae.
[0058] In one embodiment, the disclosure provides a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, wherein the GPT transgene is a plant-derived GPT, wherein the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO 24, SEQ ID NO: 30, SEQ ID NO:31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 44, SEQ ID NO: 47, an amino acid sequence that is at least 75% identical to SEQ ID NO: 2, an amino acid sequence that is at least 75% identical to SEQ ID NO: 9, an amino acid sequence that is at least 75% identical to SEQ ID NO: 15, an amino acid sequence that is at least 75% identical to SEQ ID NO: 19, an amino acid sequence that is at least 75% identical to SEQ ID NO: 21, an amino acid sequence that is at least 75% identical to SEQ ID NO 24, an amino acid sequence that is at least 75% identical to SEQ ID NO: 30, an amino acid sequence that is at least 75% identical to SEQ ID NO:31, an amino acid sequence that is at least 75% identical to SEQ ID NO: 32, an amino acid sequence that is at least 75% identical to SEQ ID NO: 33, an amino acid sequence that is at least 75% identical to SEQ ID NO: 34, an amino acid sequence that is at least 75% identical to SEQ ID NO: 35, an amino acid sequence that is at least 75% identical to SEQ ID NO: 36, an amino acid sequence that is at least 75% identical to SEQ ID NO: 44, and an amino acid sequence that is at least 75% identical to SEQ ID NO: 47, and wherein the GPT transgene is incorporated into the genome of the algae.
[0059] In one embodiment, the disclosure provides a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, and wherein the GPT is an algal-derived GPT, wherein the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 48, and an amino acid sequence that is at least 75% identical to SEQ ID NO: 48, and wherein the GPT transgene is incorporated into the genome of the algae.
[0060] In one embodiment, the disclosure provides a progeny of any generation of a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, wherein the GPT transgene is incorporated into the genome of the algae, and wherein said progeny comprises said GPT transgene.
[0061] In one embodiment, the disclosure provides a progeny of any generation of a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, wherein the algae is a green algae, wherein the GPT transgene is incorporated into the genome of the algae, and wherein said progeny comprises said GPT transgene.
[0062] In one embodiment, the disclosure provides a progeny of any generation of a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, wherein the algae is selected from a Chlorella or Chlamydomonas species, wherein the GPT transgene is incorporated into the genome of the algae, and wherein said progeny comprises said GPT transgene.
[0063] In one embodiment, the disclosure provides a progeny of any generation of a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, wherein the GPT transgene is a plant-derived GPT, wherein the GPT transgene is incorporated into the genome of the algae, and wherein said progeny comprises said GPT transgene.
[0064] In one embodiment, the disclosure provides a progeny of any generation of a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, wherein the GPT is an algal-derived GPT, wherein the GPT transgene is incorporated into the genome of the algae, and wherein said progeny comprises said GPT transgene.
[0065] In one embodiment, the disclosure provides a progeny of any generation of a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, wherein the GPT transgene is a plant-derived GPT, wherein the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO 24, SEQ ID NO: 30, SEQ ID NO:31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 44, SEQ ID NO: 47, an amino acid sequence that is at least 75% identical to SEQ ID NO: 2, an amino acid sequence that is at least 75% identical to SEQ ID NO: 9, an amino acid sequence that is at least 75% identical to SEQ ID NO: 15, an amino acid sequence that is at least 75% identical to SEQ ID NO: 19, an amino acid sequence that is at least 75% identical to SEQ ID NO: 21, an amino acid sequence that is at least 75% identical to SEQ ID NO 24, an amino acid sequence that is at least 75% identical to SEQ ID NO: 30, an amino acid sequence that is at least 75% identical to SEQ ID NO:31, an amino acid sequence that is at least 75% identical to SEQ ID NO: 32, an amino acid sequence that is at least 75% identical to SEQ ID NO: 33, an amino acid sequence that is at least 75% identical to SEQ ID NO: 34, an amino acid sequence that is at least 75% identical to SEQ ID NO: 35, an amino acid sequence that is at least 75% identical to SEQ ID NO: 36, an amino acid sequence that is at least 75% identical to SEQ ID NO: 44, and an amino acid sequence that is at least 75% identical to SEQ ID NO: 47, wherein the GPT transgene is incorporated into the genome of the algae, and wherein said progeny comprises said GPT transgene.
[0066] In one embodiment, the disclosure provides a progeny of any generation of a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, and wherein the GPT is an algal-derived GPT, wherein the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 48, and an amino acid sequence that is at least 75% identical to SEQ ID NO: 48, wherein the GPT transgene is incorporated into the genome of the algae, and wherein said progeny comprises said GPT transgene.
[0067] In one embodiment, the disclosure provides a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, wherein the GPT transgene is incorporated into the genome of the algae, and wherein the transgenic algae displays a faster growth rate, increased chlorophyll production, increased ribulose bisphosphate carboxylase (RUBISCO) level, increased nitrogen use efficiency, increased biomass yield, increased GPT activity, increased GS activity, increased 2-oxoglutaramate levels, and/or increased tolerance to salt or saline conditions when compared to an analogous wild-type or untransformed algae.
[0068] In one embodiment, the disclosure provides a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, wherein the algae is a green algae, wherein the GPT transgene is incorporated into the genome of the algae, and wherein the transgenic algae displays a faster growth rate, increased chlorophyll production, increased ribulose bisphosphate carboxylase (RUBISCO) level, increased nitrogen use efficiency, increased biomass yield, increased GPT activity, increased GS activity, increased 2-oxoglutaramate levels, and/or increased tolerance to salt or saline conditions when compared to an analogous wild-type or untransformed algae.
[0069] In one embodiment, the disclosure provides a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, wherein the algae is selected from a Chlorella or Chlamydomonas species, wherein the GPT transgene is incorporated into the genome of the algae, and wherein the transgenic algae displays a faster growth rate, increased chlorophyll production, increased ribulose bisphosphate carboxylase (RUBISCO) level, increased nitrogen use efficiency, increased biomass yield, increased GPT activity, increased GS activity, increased 2-oxoglutaramate levels, and/or increased tolerance to salt or saline conditions when compared to an analogous wild-type or untransformed algae.
[0070] In one embodiment, the disclosure provides a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, wherein the GPT transgene is a plant-derived GPT, wherein the GPT transgene is incorporated into the genome of the algae, and wherein the transgenic algae displays a faster growth rate, increased chlorophyll production, increased ribulose bisphosphate carboxylase (RUBISCO) level, increased nitrogen use efficiency, increased biomass yield, increased GPT activity, increased GS activity, increased 2-oxoglutaramate levels, and/or increased tolerance to salt or saline conditions when compared to an analogous wild-type or untransformed algae.
[0071] In one embodiment, the disclosure provides a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, wherein the GPT is an algal-derived GPT, wherein the GPT transgene is incorporated into the genome of the algae, and wherein the transgenic algae displays a faster growth rate, increased chlorophyll production, increased ribulose bisphosphate carboxylase (RUBISCO) level, increased nitrogen use efficiency, increased biomass yield, increased GPT activity, increased GS activity, increased 2-oxoglutaramate levels, and/or increased tolerance to salt or saline conditions when compared to an analogous wild-type or untransformed algae.
[0072] In one embodiment, the disclosure provides a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, wherein the GPT transgene is a plant-derived GPT, wherein the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO 24, SEQ ID NO: 30, SEQ ID NO:31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 44, SEQ ID NO: 47, an amino acid sequence that is at least 75% identical to SEQ ID NO: 2, an amino acid sequence that is at least 75% identical to SEQ ID NO: 9, an amino acid sequence that is at least 75% identical to SEQ ID NO: 15, an amino acid sequence that is at least 75% identical to SEQ ID NO: 19, an amino acid sequence that is at least 75% identical to SEQ ID NO: 21, an amino acid sequence that is at least 75% identical to SEQ ID NO 24, an amino acid sequence that is at least 75% identical to SEQ ID NO: 30, an amino acid sequence that is at least 75% identical to SEQ ID NO:31, an amino acid sequence that is at least 75% identical to SEQ ID NO: 32, an amino acid sequence that is at least 75% identical to SEQ ID NO: 33, an amino acid sequence that is at least 75% identical to SEQ ID NO: 34, an amino acid sequence that is at least 75% identical to SEQ ID NO: 35, an amino acid sequence that is at least 75% identical to SEQ ID NO: 36, an amino acid sequence that is at least 75% identical to SEQ ID NO: 44, and an amino acid sequence that is at least 75% identical to SEQ ID NO: 47, wherein the GPT transgene is incorporated into the genome of the algae, and wherein the transgenic algae displays a faster growth rate, increased chlorophyll production, increased ribulose bisphosphate carboxylase (RUBISCO) level, increased nitrogen use efficiency, increased biomass yield, increased GPT activity, increased GS activity, increased 2-oxoglutaramate levels, and/or increased tolerance to salt or saline conditions when compared to an analogous wild-type or untransformed algae.
[0073] In one embodiment, the disclosure provides a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, and wherein the GPT is an algal-derived GPT, wherein the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 48, and an amino acid sequence that is at least 75% identical to SEQ ID NO: 48, wherein the GPT transgene is incorporated into the genome of the algae, and wherein the transgenic algae displays a faster growth rate, increased chlorophyll production, increased ribulose bisphosphate carboxylase (RUBISCO) level, increased nitrogen use efficiency, increased biomass yield, increased GPT activity, increased GS activity, increased 2-oxoglutaramate levels, and/or increased tolerance to salt or saline conditions when compared to an analogous wild-type or untransformed algae.
[0074] In one embodiment, the disclosure provides a method for increasing growth characteristics of an algae relative to an wild type or progenitor algae of the same species, the method including: (a) introducing a GPT transgene into the algae; (b) expressing the GPT transgene in the algae or the progeny of the algae; and, (c) selecting an algae having an increased growth characteristic relative to an algae of the same species that does not comprise a GPT transgene.
[0075] In one embodiment, the disclosure provides a method for increasing growth characteristics of an algae relative to an wild type or progenitor algae of the same species, the method including: (a) introducing a GPT transgene into the algae; (b) expressing the GPT transgene in the algae or the progeny of the algae; and, (c) selecting an algae having an increased growth characteristic relative to an algae of the same species that does not comprise a GPT transgene, wherein the increased growth characteristic is selected from the group consisting of: faster growth rate, increased chlorophyll production, increased ribulose bisphosphate carboxylase (RUBISCO) level, increased nitrogen use efficiency, increased biomass yield, increased GPT activity, increased GS activity, increased 2-oxoglutaramate levels, and/or increased tolerance to salt or saline conditions.
BRIEF DESCRIPTION OF THE DRAWINGS
[0076] FIG. 1. Photograph of tissue culture plates, showing GPT+GS1 transformed Chlorella vulgaris on the right, and untransformed Chlorella vulgaris (control) on the left. See, Example 1, I nfra.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0077] Unless otherwise defined, all terms of art, notations and other scientific terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd. edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology (Ausbel et al., eds., John Wiley & Sons, Inc. 2001). As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer defined protocols and/or parameters unless otherwise noted.
[0078] The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof ("polynucleotides") in either single- or double-stranded form. Unless specifically limited, the term "polynucleotide" encompasses nucleic acids containing known analogues of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g. degenerate codon substitutions) and complementary sequences and as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., 1991, Nucleic Acid Res. 19:5081; Ohtsuka et al., 1985 J. Biol. Chem. 260:2605-2608; and Cassol et al., 1992; Rossolini et al., 1994, Mol. Cell. Probes 8:91-98). The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.
[0079] The term "promoter" refers to a nucleic acid control sequence or sequences that direct transcription of an operably linked nucleic acid. Promoters include necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. A "constitutive" promoter is a promoter that is active under most environmental and developmental conditions. An "inducible" promoter is a promoter that is active under environmental or developmental regulation. The term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
[0080] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.
[0081] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
[0082] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
[0083] The term "algae" refers to photosynthetic organisms of multiple phylogenetic groups, and includes numerous unicellular and multicellular species. The term "algae" as used herein includes organisms of the following phylogenetic groups: Chlorophyta (green algae; includes mostly fresh water species); Phaeophyta (brown algae; includes mostly marine species); Rhodophyta (red algae; includes mostly marine species); Chrysophyta; Xanthophyta; Bacillariophyta; Euglenophyta; Cryptophyta; Pyrrophyta; Raphidophyta; Haptophyta; Eustigmatophyta; Prasinophyta; Glaucophyta and Cyanobacteria (prokaryotic, blue-green algae). The class of algae which can be used in the methods of the invention is generally as broad as the class of algae amenable to transformation techniques.
[0084] Algae of the present disclosure include but are not limited to organisms of the following genera: Acanthoceras, Acanthococcus, Acaryochloris, Achnanthes, Achnanthidium, Actinastrum, Actinochloris, Actinocyclus, Actinotaenium, Amphichrysis, Amphidinium, Amphikrikos, Amphipleura, Amphiprora, Amphithrix, Amphora, Anabaena, Anabaenopsis, Aneumastus, Ankistrodesmus, Ankyra, Anomoeoneis, Apatococcus, Aphanizomenon, Aphanocapsa, Aphanochaete, Aphanothece, Apiocystis, Apistonema, Arthrodesmus, Artherospira, Ascochloris, Asterionella, Asterococcus, Audouinella, Aulacoseira, Bacillaria, Balbiania, Bambusina, Bangia, Basichlamys, Batrachospermum, Binuclearia, Bitrichia, Blidingia, Botrdiopsis, Botrydium, Botryococcus, Botryosphaerella, Brachiomonas, Brachysira, Brachytrichia, Brebissonia, Bulbochaete, Bumilleria, Bumilleriopsis, Caloneis, Calothrix, Campylodiscus, Capsosiphon, Carteria, Catena, Cavinula, Centritractus, Centronella, Ceratium, Chaetoceros, Chaetochloris, Chaetomorpha, Chaetonella, Chaetonema, Chaetopeltis, Chaetophora, Chaetosphaeridium, Chamaesiphon, Chara, Characiochloris, Characiopsis, Characium, Charales, Chilomonas, Chlainomonas, Chlamydoblepharis, Chlamydocapsa, Chlamydomonas, Chlamydomonopsis, Chlamydomyxa, Chlamydonephris, Chlorangiella, Chlorangiopsis, Chlorella, Chlorobotrys, Chlorobrachis, Chlorochytrium, Chlorococcum, Chlorogloea, Chlorogloeopsis, Chlorogonium, Chlorolobion, Chloromonas, Chlorophysema, Chlorophyta, Chlorosaccus, Chlorosarcina, Choricystis, Chromophyton, Chromulina, Chroococcidiopsis, Chroococcus, Chroodactylon, Chroomonas, Chroothece, Chrysamoeba, Chrysapsis, Chrysidiastrum, Chrysocapsa, Chrysocapsella, Chrysochaete, Chrysochromulina, Chrysococcus, Chrysocrinus, Chrysolepidomonas, Chrysolykos, Chrysonebula, Chrysophyta, Chrysopyxis, Chrysosaccus, Chrysophaerella, Chrysostephanosphaera, Clodophora, Clastidium, Closteriopsis, Closterium, Coccomyxa, Cocconeis, Coelastrella, Coelastrum, Coelosphaerium, Coenochloris, Coenococcus, Coenocystis, Colacium, Coleochaete, Collodictyon, Compsogonopsis, Compsopogon, Conjugatophyta, Conochaete, Coronastrum, Cosmarium, Cosmioneis, Cosmocladium, Crateriportula, Craticula, Crinalium, Crucigenia, Crucigeniella, Cryptoaulax, Cryptomonas, Cryptophyta, Ctenophora, Cyanidioschyzon, Cyanodictyon, Cyanonephron, Cyanophora, Cyanophyta, Cyanothece, Cyanothomonas, Cyclonexis, Cyclostephanos, Cyclotella, Cylindrocapsa, Cylindrocystis, Cylindrospermum, Cylindrotheca, Cymatopleura, Cymbella, Cymbellonitzschia, Cystodinium Dactylococcopsis, Debarya, Denticula, Dermatochrysis, Dermocarpa, Dermocarpella, Desmatractum, Desmidium, Desmococcus, Desmonema, Desmosiphon, Diacanthos, Diacronema, Diadesmis, Diatoma, Diatomella, Dicellula, Dichothrix, Dichotomococcus, Dicranochaete, Dictyochloris, Dictyococcus, Dictyosphaerium, Didymocystis, Didymogenes, Didymosphenia, Dilabifilum, Dimorphococcus, Dinobryon, Dinococcus, Diplochloris, Diploneis, Diplostauron, Distrionella, Docidium, Draparnaldia, Dunaliella, Dysmorphococcus, Ecballocystis, Elakatothrix, Ellerbeckia, Encyonema, Enteromorpha, Entocladia, Entomoneis, Entophysalis, Epichrysis, Epipyxis, Epithemia, Eremosphaera, Euastropsis, Euastrum, Eucapsis, Eucocconeis, Eudorina, Euglena, Euglenophyta, Eunotia, Eustigmatophyta, Eutreptia, Fallacia, Fischerella, Fragilaria, Fragilariforma, Franceia, Frustulia, Curcilla, Geminella, Genicularia, Glaucocystis, Glaucophyta, Glenodiniopsis, Glenodinium, Gloeocapsa, Gloeochaete, Gloeochrysis, Gloeococcus, Gloeocystis, Gloeodendron, Gloeomonas, Gloeoplax, Gloeothece, Gloeotila, Gloeotrichia, Gloiodictyon, Golenkinia, Golenkiniopsis, Gomontia, Gomphocymbella, Gomphonema, Gomphosphaeria, Gonatozygon, Gongrosia, Gongrosira, Goniochloris, Gonium, Gonyostomum, Granulochloris, Granulocystopsis, Groenbladia, Gymnodinium, Gymnozyga, Gyrosigma, Haematococcus, Hafniomonas, Hallassia, Hammatoidea, Hannaea, Hantzschia, Hapalosiphon, Haplotaenium, Haptophyta, Haslea, Hemidinium, Hemitoma, Heribaudiella, Heteromastix, Heterothrix, Hibberdia, Hildenbrandia, Hillea, Holopedium, Homoeothrix, Hormanthonema, Hormotila, Hyalobrachion, Hyalocardium, Hyalodiscus, Hyalogonium, Hyalotheca, Hydrianum, Hydrococcus, Hydrocoleum, Hydrocoryne, Hydrodictyon, Hydrosera, Hydrurus, Hyella, Hymenomonas, Isthmochloron, Johannesbaptistia, Juranyiella, Kappaphycus Karayevia, Kathablepharis, Katodinium, Kephyrion, Keratococcus, Kirchneriella, Klebsormidium, Kolbesia, Koliella, Komarekia, Korshikoviella, Kraskella, Lagerheimia, Lagynion, Laminaria, Lamprothamnium, Lemanea, Lepocinclis, Leptosira, Lobococcus, Lobocystis, Lobomonas, Luticola, Lyngbya, Malleochloris, Mallomonas, Mantoniella, Marssoniella, Martyana, Mastigocoleus, Gastogloia, Melosira, Merismopedia, Mesostigma, Mesotaenium, Micractinium, Micrasterias, Microchaete, Microcoleus, Microcystis, Microglena, Micromonas, Microspora, Microthamnion, Mischococcus, Monochrysis, Monodus, Monomastix, Monoraphidium, Monostroma, Mougeotia, Mougeotiopsis, Myochloris, Myromecia, Myxosarcina, Naegeliella, Nannochloris, Nautococcus, Navicula, Neglectella, Neidium, Nephroclamys, Nephrocytium, Nephrodiella, Nephroselmis, Netrium, Nitella, Nitellopsis, Nitzschia, Nodularia, Nostoc, Ochromonas, Oedogonium, Oligochaetophora, Onychonema, Oocardium, Oocystis, Opephora, Ophiocytium, Orthoseira, Oscillatoria, Ostreococcus, Oxyneis, Pachycladella, Palmella, Palmodictyon, Pnadorina, Pannus, Paralia, Pascherina, Paulschulzia, Pediastrum, Pedinella, Pedinomonas, Pedinopera, Pelagodictyon, Penium, Peranema, Peridiniopsis, Peridinium, Peronia, Petroneis, Phacotus, Phacus, Phaeaster, Phaeodactylum Phaeodermatium, Phaeophyta, Phaeosphaera, Phaeothamnion, Phormidium, Phycopeltis, Phyllariochloris, Phyllocardium, Phyllomitas, Pinnularia, Pitophora, Placoneis, Planctonema, Planktosphaeria, Planothidium, Plectonema, Pleodorina, Pleurastrum, Pleurocapsa, Pleurocladia, Pleurodiscus, Pleurosigma, Pleurosira, Pleurotaenium, Pocillomonas, Podohedra, Polyblepharides, Polychaetophora, Polyedriella, Polyedriopsis, Polygoniochloris, Polyepidomonas, Polytaenia, Polytoma, Polytomella, Porphyra, Porphyridium, Posteriochromonas, Prasinochloris, Prasinocladus, Prasinophyta, Prasiola, Prochlorphyta, Prochlorothrix, Protoderma, Protosiphon, Provasoliella, Prymnesium, Psammodictyon, Psammothidium, Pseudanabaena, Pseudenoclonium, Psuedocarteria, Pseudochate, Pseudocharacium, Pseudococcomyxa, Pseudodictyosphaerium, Pseudokephyrion, Pseudoncobyrsa, Pseudoquadrigula, Pseudosphaerocystis, Pseudostaurastrum, Pseudostaurosira, Pseudotetrastrum, Pteromonas, Punctastruata, Pyramichlamys, Pyramimonas, Pyrrophyta, Quadrichloris, Quadricoccus, Quadrigula, Radiococcus, Radiofilum, Raphidiopsis, Raphidocelis, Raphidonema, Raphidophyta, Peimeria, Rhabdoderma, Rhabdomonas, Rhizoclonium, Rhodomonas, Rhodophyta, Rhoicosphenia, Rhopalodia, Rivularia, Rosenvingiella, Rossithidium, Roya, Scenedesmus, Scherffelia, Schizochlamydella, Schizochlamys, Schizomeris, Schizothrix, Schroederia, Scolioneis, Scotiella, Scotiellopsis, Scourfieldia, Scytonema, Selenastrum, Selenochloris, Sellaphora, Semiorbis, Siderocelis, Diderocystopsis, Dimonsenia, Siphononema, Sirocladium, Sirogonium, Skeletonema, Sorastrum, Spermatozopsis, Sphaerellocystis, Sphaerellopsis, Sphaerodinium, Sphaeroplea, Sphaerozosma, Spiniferomonas, Spirogyra, Spirotaenia, Spirulina, Spondylomorum, Spondylosium, Sporotetras, Spumella, Staurastrum, Stauerodesmus, Stauroneis, Staurosira, Staurosirella, Stenopterobia, Stephanocostis, Stephanodiscus, Stephanoporos, Stephanosphaera, Stichococcus, Stichogloea, Stigeoclonium, Stigonema, Stipitococcus, Stokesiella, Strombomonas, Stylochrysalis, Stylodinium, Styloyxis, Stylosphaeridium, Surirella, Sykidion, Symbiodinium, Symploca, Synechococcus, Synechocystis, Synedra, Synochromonas, Synura, Tabellaria, Tabularia, Teilingia, Temnogametum, Tetmemorus, Tetrachlorella, Tetracyclus, Tetradesmus, Tetraedriella, Tetraedron, Tetraselmis, Tetraspora, Tetrastrum, Thalassiosira, Thamniochaete, Thorakochloris, Thorea, Tolypella, Tolypothrix, Trachelomonas, Trachydiscus, Trebouxia, Trentepholia, Treubaria, Tribonema, Trichodesmium, Trichodiscus, Trochiscia, Tryblionella, Ulothrix, Uroglena, Uronema, Urosolenia, Urospora, Uva, Vacuolaria, Vaucheria, Volvox, Volvulina, Westella, Woloszynskia, Xanthidium, Xanthophyta, Xenococcus, Zygnema, Zygnemopsis, and Zygonium.
[0085] The terms "GPT polynucleotide" and "GPT nucleic acid" are used interchangeably herein, and refer to a full length or partial length polynucleotide sequence of a gene which encodes a polypeptide involved in catalyzing the synthesis of 2-oxoglutaramate, and includes polynucleotides containing both translated (coding) and un-translated sequences, as well as the complements thereof. The term "GPT coding sequence" refers to the part of the gene which is transcribed and encodes a GPT protein. The term "targeting sequence" refers to the amino terminal part of a protein which directs the protein into a subcellular compartment of a cell, such as a chloroplast. GPT polynucleotides are further defined by their ability to hybridize under defined conditions to the GPT polynucleotides specifically disclosed herein, or to PCR products derived therefrom.
[0086] A "GPT transgene" is a nucleic acid molecule including a GPT polynucleotide which is exogenous to a transgenic algae harboring the nucleic acid molecule, or which is exogenous to an ancestor algae, of a transgenic algae harboring the GPT polynucleotide. More particularly, the exogenous GPT transgene will be heterogeneous with any GPT polynucleotide sequence present in wild-type algae into which the GPT transgene is inserted. To this extent, the scope of the heterogeneity required need only be a single nucleotide difference. However, preferably the heterogeneity will be in the order of an identity between sequences selected from the following identities: 0.01%, 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, 15%, and 20%.
[0087] The terms "GS polynucleotide" and "GS nucleic acid" are used interchangeably herein, and refer to a full length or partial length polynucleotide sequence of a gene which encodes a glutamine synthetase protein, and includes polynucleotides containing both translated (coding) and un-translated sequences, as well as the complements thereof. The term "GS coding sequence" refers to the part of the gene which is transcribed and encodes a GS protein. The terms "GS1 polynucleotide" and "GS1 nucleic acid" are used interchangeably herein, and refer to a full length or partial length polynucleotide sequence of a gene which encodes a glutamine synthetase isoform 1 protein, and includes polynucleotides containing both translated (coding) and un-translated sequences, as well as the complements thereof. The term "GS1 coding sequence" refers to the part of the gene which is transcribed and encodes a GS1 protein.
[0088] A "GS transgene" is a nucleic acid molecule including a GS polynucleotide which is exogenous to a transgenic algae, or which is exogenous to an ancestor algae of a transgenic algae harboring the GS polynucleotide. A "GS1 transgene" is a nucleic acid molecule including a GS1 polynucleotide which is exogenous to a transgenic algae harboring the nucleic acid molecule, or which is exogenous to an ancestor algae of a transgenic algae harboring the GS1 polynucleotide. More particularly, the exogenous GS or GS1 transgene will be heterogeneous with any GS or GS1 polynucleotide sequence present in wild-type algae into which the GS or GS1 transgene is inserted. To this extent the scope of the heterogeneity required need only be a single nucleotide difference. However, preferably the heterogeneity will be in the order of an identity between sequences selected from the following identities: 0.01%, 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, 15%, and 20%.
[0089] Exemplary GPT from algae, higher plants and a fish are presented herein, and include GPT sequences derived from Chlorella, Arabidopsis, rice, barley, bamboo, soybean, grape, and zebra fish. GS and GS1 proteins are known; exemplary GS1 sequences provided herein include Arabidopsis and Hordeum.
[0090] Partial length GPT polynucleotides include polynucleotide sequences encoding N- or C-terminal truncations of GPT, mature GPT (without targeting sequence) as well as sequences encoding domains of GPT. Exemplary GPT polynucleotides encoding N-terminal truncations of GPT include Arabidopsis-30, -45 and -56 constructs, in which coding sequences for the first 30, 45, and 56, respectively, amino acids of the full length GPT structure of SEQ ID NO: 2 are eliminated.
[0091] In employing the GPT polynucleotides of the invention in the generation of transformed algal cells and transgenic algae, one of skill will recognize that the inserted polynucleotide sequence need not be identical, but may be only "substantially identical" to a sequence of the gene from which it was derived, as further defined below. The term "GPT polynucleotide" specifically encompasses such substantially identical variants. Similarly, one of skill will recognize that because of codon degeneracy, a number of polynucleotide sequences will encode the same polypeptide, and all such polynucleotide sequences are meant to be included in the term GPT polynucleotide. In addition, the term specifically includes those sequences substantially identical (determined as described below) with a GPT polynucleotide sequence disclosed herein and that encode polypeptides that are either mutants of wild type GPT polypeptides or retain the function of the GPT polypeptide (e.g., resulting from conservative substitutions of amino acids in a GPT polypeptide). The term "GPT polynucleotide" therefore also includes such substantially identical variants.
[0092] The term "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.
[0093] As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.
[0094] The following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (1), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).
[0095] Macromolecular structures such as polypeptide structures can be described in terms of various levels of organization. For a general discussion of this organization, see, e.g., Alberts et al., Molecular Biology of the Cell (3rd ed., 1994) and Cantor and Schimmel, Biophysical Chemistry Part 1: The Conformation of Biological Macromolecules (1980). "Primary structure" refers to the amino acid sequence of a particular peptide. "Secondary structure" refers to locally ordered, three dimensional structures within a polypeptide. These structures are commonly known as domains. Domains are portions of a polypeptide that form a compact unit of the polypeptide and are typically 25 to approximately 500 amino acids long. Typical domains are made up of sections of lesser organization such as stretches of β-sheet and α-helices. "Tertiary structure" refers to the complete three dimensional structure of a polypeptide monomer. "Quaternary structure" refers to the three dimensional structure formed by the noncovalent association of independent tertiary units. Anisotropic terms are also known as energy terms.
[0096] The term "isolated" refers to material which is substantially or essentially free from components which normally accompany the material as it is found in its native or natural state. However, the term "isolated" is not intended refer to the components present in an electrophoretic gel or other separation medium. An isolated component is free from such separation media and in a form ready for use in another application or already in use in the new application/milieu. An "isolated" antibody is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
[0097] The term "heterologous" when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For instance, a nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a nucleic acid encoding a protein from one source and a nucleic acid encoding a peptide sequence from another source. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).
[0098] The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 70% identity, preferably 75%, 80%, 85%, 90%, or 95% identity over a specified region, when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using a sequence comparison algorithms, or by manual alignment and visual inspection. This definition also refers to the complement of a test sequence, which has substantial sequence or subsequence complementarity when the test sequence has substantial identity to a reference sequence. This definition also refers to the complement of a test sequence, which has substantial sequence or subsequence complementarity when the test sequence has substantial identity to a reference sequence.
[0099] When percentage of sequence identity is used in reference to polypeptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, where amino acids residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the polypeptide. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution.
[0100] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
[0101] A "comparison window", as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
[0102] Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, 1981, Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman & Wunsch, 1970, J. Mol. Biol. 48:443, by the search for similarity method of Pearson & Lipman, 1988, Proc. Narl. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).
[0103] A preferred example of algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., 1977, Nue. Acids Res. 25:3389-3402 and Altschul et al., 1990, J. Mol. Biol. 215:403-410, respectively. BLAST and BLAST 2.0 are used, typically with the default parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.
[0104] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, 1993, Proc. Nat'l. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
[0105] The phrase "stringent hybridization conditions" refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acid, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993). Generally, highly stringent conditions are selected to be about 5-10° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. Low stringency conditions are generally selected to be about 15-30° C. below the Tm. Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions will be those in which the salt concentration is less than about 1.0M sodium ion, typically about 0.01 to 1.0M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide For selective or specific hybridization, a positive signal is at least two times background, preferably 10 times background hybridization.
[0106] Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cased, the nucleic acids typically hybridize under moderately stringent hybridization conditions.
[0107] Genomic DNA or cDNA including GPT polynucleotides may be identified in standard Southern blots under stringent conditions using the GPT polynucleotide sequences disclosed here. For this purpose, suitable stringent conditions for such hybridizations are those which include a hybridization in a buffer of 40% formamide, 1M NaCl, 1% SDS at 37° C., and at least one wash in 0.2×SSC at a temperature of at least about 50° C., usually about 55° C. to about 60° C., for 20 minutes, or equivalent conditions. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions may be utilized to provide conditions of similar stringency.
[0108] A further indication that two polynucleotides are substantially identical is if the reference sequence, amplified by a pair of oligonucleotide primers, can then be used as a probe under stringent hybridization conditions to isolate the test sequence from a cDNA or genomic library, or to identify the test sequence in, e.g., a northern or Southern blot.
Transgenic Algae:
[0109] The invention provides novel transgenic algae exhibiting faster growth and increased chlorophyll production. The transgenic algae of the invention are generated by introducing into algae cells one or more expressible genetic constructs capable of driving the expression of one or more polynucleotides encoding glutamine phenylpyruvate transaminase (GPT), and in some embodiments, one or more polynucleotides encoding glutamine synthetase (GS) and GPT.
[0110] The transgenic algae of the invention may be of any species capable of transformation, including those from the subphyla Chlorophyta, Chrysophyta, Phaeophyta, Rhodophyta, as well as the Cyanobacteria. In the last few years, successful genetic transformation of ˜25 algal species has been demonstrated, mostly via nuclear transformation (Hallmann, 2007, Transgenic Plant Journal 1:81-98). Among these, at least ten green algae species have been successfully transformed (mostly all unicellular species). Several species of red algae, brown algae and diatom species has also been reported (Hallmann, 2007, supra).
[0111] Improving the production step of algal biofuels production is being approached in more or less the standard ways of improving biological production, i.e., by optimizing nutrients, growth conditions and light/energy supply. Mineral nutrient supplies are being optimized through detailed studies of the need for each nutrient, and at what stoichiometry relative to other nutrients. The availability of carbon dioxide is being maximized typically with various delivery systems, each designed to maximize the amount of carbon dioxide dissolved in the water. As well, pH is being maintained at or near the optimum for growth.
[0112] Some attempts have been made to grow algal faster and more economically by establishing heterotrophic growth conditions. This has been achieved by providing fixed carbon, such as sugars, for the algae to use as carbon and energy sources. Algae also require an optimal amount of light to provide the energy for growth. The light impinging upon an algal cell is converted to chemical energy and used to drive the algal metabolism and cell growth. The amount of light striking algal cells in growing cultures is impacted by culture density, the distance the light must penetrate into the culture (i.e., the cells at the surface receive more light than cells further from the surface), and the amount of light receptors within an algal cell. Algal bioreactor designs are being tested that range from deep or shallow horizontal ponds to vertical glass/plastic reactors.
[0113] Alga cells over-expressing GPT and GS transgenes can be expected to take much better advantage of the optimized nutrients and the high availability of carbon dioxide, because they can be expected to increase their carbon dioxide fixing machinery, the ribulose bisphosphate carboxylase (RUBISCO) enzyme protein and activity state. If algae respond in the same way GPT+GS1 transgenic plants do (see co-owned, co-pending U.S. patent application Ser. No. 12/551,271), the over-expression of the GPT and GS transgenes in algae may be accompanied by increased expression of genes encoding RUBISCO subunits and the RUBISCO activase enzyme which controls the activity state of RUBISCO. The transgenic algae of the invention may also increase their capacity for and rate of uptake of nitrogen-based nutrients such as nitrate and ammonia. Such increased carbon fixation will result in increased flux through central metabolism and the incumbent increase in the concentration of organic acids that are known to induce production of the nitrogen uptake transporters.
[0114] In stable transformation embodiments of the invention, one or more copies of the expressible genetic construct become integrated into the host algae genome, thereby providing increased GS and/or GPT enzyme capacity into the algae, which may serve to mediate increased synthesis of 2-oxoglutaramate therein, which in turn signals metabolic gene expression, resulting in increased algal growth. 2-oxoglutaramate is a metabolite which is an extremely potent effector of gene expression, metabolism and plant growth (U.S. Pat. No. 6,555,500), and which may play a pivotal role in the coordination of the carbon and nitrogen metabolism systems in plants (Lancien et al., 2000, Enzyme Redundancy and the Importance of 2-Oxoglutarate in Higher Plants Ammonium Assimilation, Plant Physiol. 123:817-824).
[0115] The invention also provides methods of generating transgenic algae having faster growth rates. In one embodiment, a method of generating a transgenic algae having a faster growth rate, introducing into an algal cell an expression cassette including a nucleic acid molecule encoding a GPT transgene, under the control of a suitable promoter capable of driving the expression of the transgene, so as to yield a transformed algal cell, and obtaining a transgenic algae which expresses the encoded GPT. In another embodiment, a method of generating a transgenic algae having a faster growth rate comprises introducing into an algal cell one or more nucleic acid constructs or expression cassettes including nucleic acid molecules encoding a GPT transgene and an GS transgene, under the control of one or more suitable promoters (and, optionally, other regulatory elements) capable of driving the expression of the transgenes, so as to yield an algal cell transformed thereby, and obtaining a transgenic algae which expresses the GPT and GS transgenes.
[0116] GPT and GS transgenes suitable for use in generating the transgenic algae of the invention are described in co-owned, co-pending U.S. patent application Ser. No. 12/551,271. Other GPT polynucleotides suitable for use as GPT transgenes in the practice of the invention may be obtained by various means, as will be appreciated by one skilled in the art, tested for the ability to direct the expression of a GPT with GPT activity in a recombinant expression, or in a transient in planta expression system (U.S. Ser. No. 12/551,271, supra), or preferably in a transgenic algae.
Transgene Constructs/Algal Expression Vectors
[0117] In order to generate the transgenic algae of the invention, the gene coding sequence for the desired transgene(s) must be incorporated into a nucleic acid construct (also interchangeably referred to herein as a/an (transgene) expression vector, expression cassette, expression construct or expressible genetic construct), which can direct the expression of the transgene sequence in transformed algal cells. Such nucleic acid constructs carrying the transgene(s) of interest may be introduced into an algal cell or cells using a number of methods known in the art, including but not limited to electroporation, DNA bombardment or biolistic approaches, microinjection, and via the use of various DNA-based vectors. Once introduced into the transformed algal cell, the nucleic acid construct may direct the expression of the incorporated transgene(s) (i.e., GPT), either in a transient or stable fashion. Stable expression is preferred, and is achieved by utilizing transformation vectors which are able to direct the chromosomal integration of the transgene construct.
[0118] The basic elements of a nucleic acid construct for use in generating the transgenic algae of the invention are: a suitable promoter capable of directing the functional expression of the transgene(s) in a transformed algae cell, the transgene(s) (i.e., GPT coding sequence) operably linked to the promoter, preferably a suitable transcription termination sequence (i.e., nopaline synthetic enzyme gene terminator) operably linked to the transgene, and sometimes other elements useful for controlling the expression of the transgene, as well as one or more selectable marker genes suitable for selecting the desired transgenic product (i.e., antibiotic resistance genes).
[0119] Various plant, algae and animal GPT protein sequences and encoding DNA and GPT transgene expression constructs are presented in the Table of Sequences, infra. Similarly, various GS1 protein sequences and encoding DNA and GS1 transgene expression constructs are provided. These sequences are provided as examples and should not be considered limiting.
[0120] Typically, algae is transformed by causing the temporal permeabilization of the cell membrane, there by enabling vector DNA to enter the algal cell. DNA integration occurs naturally, by recombination events, resulting in ectopic integration into the algal genome, resulting in stable events. Biolistic approaches, such as particle bombardment, are typically used to transform algal cells. Polyethylene glycol mixtures using DNA coated particle has also been successfully utilized. Additionally, certain wall-reduced algae strains have been employed, in order to achieve protoplast transformation in the presence of polyethylene glycol and the transgene construct. Electroporation and even Agrobacterium-mediated transformation of algae has been reported. For details, and references to published reports of various transformation protocols and vector systems, see Hallmann, 2007, supra for review.
[0121] Any promoter capable of being functional in algae may be used to direct the expression of the GPT or GPT+GS transgene constructs. In preferred embodiments, the promoter may be an endogenous algal promoter. Various vectors used to transform algae are known, including plasmid vectors which become integrated into the nucleus of algal cells and there direct the cytoplasmic expression of the transgene products (i.e., plasmid pSSCR7, Davies et al., 1994, Plant Cell 6:53-63). Other nuclear-directed vectors direct transgene expression products to the periplasm. One such vector is a derivative of pSSCR7, modified to incorporate a 5' aryl sulfatase periplasmic targeting transit sequence (Davies et al., 1994, supra). Still other vectors direct transgene integration to the chloroplast plastome by homologous recombination, whereby transgene expression is localized to the chloroplast (Hutchinson, et al., 1996, In: Molecular Genetics of Photosynthesis, Frontiers in Molecular Biology. Anderson B., Salter A H, and Barber J. eds., Oxford University Press, pp. 180-196).
EXAMPLES
[0122] Various aspects of the invention are further described and illustrated by way of the example which follows, which is not intended to limit the scope of the invention.
Example 1
Generation of Transgenic Chlorella
Materials and Methods:
[0123] Chlorella vulgaris strain #259 was purchased from Culture Collection of Algae at the University of Texas (UTEX, Austin, Tex.), and maintained as recommended on Bristol medium.
[0124] Two transgene expression vectors were assembled and used to transform the algae with electroporation. The first vector was the Cambia 1201 vector containing Tomato Rubisco small subunit promoter and Arabidopsis GS (SEQ ID NO: 45). The second vector was the Cambia 1305.1 vector containing 35S CMV promoter Zea mays full length GPT gene sequence [SEQ ID NO: 46]
[0125] Electrotransformation was used to insert these vectors simultaneously into the Chlorella culture; this was carried out according to the method of K. C. Chow and W. L. Tung. (Electrotransformation of Chlorella vulgaris. Plant Cell Reports 1999 18:778-780). The antibiotic selection was Hygromycin B at a concentration of 20 micrograms/ml. Bristol peptone digest media (UTEX web site) was used to culture the cells on plates.
Results:
[0126] Transformed and control algae were plated out identically. Twelve days later, 15 dark green colonies were observed growing on the transformed plate and none on the control plate. FIG. 1 shows a photograph of the transformed (right) and untransformed (control, left) Chlorella, taken 23 days post-plating. Vigorous green cultures were seen only on the transformed algae plate. In addition, the transformed algae colonies showed far greener and darker coloration compared the control algae. The dark green is chlorophyll, and the increase in chlorophyll per cell has been observed as a characteristic of the faster growing plants. Finally, the colony count at day 15 post-plating, and the much greater colony numbers at 23 days post-plating, are consistent with faster growth.
[0127] All publications, patents, and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.
[0128] The present invention is not to be limited in scope by the embodiments disclosed herein, which are intended as single illustrations of individual aspects of the invention, and any which are functionally equivalent are within the scope of the invention. Various modifications to the models and methods of the invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and teachings, and are similarly intended to fall within the scope of the invention. Such modifications or other embodiments can be practiced without departing from the true scope and spirit of the invention.
Sequence CWU
1
1
4811323DNAArabidopsis thalianaglutamine phenylpyruvate transaminase (GPT)
1atgtacctgg acataaatgg tgtgatgatc aaacagttta gcttcaaagc ctctcttctc
60ccattctctt ctaatttccg acaaagctcc gccaaaatcc atcgtcctat cggagccacc
120atgaccacag tttcgactca gaacgagtct actcaaaaac ccgtccaggt ggcgaagaga
180ttagagaagt tcaagactac tattttcact caaatgagca tattggcagt taaacatgga
240gcgatcaatt taggccaagg ctttcccaat ttcgacggtc ctgattttgt taaagaagct
300gcgatccaag ctattaaaga tggtaaaaac cagtatgctc gtggatacgg cattcctcag
360ctcaactctg ctatagctgc gcggtttcgt gaagatacgg gtcttgttgt tgatcctgag
420aaagaagtta ctgttacatc tggttgcaca gaagccatag ctgcagctat gttgggttta
480ataaaccctg gtgatgaagt cattctcttt gcaccgtttt atgattccta tgaagcaaca
540ctctctatgg ctggtgctaa agtaaaagga atcactttac gtccaccgga cttctccatc
600cctttggaag agcttaaagc tgcggtaact aacaagactc gagccatcct tatgaacact
660ccgcacaacc cgaccgggaa gatgttcact agggaggagc ttgaaaccat tgcatctctc
720tgcattgaaa acgatgtgct tgtgttctcg gatgaagtat acgataagct tgcgtttgaa
780atggatcaca tttctatagc ttctcttccc ggtatgtatg aaagaactgt gaccatgaat
840tccctgggaa agactttctc tttaaccgga tggaagatcg gctgggcgat tgcgccgcct
900catctgactt ggggagttcg acaagcacac tcttacctca cattcgccac atcaacacca
960gcacaatggg cagccgttgc agctctcaag gcaccagagt cttacttcaa agagctgaaa
1020agagattaca atgtgaaaaa ggagactctg gttaagggtt tgaaggaagt cggatttaca
1080gtgttcccat cgagcgggac ttactttgtg gttgctgatc acactccatt tggaatggag
1140aacgatgttg ctttctgtga gtatcttatt gaagaagttg gggtcgttgc gatcccaacg
1200agcgtctttt atctgaatcc agaagaaggg aagaatttgg ttaggtttgc gttctgtaaa
1260gacgaagaga cgttgcgtgg tgcaattgag aggatgaagc agaagcttaa gagaaaagtc
1320tga
13232440PRTArabidopsis thalianaglutamine phenylpyruvate transaminase
(GPT) 2Met Tyr Leu Asp Ile Asn Gly Val Met Ile Lys Gln Phe Ser Phe Lys1
5 10 15 Ala Ser Leu
Leu Pro Phe Ser Ser Asn Phe Arg Gln Ser Ser Ala Lys 20
25 30 Ile His Arg Pro Ile Gly Ala Thr
Met Thr Thr Val Ser Thr Gln Asn 35 40
45 Glu Ser Thr Gln Lys Pro Val Gln Val Ala Lys Arg Leu
Glu Lys Phe 50 55 60
Lys Thr Thr Ile Phe Thr Gln Met Ser Ile Leu Ala Val Lys His Gly65
70 75 80 Ala Ile Asn Leu Gly
Gln Gly Phe Pro Asn Phe Asp Gly Pro Asp Phe 85
90 95 Val Lys Glu Ala Ala Ile Gln Ala Ile Lys
Asp Gly Lys Asn Gln Tyr 100 105
110 Ala Arg Gly Tyr Gly Ile Pro Gln Leu Asn Ser Ala Ile Ala Ala
Arg 115 120 125 Phe
Arg Glu Asp Thr Gly Leu Val Val Asp Pro Glu Lys Glu Val Thr 130
135 140 Val Thr Ser Gly Cys Thr
Glu Ala Ile Ala Ala Ala Met Leu Gly Leu145 150
155 160 Ile Asn Pro Gly Asp Glu Val Ile Leu Phe Ala
Pro Phe Tyr Asp Ser 165 170
175 Tyr Glu Ala Thr Leu Ser Met Ala Gly Ala Lys Val Lys Gly Ile Thr
180 185 190 Leu Arg Pro
Pro Asp Phe Ser Ile Pro Leu Glu Glu Leu Lys Ala Ala 195
200 205 Val Thr Asn Lys Thr Arg Ala Ile
Leu Met Asn Thr Pro His Asn Pro 210 215
220 Thr Gly Lys Met Phe Thr Arg Glu Glu Leu Glu Thr Ile
Ala Ser Leu225 230 235
240 Cys Ile Glu Asn Asp Val Leu Val Phe Ser Asp Glu Val Tyr Asp Lys
245 250 255 Leu Ala Phe Glu
Met Asp His Ile Ser Ile Ala Ser Leu Pro Gly Met 260
265 270 Tyr Glu Arg Thr Val Thr Met Asn Ser
Leu Gly Lys Thr Phe Ser Leu 275 280
285 Thr Gly Trp Lys Ile Gly Trp Ala Ile Ala Pro Pro His Leu
Thr Trp 290 295 300
Gly Val Arg Gln Ala His Ser Tyr Leu Thr Phe Ala Thr Ser Thr Pro305
310 315 320 Ala Gln Trp Ala Ala
Val Ala Ala Leu Lys Ala Pro Glu Ser Tyr Phe 325
330 335 Lys Glu Leu Lys Arg Asp Tyr Asn Val Lys
Lys Glu Thr Leu Val Lys 340 345
350 Gly Leu Lys Glu Val Gly Phe Thr Val Phe Pro Ser Ser Gly Thr
Tyr 355 360 365 Phe
Val Val Ala Asp His Thr Pro Phe Gly Met Glu Asn Asp Val Ala 370
375 380 Phe Cys Glu Tyr Leu Ile
Glu Glu Val Gly Val Val Ala Ile Pro Thr385 390
395 400 Ser Val Phe Tyr Leu Asn Pro Glu Glu Gly Lys
Asn Leu Val Arg Phe 405 410
415 Ala Phe Cys Lys Asp Glu Glu Thr Leu Arg Gly Ala Ile Glu Arg Met
420 425 430 Lys Gln Lys
Leu Lys Arg Lys Val 435 440 31374DNAMedicago
sativaalfalfa glutamine synthetase (GS1) with 5' and 3'
untranslated sequences 3atttccgttt tcgttttcat ttgattcatt gaatcaaatc
gaatcgaatc tttaggattc 60aatacagatt ccttagattt tactaagttt gaaaccaaaa
ccaaaacatg tctctccttt 120cagatcttat caaccttgac ctctccgaaa ccaccgagaa
aatcatcgcc gaatacatat 180ggattggtgg atctggtttg gacttgagga gcaaagcaag
gactctacca ggaccagtta 240ctgacccttc acagcttccc aagtggaact atgatggttc
cagcacaggt caagctcctg 300gagaagatag tgaagttatt atctacccac aagccatttt
caaggaccca tttagaaggg 360gtaacaatat cttggttatg tgtgatgcat acactccagc
tggagagccc attcccacca 420acaagagaca tgcagctgcc aagattttca gccatcctga
tgttgttgct gaagtaccat 480ggtatggtat tgagcaagaa tacaccttgt tgcagaaaga
catcaattgg cctcttggtt 540ggccagttgg tggttttcct ggacctcagg gaccatacta
ttgtggagct ggtgctgaca 600aggcatttgg ccgtgacatt gttgactcac attacaaagc
ctgtctttat gccggcatca 660acatcagtgg aatcaatggt gaagtgatgc ctggtcaatg
ggaattccaa gttggtccct 720cagttggtat ctctgctggt gatgagatat gggttgctcg
ttacattttg gagaggatca 780ctgaggttgc tggtgtggtg ctttcctttg acccaaaacc
aattaagggt gattggaatg 840gtgctggtgc tcacacaaat tacagcacca agtctatgag
agaagatggt ggctatgaag 900tcatcttgaa agcaattgag aagcttggga agaagcacaa
ggagcacatt gctgcttatg 960gagaaggcaa cgagcgtaga ttgacagggc gacatgagac
agctgacatt aacaccttct 1020tatggggtgt tgcaaaccgt ggtgcgtcga ttagagttgg
aagggacaca gagaaagcag 1080ggaaaggtta tttcgaggat aggaggccat catctaacat
ggatccatat gttgttactt 1140ccatgattgc agacaccacc attctctgga aaccataagc
caccacacac acatgcattg 1200aagtatttga aagtcattgt tgattccgca ttagaatttg
gtcattgttt tttctaggat 1260ttggatttgt gttattgtta tggttcacac tttgtttgtt
tgaatttgag gccttgttat 1320aggtttcata tttctttctc ttgttctaag taaatgtcag
aataataatg taat 13744356PRTMedicago sativaalfalfa glutamine
synthetase (GS1) 4Met Ser Leu Leu Ser Asp Leu Ile Asn Leu Asp Leu Ser Glu
Thr Thr1 5 10 15
Glu Lys Ile Ile Ala Glu Tyr Ile Trp Ile Gly Gly Ser Gly Leu Asp
20 25 30 Leu Arg Ser Lys Ala
Arg Thr Leu Pro Gly Pro Val Thr Asp Pro Ser 35 40
45 Gln Leu Pro Lys Trp Asn Tyr Asp Gly Ser
Ser Thr Gly Gln Ala Pro 50 55 60
Gly Glu Asp Ser Glu Val Ile Ile Tyr Pro Gln Ala Ile Phe Lys
Asp65 70 75 80 Pro
Phe Arg Arg Gly Asn Asn Ile Leu Val Met Cys Asp Ala Tyr Thr
85 90 95 Pro Ala Gly Glu Pro Ile
Pro Thr Asn Lys Arg His Ala Ala Ala Lys 100
105 110 Ile Phe Ser His Pro Asp Val Val Ala Glu
Val Pro Trp Tyr Gly Ile 115 120
125 Glu Gln Glu Tyr Thr Leu Leu Gln Lys Asp Ile Asn Trp Pro
Leu Gly 130 135 140
Trp Pro Val Gly Gly Phe Pro Gly Pro Gln Gly Pro Tyr Tyr Cys Gly145
150 155 160 Ala Gly Ala Asp Lys
Ala Phe Gly Arg Asp Ile Val Asp Ser His Tyr 165
170 175 Lys Ala Cys Leu Tyr Ala Gly Ile Asn Ile
Ser Gly Ile Asn Gly Glu 180 185
190 Val Met Pro Gly Gln Trp Glu Phe Gln Val Gly Pro Ser Val Gly
Ile 195 200 205 Ser
Ala Gly Asp Glu Ile Trp Val Ala Arg Tyr Ile Leu Glu Arg Ile 210
215 220 Thr Glu Val Ala Gly Val
Val Leu Ser Phe Asp Pro Lys Pro Ile Lys225 230
235 240 Gly Asp Trp Asn Gly Ala Gly Ala His Thr Asn
Tyr Ser Thr Lys Ser 245 250
255 Met Arg Glu Asp Gly Gly Tyr Glu Val Ile Leu Lys Ala Ile Glu Lys
260 265 270 Leu Gly Lys
Lys His Lys Glu His Ile Ala Ala Tyr Gly Glu Gly Asn 275
280 285 Glu Arg Arg Leu Thr Gly Arg His
Glu Thr Ala Asp Ile Asn Thr Phe 290 295
300 Leu Trp Gly Val Ala Asn Arg Gly Ala Ser Ile Arg Val
Gly Arg Asp305 310 315
320 Thr Glu Lys Ala Gly Lys Gly Tyr Phe Glu Asp Arg Arg Pro Ser Ser
325 330 335 Asn Met Asp Pro
Tyr Val Val Thr Ser Met Ile Ala Asp Thr Thr Ile 340
345 350 Leu Trp Lys Pro 355
51419DNAArtificial Sequencesynthetic Medicago sativa (barley) glutamine
synthetase (GS1) with 5' and 3' untranslated sequences and vector
sequences 5atcgatgaat tcgagctcgg tacccatttc cgttttcgtt ttcatttgat
tcattgaatc 60aaatcgaatc gaatctttag gattcaatac agattcctta gattttacta
agtttgaaac 120caaaaccaaa acatgtctct cctttcagat cttatcaacc ttgacctctc
cgaaaccacc 180gagaaaatca tcgccgaata catatggatt ggtggatctg gtttggactt
gaggagcaaa 240gcaaggactc taccaggacc agttactgac ccttcacagc ttcccaagtg
gaactatgat 300ggttccagca caggtcaagc tcctggagaa gatagtgaag ttattatcta
cccacaagcc 360attttcaagg acccatttag aaggggtaac aatatcttgg ttatgtgtga
tgcatacact 420ccagctggag agcccattcc caccaacaag agacatgcag ctgccaagat
tttcagccat 480cctgatgttg ttgctgaagt accatggtat ggtattgagc aagaatacac
cttgttgcag 540aaagacatca attggcctct tggttggcca gttggtggtt ttcctggacc
tcagggacca 600tactattgtg gagctggtgc tgacaaggca tttggccgtg acattgttga
ctcacattac 660aaagcctgtc tttatgccgg catcaacatc agtggaatca atggtgaagt
gatgcctggt 720caatgggaat tccaagttgg tccctcagtt ggtatctctg ctggtgatga
gatatgggtt 780gctcgttaca ttttggagag gatcactgag gttgctggtg tggtgctttc
ctttgaccca 840aaaccaatta agggtgattg gaatggtgct ggtgctcaca caaattacag
caccaagtct 900atgagagaag atggtggcta tgaagtcatc ttgaaagcaa ttgagaagct
tgggaagaag 960cacaaggagc acattgctgc ttatggagaa ggcaacgagc gtagattgac
agggcgacat 1020gagacagctg acattaacac cttcttatgg ggtgttgcaa accgtggtgc
gtcgattaga 1080gttggaaggg acacagagaa agcagggaaa ggttatttcg aggataggag
gccatcatct 1140aacatggatc catatgttgt tacttccatg attgcagaca ccaccattct
ctggaaacca 1200taagccacca cacacacatg cattgaagta tttgaaagtc attgttgatt
ccgcattaga 1260atttggtcat tgttttttct aggatttgga tttgtgttat tgttatggtt
cacactttgt 1320ttgtttgaat ttgaggcctt gttataggtt tcatatttct ttctcttgtt
ctaagtaaat 1380gtcagaataa taatgtaatg gggatcctct agagtcgag
141961302DNAArtificial Sequencesynthetic Arabidopsis glutamine
synthetase (GS1) coding sequence (Cambia 1201
vector+rbcS3C+arabidopsis GS1), plasmid vector 6aaaaaagaaa
aaaaaaacat atcttgtttg tcagtatggg aagtttgaga taaggacgag 60tgaggggtta
aaattcagtg gccattgatt ttgtaatgcc aagaaccaca aaatccaatg 120gttaccattc
ctgtaagatg aggtttgcta actctttttg tccgttagat aggaagcctt 180atcactatat
atacaaggcg tcctaataac ctcttagtaa ccaattattt cagcaccatg 240tctctgctct
cagatctcgt taacctcaac ctcaccgatg ccaccgggaa aatcatcgcc 300gaatacatat
ggatcggtgg atctggaatg gatatcagaa gcaaagccag gacactacca 360ggaccagtga
ctgatccatc aaagcttccc aagtggaact acgacggatc cagcaccggt 420caggctgctg
gagaagacag tgaagtcatt ctataccctc aggcaatatt caaggatccc 480ttcaggaaag
gcaacaacat cctggtgatg tgtgatgctt acacaccagc tggtgatcct 540attccaacca
acaagaggca caacgctgct aagatcttca gccaccccga cgttgccaag 600gaggagcctt
ggtatgggat tgagcaagaa tacactttga tgcaaaagga tgtgaactgg 660ccaattggtt
ggcctgttgg tggctaccct ggccctcagg gaccttacta ctgtggtgtg 720ggagctgaca
aagccattgg tcgtgacatt gtggatgctc actacaaggc ctgtctttac 780gccggtattg
gtatttctgg tatcaatgga gaagtcatgc caggccagtg ggagttccaa 840gtcggccctg
ttgagggtat tagttctggt gatcaagtct gggttgctcg ataccttctc 900gagaggatca
ctgagatctc tggtgtaatt gtcagcttcg acccgaaacc agtcccgggt 960gactggaatg
gagctggagc tcactgcaac tacagcacta agacaatgag aaacgatgga 1020ggattagaag
tgatcaagaa agcgataggg aagcttcagc tgaaacacaa agaacacatt 1080gctgcttacg
gtgaaggaaa cgagcgtcgt ctcactggaa agcacgaaac cgcagacatc 1140aacacattct
cttggggagt cgcgaaccgt ggagcgtcag tgagagtggg acgtgacaca 1200gagaaggaag
gtaaagggta cttcgaagac agaaggccag cttctaacat ggatccttac 1260gttgtcacct
ccatgatcgc tgagacgacc atactcggtt ga
13027364PRTArtificial Sequencesynthetic Arabidopsis glutamine synthetase
(GS1) and vector sequences at N-terminus 7Met Val Asp Leu Arg Asn
Arg Arg Thr Ser Met Ser Leu Leu Ser Asp1 5
10 15 Leu Val Asn Leu Asn Leu Thr Asp Ala Thr Gly
Lys Ile Ile Ala Glu 20 25 30
Tyr Ile Trp Ile Gly Gly Ser Gly Met Asp Ile Arg Ser Lys Ala Arg
35 40 45 Thr Leu Pro
Gly Pro Val Thr Asp Pro Ser Lys Leu Pro Lys Trp Asn 50
55 60 Tyr Asp Gly Ser Ser Thr Gly Gln
Ala Ala Gly Glu Asp Ser Glu Val65 70 75
80 Ile Leu Tyr Pro Gln Ala Ile Phe Lys Asp Pro Phe Arg
Lys Gly Asn 85 90 95
Asn Ile Leu Val Met Cys Asp Ala Tyr Thr Pro Ala Gly Asp Pro Ile
100 105 110 Pro Thr Asn Lys Arg
His Asn Ala Ala Lys Ile Phe Ser His Pro Asp 115
120 125 Val Ala Lys Glu Glu Pro Trp Tyr Gly
Ile Glu Gln Glu Tyr Thr Leu 130 135
140 Met Gln Lys Asp Val Asn Trp Pro Ile Gly Trp Pro Val
Gly Gly Tyr145 150 155
160 Pro Gly Pro Gln Gly Pro Tyr Tyr Cys Gly Val Gly Ala Asp Lys Ala
165 170 175 Ile Gly Arg Asp
Ile Val Asp Ala His Tyr Lys Ala Cys Leu Tyr Ala 180
185 190 Gly Ile Gly Ile Ser Gly Ile Asn Gly
Glu Val Met Pro Gly Gln Trp 195 200
205 Glu Phe Gln Val Gly Pro Val Glu Gly Ile Ser Ser Gly Asp
Gln Val 210 215 220
Trp Val Ala Arg Tyr Leu Leu Glu Arg Ile Thr Glu Ile Ser Gly Val225
230 235 240 Ile Val Ser Phe Asp
Pro Lys Pro Val Pro Gly Asp Trp Asn Gly Ala 245
250 255 Gly Ala His Cys Asn Tyr Ser Thr Lys Thr
Met Arg Asn Asp Gly Gly 260 265
270 Leu Glu Val Ile Lys Lys Ala Ile Gly Lys Leu Gln Leu Lys His
Lys 275 280 285 Glu
His Ile Ala Ala Tyr Gly Glu Gly Asn Glu Arg Arg Leu Thr Gly 290
295 300 Lys His Glu Thr Ala Asp
Ile Asn Thr Phe Ser Trp Gly Val Ala Asn305 310
315 320 Arg Gly Ala Ser Val Arg Val Gly Arg Asp Thr
Glu Lys Glu Gly Lys 325 330
335 Gly Tyr Phe Glu Asp Arg Arg Pro Ala Ser Asn Met Asp Pro Tyr Val
340 345 350 Val Thr Ser
Met Ile Ala Glu Thr Thr Ile Leu Gly 355 360
81817DNAArtificial Sequencesynthetic Vitis vinifera (grape)
glutamine phenylpyruvate transaminase (GPT) with Cambia 1305.1 and
(3' end of) rbcS3C+Vitis vinifera GPT + catl intron, plasmid
vector 8aaaaaagaaa aaaaaaacat atcttgtttg tcagtatggg aagtttgaga taaggacgag
60tgaggggtta aaattcagtg gccattgatt ttgtaatgcc aagaaccaca aaatccaatg
120gttaccattc ctgtaagatg aggtttgcta actctttttg tccgttagat aggaagcctt
180atcactatat atacaaggcg tcctaataac ctcttagtaa ccaattattt cagcaccatg
240gtagatctga gggtaaattt ctagtttttc tccttcattt tcttggttag gacccttttc
300tctttttatt tttttgagct ttgatctttc tttaaactga tctatttttt aattgattgg
360ttatggtgta aatattacat agctttaact gataatctga ttactttatt tcgtgtgtct
420atgatgatga tgatagttac agaaccgacg aactagtatg cagctctctc aatgtacctg
480gacattccca gagttgctta aaagaccagc ctttttaagg aggagtattg atagtatttc
540gagtagaagt aggtccagct ccaagtatcc atctttcatg gcgtccgcat caacggtctc
600cgctccaaat acggaggctg agcagaccca taacccccct caacctctac aggttgcaaa
660gcgcttggag aaattcaaaa caacaatctt tactcaaatg agcatgcttg ccatcaaaca
720tggagcaata aaccttggcc aagggtttcc caactttgat ggtcctgagt ttgtcaaaga
780agcagcaatt caagccatta aggatgggaa aaaccaatat gctcgtggat atggagttcc
840tgatctcaac tctgctgttg ctgatagatt caagaaggat acaggactcg tggtggaccc
900cgagaaggaa gttactgtta cttctggatg tacagaagca attgctgcta ctatgctagg
960cttgataaat cctggtgatg aggtgatcct ctttgctcca ttttatgatt cctatgaagc
1020cactctatcc atggctggtg cccaaataaa atccatcact ttacgtcctc cggattttgc
1080tgtgcccatg gatgagctca agtctgcaat ctcaaagaat acccgtgcaa tccttataaa
1140cactccccat aaccccacag gaaagatgtt cacaagggag gaactgaatg tgattgcatc
1200cctctgcatt gagaatgatg tgttggtgtt tactgatgaa gtttacgaca agttggcttt
1260cgaaatggat cacatttcca tggcttctct tcctgggatg tacgagagga ccgtgactat
1320gaattcctta gggaaaactt tctccctgac tggatggaag attggttgga cagtagctcc
1380cccacacctg acatggggag tgaggcaagc ccactcattc ctcacgtttg ctacctgcac
1440cccaatgcaa tgggcagctg caacagccct ccgggcccca gactcttact atgaagagct
1500aaagagagat tacagtgcaa agaaggcaat cctggtggag ggattgaagg ctgtcggttt
1560cagggtatac ccatcaagtg ggacctattt tgtggtggtg gatcacaccc catttgggtt
1620gaaagacgat attgcgtttt gtgagtatct gatcaaggaa gttggggtgg tagcaattcc
1680gacaagcgtt ttctacttac acccagaaga tggaaagaac cttgtgaggt ttaccttctg
1740taaagacgag ggaactctga gagctgcagt tgaaaggatg aaggagaaac tgaagcctaa
1800acaatagggg cacgtga
18179459PRTVitis viniferagrape glutamine phenylpyruvate transaminase
(GPT) 9Met Val Asp Leu Arg Asn Arg Arg Thr Ser Met Gln Leu Ser Gln Cys1
5 10 15 Thr Trp Thr
Phe Pro Glu Leu Leu Lys Arg Pro Ala Phe Leu Arg Arg 20
25 30 Ser Ile Asp Ser Ile Ser Ser Arg
Ser Arg Ser Ser Ser Lys Tyr Pro 35 40
45 Ser Phe Met Ala Ser Ala Ser Thr Val Ser Ala Pro Asn
Thr Glu Ala 50 55 60
Glu Gln Thr His Asn Pro Pro Gln Pro Leu Gln Val Ala Lys Arg Leu65
70 75 80 Glu Lys Phe Lys Thr
Thr Ile Phe Thr Gln Met Ser Met Leu Ala Ile 85
90 95 Lys His Gly Ala Ile Asn Leu Gly Gln Gly
Phe Pro Asn Phe Asp Gly 100 105
110 Pro Glu Phe Val Lys Glu Ala Ala Ile Gln Ala Ile Lys Asp Gly
Lys 115 120 125 Asn
Gln Tyr Ala Arg Gly Tyr Gly Val Pro Asp Leu Asn Ser Ala Val 130
135 140 Ala Asp Arg Phe Lys Lys
Asp Thr Gly Leu Val Val Asp Pro Glu Lys145 150
155 160 Glu Val Thr Val Thr Ser Gly Cys Thr Glu Ala
Ile Ala Ala Thr Met 165 170
175 Leu Gly Leu Ile Asn Pro Gly Asp Glu Val Ile Leu Phe Ala Pro Phe
180 185 190 Tyr Asp Ser
Tyr Glu Ala Thr Leu Ser Met Ala Gly Ala Gln Ile Lys 195
200 205 Ser Ile Thr Leu Arg Pro Pro Asp
Phe Ala Val Pro Met Asp Glu Leu 210 215
220 Lys Ser Ala Ile Ser Lys Asn Thr Arg Ala Ile Leu Ile
Asn Thr Pro225 230 235
240 His Asn Pro Thr Gly Lys Met Phe Thr Arg Glu Glu Leu Asn Val Ile
245 250 255 Ala Ser Leu Cys
Ile Glu Asn Asp Val Leu Val Phe Thr Asp Glu Val 260
265 270 Tyr Asp Lys Leu Ala Phe Glu Met Asp
His Ile Ser Met Ala Ser Leu 275 280
285 Pro Gly Met Tyr Glu Arg Thr Val Thr Met Asn Ser Leu Gly
Lys Thr 290 295 300
Phe Ser Leu Thr Gly Trp Lys Ile Gly Trp Thr Val Ala Pro Pro His305
310 315 320 Leu Thr Trp Gly Val
Arg Gln Ala His Ser Phe Leu Thr Phe Ala Thr 325
330 335 Cys Thr Pro Met Gln Trp Ala Ala Ala Thr
Ala Leu Arg Ala Pro Asp 340 345
350 Ser Tyr Tyr Glu Glu Leu Lys Arg Asp Tyr Ser Ala Lys Lys Ala
Ile 355 360 365 Leu
Val Glu Gly Leu Lys Ala Val Gly Phe Arg Val Tyr Pro Ser Ser 370
375 380 Gly Thr Tyr Phe Val Val
Val Asp His Thr Pro Phe Gly Leu Lys Asp385 390
395 400 Asp Ile Ala Phe Cys Glu Tyr Leu Ile Lys Glu
Val Gly Val Val Ala 405 410
415 Ile Pro Thr Ser Val Phe Tyr Leu His Pro Glu Asp Gly Lys Asn Leu
420 425 430 Val Arg Phe
Thr Phe Cys Lys Asp Glu Gly Thr Leu Arg Ala Ala Val 435
440 445 Glu Arg Met Lys Glu Lys Leu Lys
Pro Lys Gln 450 455
101446DNAArtificial Sequencesynthetic Oryza sativa (rice) glutamine
phenylpyruvate transaminase (GPT) codon optimized for E. coli
expression with untranslated sequences 10atgtggatga acctggcagg ctttctggca
accccggcaa ccgcaaccgc aacccgtcat 60gaaatgccgc tgaacccgag cagcagcgcg
agctttctgc tgagcagcct gcgtcgtagc 120ctggtggcga gcctgcgtaa agcgagcccg
gcagcagcag cagcactgag cccgatggca 180agcgcaagca ccgtggcagc agaaaacggt
gcagcaaaag cagcagcaga aaaacagcag 240cagcagccgg tgcaggtggc gaaacgtctg
gaaaaattta aaaccaccat ttttacccag 300atgagcatgc tggcgattaa acatggcgcg
attaacctgg gccagggctt tccgaacttt 360gatggcccgg attttgtgaa agaagcggcg
attcaggcga ttaacgcggg caaaaaccag 420tatgcgcgtg gctatggcgt gccggaactg
aacagcgcga ttgcggaacg ttttctgaaa 480gatagcggcc tgcaggtgga tccggaaaaa
gaagtgaccg tgaccagcgg ctgcaccgaa 540gcgattgcgg cgaccattct gggcctgatt
aacccgggcg atgaagtgat tctgtttgcg 600ccgttttatg atagctatga agcgaccctg
agcatggcgg gcgcgaacgt gaaagcgatt 660accctgcgtc cgccggattt tagcgtgccg
ctggaagaac tgaaagcggc cgtgagcaaa 720aacacccgtg cgattatgat taacaccccg
cataacccga ccggcaaaat gtttacccgt 780gaagaactgg aatttattgc gaccctgtgc
aaagaaaacg atgtgctgct gtttgcggat 840gaagtgtatg ataaactggc gtttgaagcg
gatcatatta gcatggcgag cattccgggc 900atgtatgaac gtaccgtgac catgaacagc
ctgggcaaaa cctttagcct gaccggctgg 960aaaattggct gggcgattgc gccgccgcat
ctgacctggg gcgtgcgtca ggcacatagc 1020tttctgacct ttgcaacctg caccccgatg
caggcagccg ccgcagcagc actgcgtgca 1080ccggatagct attatgaaga actgcgtcgt
gattatggcg cgaaaaaagc gctgctggtg 1140aacggcctga aagatgcggg ctttattgtg
tatccgagca gcggcaccta ttttgtgatg 1200gtggatcata ccccgtttgg ctttgataac
gatattgaat tttgcgaata tctgattcgt 1260gaagtgggcg tggtggcgat tccgccgagc
gtgttttatc tgaacccgga agatggcaaa 1320aacctggtgc gttttacctt ttgcaaagat
gatgaaaccc tgcgtgcggc ggtggaacgt 1380atgaaaacca aactgcgtaa aaaaaagctt
gcggccgcac tcgagcacca ccaccaccac 1440cactga
144611481PRTArtificial Sequencesynthetic
Oryza sativa (rice) glutamine phenylpyruvate transaminase (GPT)
with amino terminal cloning and N-terminus His tag sequences from
pet28 vector 11Met Trp Met Asn Leu Ala Gly Phe Leu Ala Thr Pro Ala Thr
Ala Thr1 5 10 15
Ala Thr Arg His Glu Met Pro Leu Asn Pro Ser Ser Ser Ala Ser Phe
20 25 30 Leu Leu Ser Ser Leu
Arg Arg Ser Leu Val Ala Ser Leu Arg Lys Ala 35 40
45 Ser Pro Ala Ala Ala Ala Ala Leu Ser Pro
Met Ala Ser Ala Ser Thr 50 55 60
Val Ala Ala Glu Asn Gly Ala Ala Lys Ala Ala Ala Glu Lys Gln
Gln65 70 75 80 Gln
Gln Pro Val Gln Val Ala Lys Arg Leu Glu Lys Phe Lys Thr Thr
85 90 95 Ile Phe Thr Gln Met Ser
Met Leu Ala Ile Lys His Gly Ala Ile Asn 100
105 110 Leu Gly Gln Gly Phe Pro Asn Phe Asp Gly
Pro Asp Phe Val Lys Glu 115 120
125 Ala Ala Ile Gln Ala Ile Asn Ala Gly Lys Asn Gln Tyr Ala
Arg Gly 130 135 140
Tyr Gly Val Pro Glu Leu Asn Ser Ala Ile Ala Glu Arg Phe Leu Lys145
150 155 160 Asp Ser Gly Leu Gln
Val Asp Pro Glu Lys Glu Val Thr Val Thr Ser 165
170 175 Gly Cys Thr Glu Ala Ile Ala Ala Thr Ile
Leu Gly Leu Ile Asn Pro 180 185
190 Gly Asp Glu Val Ile Leu Phe Ala Pro Phe Tyr Asp Ser Tyr Glu
Ala 195 200 205 Thr
Leu Ser Met Ala Gly Ala Asn Val Lys Ala Ile Thr Leu Arg Pro 210
215 220 Pro Asp Phe Ser Val Pro
Leu Glu Glu Leu Lys Ala Ala Val Ser Lys225 230
235 240 Asn Thr Arg Ala Ile Met Ile Asn Thr Pro His
Asn Pro Thr Gly Lys 245 250
255 Met Phe Thr Arg Glu Glu Leu Glu Phe Ile Ala Thr Leu Cys Lys Glu
260 265 270 Asn Asp Val
Leu Leu Phe Ala Asp Glu Val Tyr Asp Lys Leu Ala Phe 275
280 285 Glu Ala Asp His Ile Ser Met Ala
Ser Ile Pro Gly Met Tyr Glu Arg 290 295
300 Thr Val Thr Met Asn Ser Leu Gly Lys Thr Phe Ser Leu
Thr Gly Trp305 310 315
320 Lys Ile Gly Trp Ala Ile Ala Pro Pro His Leu Thr Trp Gly Val Arg
325 330 335 Gln Ala His Ser
Phe Leu Thr Phe Ala Thr Cys Thr Pro Met Gln Ala 340
345 350 Ala Ala Ala Ala Ala Leu Arg Ala Pro
Asp Ser Tyr Tyr Glu Glu Leu 355 360
365 Arg Arg Asp Tyr Gly Ala Lys Lys Ala Leu Leu Val Asn Gly
Leu Lys 370 375 380
Asp Ala Gly Phe Ile Val Tyr Pro Ser Ser Gly Thr Tyr Phe Val Met385
390 395 400 Val Asp His Thr Pro
Phe Gly Phe Asp Asn Asp Ile Glu Phe Cys Glu 405
410 415 Tyr Leu Ile Arg Glu Val Gly Val Val Ala
Ile Pro Pro Ser Val Phe 420 425
430 Tyr Leu Asn Pro Glu Asp Gly Lys Asn Leu Val Arg Phe Thr Phe
Cys 435 440 445 Lys Asp
Asp Glu Thr Leu Arg Ala Ala Val Glu Arg Met Lys Thr Lys 450
455 460 Leu Arg Lys Lys Lys Leu Ala
Ala Ala Leu Glu His His His His His465 470
475 480 His121251DNAArtificial Sequencesynthetic Glycine
max (soybean) glutamine phenylpyruvate transaminase (GPT) TOPO 151D
WITH SOYBEAN codon optimized for E. coli expression and vector
sequences 12atgcatcatc accatcacca tggtaagcct atccctaacc ctctcctcgg
tctcgattct 60acggaaaacc tgtattttca gggaattgat cccttcaccg cgaaacgtct
ggaaaaattt 120cagaccacca tttttaccca gatgagcctg ctggcgatta aacatggcgc
gattaacctg 180ggccagggct ttccgaactt tgatggcccg gaatttgtga aagaagcggc
gattcaggcg 240attcgtgatg gcaaaaacca gtatgcgcgt ggctatggcg tgccggatct
gaacattgcg 300attgcggaac gttttaaaaa agataccggc ctggtggtgg atccggaaaa
agaaattacc 360gtgaccagcg gctgcaccga agcgattgcg gcgaccatga ttggcctgat
taacccgggc 420gatgaagtga ttatgtttgc gccgttttat gatagctatg aagcgaccct
gagcatggcg 480ggcgcgaaag tgaaaggcat taccctgcgt ccgccggatt ttgcggtgcc
gctggaagaa 540ctgaaaagca ccattagcaa aaacacccgt gcgattctga ttaacacccc
gcataacccg 600accggcaaaa tgtttacccg tgaagaactg aactgcattg cgagcctgtg
cattgaaaac 660gatgtgctgg tgtttaccga tgaagtgtat gataaactgg cgtttgatat
ggaacatatt 720agcatggcga gcctgccggg catgtttgaa cgtaccgtga ccctgaacag
cctgggcaaa 780acctttagcc tgaccggctg gaaaattggc tgggcgattg cgccgccgca
tctgagctgg 840ggcgtgcgtc aggcgcatgc gtttctgacc tttgcaaccg cacatccgtt
tcagtgcgca 900gcagcagcag cactgcgtgc accggatagc tattatgtgg aactgaaacg
tgattatatg 960gcgaaacgtg cgattctgat tgaaggcctg aaagcggtgg gctttaaagt
gtttccgagc 1020agcggcacct attttgtggt ggtggatcat accccgtttg gcctggaaaa
cgatgtggcg 1080ttttgcgaat atctggtgaa agaagtgggc gtggtggcga ttccgaccag
cgtgttttat 1140ctgaacccgg aagaaggcaa aaacctggtg cgttttacct tttgcaaaga
tgaagaaacc 1200attcgtagcg cggtggaacg tatgaaagcg aaactgcgta aagtcgacta a
125113416PRTArtificial Sequencesynthetic Glycine max (soybean)
glutamine phenylpyruvate transaminase (GPT) and amino-terminal
vector sequences 13Met His His His His His His Gly Lys Pro Ile Pro
Asn Pro Leu Leu1 5 10 15
Gly Leu Asp Ser Thr Glu Asn Leu Tyr Phe Gln Gly Ile Asp Pro Phe
20 25 30 Thr Ala Lys Arg
Leu Glu Lys Phe Gln Thr Thr Ile Phe Thr Gln Met 35
40 45 Ser Leu Leu Ala Ile Lys His Gly Ala
Ile Asn Leu Gly Gln Gly Phe 50 55 60
Pro Asn Phe Asp Gly Pro Glu Phe Val Lys Glu Ala Ala Ile
Gln Ala65 70 75 80
Ile Arg Asp Gly Lys Asn Gln Tyr Ala Arg Gly Tyr Gly Val Pro Asp
85 90 95 Leu Asn Ile Ala Ile
Ala Glu Arg Phe Lys Lys Asp Thr Gly Leu Val 100
105 110 Val Asp Pro Glu Lys Glu Ile Thr Val Thr
Ser Gly Cys Thr Glu Ala 115 120
125 Ile Ala Ala Thr Met Ile Gly Leu Ile Asn Pro Gly Asp Glu
Val Ile 130 135 140
Met Phe Ala Pro Phe Tyr Asp Ser Tyr Glu Ala Thr Leu Ser Met Ala145
150 155 160 Gly Ala Lys Val Lys
Gly Ile Thr Leu Arg Pro Pro Asp Phe Ala Val 165
170 175 Pro Leu Glu Glu Leu Lys Ser Thr Ile Ser
Lys Asn Thr Arg Ala Ile 180 185
190 Leu Ile Asn Thr Pro His Asn Pro Thr Gly Lys Met Phe Thr Arg
Glu 195 200 205 Glu
Leu Asn Cys Ile Ala Ser Leu Cys Ile Glu Asn Asp Val Leu Val 210
215 220 Phe Thr Asp Glu Val Tyr
Asp Lys Leu Ala Phe Asp Met Glu His Ile225 230
235 240 Ser Met Ala Ser Leu Pro Gly Met Phe Glu Arg
Thr Val Thr Leu Asn 245 250
255 Ser Leu Gly Lys Thr Phe Ser Leu Thr Gly Trp Lys Ile Gly Trp Ala
260 265 270 Ile Ala Pro
Pro His Leu Ser Trp Gly Val Arg Gln Ala His Ala Phe 275
280 285 Leu Thr Phe Ala Thr Ala His Pro
Phe Gln Cys Ala Ala Ala Ala Ala 290 295
300 Leu Arg Ala Pro Asp Ser Tyr Tyr Val Glu Leu Lys Arg
Asp Tyr Met305 310 315
320 Ala Lys Arg Ala Ile Leu Ile Glu Gly Leu Lys Ala Val Gly Phe Lys
325 330 335 Val Phe Pro Ser
Ser Gly Thr Tyr Phe Val Val Val Asp His Thr Pro 340
345 350 Phe Gly Leu Glu Asn Asp Val Ala Phe
Cys Glu Tyr Leu Val Lys Glu 355 360
365 Val Gly Val Val Ala Ile Pro Thr Ser Val Phe Tyr Leu Asn
Pro Glu 370 375 380
Glu Gly Lys Asn Leu Val Arg Phe Thr Phe Cys Lys Asp Glu Glu Thr385
390 395 400 Ile Arg Ser Ala Val
Glu Arg Met Lys Ala Lys Leu Arg Lys Val Asp 405
410 415 141278DNAHordeum vulgarebarley glutamine
phenylpyruvate transaminase (GPT) 14atggtagatc tgaggaaccg
acgaactagt atggcatccg cccccgcctc cgcctccgcg 60gccctctcca ccgccgcccc
cgccgacaac ggggccgcca agcccacgga gcagcggccg 120gtacaggtgg ctaagcgatt
ggagaagttc aaaacaacaa ttttcacaca gatgagcatg 180ctcgcagtga agcatggagc
aataaacctt ggacaggggt ttcccaattt tgatggccct 240gactttgtca aagatgctgc
tattgaggct atcaaagctg gaaagaatca gtatgcaaga 300ggatatggtg tgcctgaatt
gaactcagct gttgctgaga gatttctcaa ggacagtgga 360ttgcacatcg atcctgataa
ggaagttact gttacatctg ggtgcacaga agcaatagct 420gcaacgatat tgggtctgat
caaccctggg gatgaagtca tactgtttgc tccattctat 480gattcttatg aggctacact
gtccatggct ggtgcgaatg tcaaagccat tacactccgc 540cctccggact ttgcagtccc
tcttgaagag ctaaaggctg cagtctcgaa gaataccaga 600gcaataatga ttaatacacc
tcacaaccct accgggaaaa tgttcacaag ggaggaactt 660gagttcattg ctgatctctg
caaggaaaat gacgtgttgc tctttgccga tgaggtctac 720gacaagctgg cgtttgaggc
ggatcacata tcaatggctt ctattcctgg catgtatgag 780aggaccgtca ctatgaactc
cctggggaag acgttctcct tgaccggatg gaagatcggc 840tgggcgatag caccaccgca
cctgacatgg ggcgtaaggc aggcacactc cttcctcaca 900ttcgccacct ccacgccgat
gcaatcagca gcggcggcgg ccctgagagc accggacagc 960tactttgagg agctgaagag
ggactacggc gcaaagaaag cgctgctggt ggacgggctc 1020aaggcggcgg gcttcatcgt
ctacccttcg agcggaacct acttcatcat ggtcgaccac 1080accccgttcg ggttcgacaa
cgacgtcgag ttctgcgagt acttgatccg cgaggtcggc 1140gtcgtggcca tcccgccaag
cgtgttctac ctgaacccgg aggacgggaa gaacctggtg 1200aggttcacct tctgcaagga
cgacgacacg ctaagggcgg cggtggacag gatgaaggcc 1260aagctcagga agaaatga
127815425PRTHordeum
vulgarebarley glutamine phenylpyruvate transaminase (GPT) 15Met Val
Asp Leu Arg Asn Arg Arg Thr Ser Met Ala Ser Ala Pro Ala1 5
10 15 Ser Ala Ser Ala Ala Leu Ser
Thr Ala Ala Pro Ala Asp Asn Gly Ala 20 25
30 Ala Lys Pro Thr Glu Gln Arg Pro Val Gln Val Ala
Lys Arg Leu Glu 35 40 45
Lys Phe Lys Thr Thr Ile Phe Thr Gln Met Ser Met Leu Ala Val Lys
50 55 60 His Gly Ala
Ile Asn Leu Gly Gln Gly Phe Pro Asn Phe Asp Gly Pro65 70
75 80 Asp Phe Val Lys Asp Ala Ala Ile
Glu Ala Ile Lys Ala Gly Lys Asn 85 90
95 Gln Tyr Ala Arg Gly Tyr Gly Val Pro Glu Leu Asn Ser
Ala Val Ala 100 105 110
Glu Arg Phe Leu Lys Asp Ser Gly Leu His Ile Asp Pro Asp Lys Glu
115 120 125 Val Thr Val Thr
Ser Gly Cys Thr Glu Ala Ile Ala Ala Thr Ile Leu 130
135 140 Gly Leu Ile Asn Pro Gly Asp Glu
Val Ile Leu Phe Ala Pro Phe Tyr145 150
155 160 Asp Ser Tyr Glu Ala Thr Leu Ser Met Ala Gly Ala
Asn Val Lys Ala 165 170
175 Ile Thr Leu Arg Pro Pro Asp Phe Ala Val Pro Leu Glu Glu Leu Lys
180 185 190 Ala Ala Val
Ser Lys Asn Thr Arg Ala Ile Met Ile Asn Thr Pro His 195
200 205 Asn Pro Thr Gly Lys Met Phe Thr
Arg Glu Glu Leu Glu Phe Ile Ala 210 215
220 Asp Leu Cys Lys Glu Asn Asp Val Leu Leu Phe Ala Asp
Glu Val Tyr225 230 235
240 Asp Lys Leu Ala Phe Glu Ala Asp His Ile Ser Met Ala Ser Ile Pro
245 250 255 Gly Met Tyr Glu
Arg Thr Val Thr Met Asn Ser Leu Gly Lys Thr Phe 260
265 270 Ser Leu Thr Gly Trp Lys Ile Gly Trp
Ala Ile Ala Pro Pro His Leu 275 280
285 Thr Trp Gly Val Arg Gln Ala His Ser Phe Leu Thr Phe Ala
Thr Ser 290 295 300
Thr Pro Met Gln Ser Ala Ala Ala Ala Ala Leu Arg Ala Pro Asp Ser305
310 315 320 Tyr Phe Glu Glu Leu
Lys Arg Asp Tyr Gly Ala Lys Lys Ala Leu Leu 325
330 335 Val Asp Gly Leu Lys Ala Ala Gly Phe Ile
Val Tyr Pro Ser Ser Gly 340 345
350 Thr Tyr Phe Ile Met Val Asp His Thr Pro Phe Gly Phe Asp Asn
Asp 355 360 365 Val Glu
Phe Cys Glu Tyr Leu Ile Arg Glu Val Gly Val Val Ala Ile 370
375 380 Pro Pro Ser Val Phe Tyr Leu
Asn Pro Glu Asp Gly Lys Asn Leu Val385 390
395 400 Arg Phe Thr Phe Cys Lys Asp Asp Asp Thr Leu Arg
Ala Ala Val Asp 405 410
415 Arg Met Lys Ala Lys Leu Arg Lys Lys 420
425 161200DNAArtificial Sequencesynthetic Danio rerio (zebra fish)
glutamine phenylpyruvate transaminase (GPT) codon optimized for E.
coli expression with 5' nucleotides added for cloning and 3' vector
sequences from pET28b 16atgtccgtgg cgaaacgtct ggaaaaattt aaaaccacca
tttttaccca gatgagcatg 60ctggcgatta aacatggcgc gattaacctg ggccagggct
ttccgaactt tgatggcccg 120gattttgtga aagaagcggc gattcaggcg attcgtgatg
gcaacaacca gtatgcgcgt 180ggctatggcg tgccggatct gaacattgcg attagcgaac
gttataaaaa agataccggc 240ctggcggtgg atccggaaaa agaaattacc gtgaccagcg
gctgcaccga agcgattgcg 300gcgaccgtgc tgggcctgat taacccgggc gatgaagtga
ttgtgtttgc gccgttttat 360gatagctatg aagcgaccct gagcatggcg ggcgcgaaag
tgaaaggcat taccctgcgt 420ccgccggatt ttgcgctgcc gattgaagaa ctgaaaagca
ccattagcaa aaacacccgt 480gcgattctgc tgaacacccc gcataacccg accggcaaaa
tgtttacccc ggaagaactg 540aacaccattg cgagcctgtg cattgaaaac gatgtgctgg
tgtttagcga tgaagtgtat 600gataaactgg cgtttgatat ggaacatatt agcattgcga
gcctgccggg catgtttgaa 660cgtaccgtga ccatgaacag cctgggcaaa acctttagcc
tgaccggctg gaaaattggc 720tgggcgattg cgccgccgca tctgacctgg ggcgtgcgtc
aggcgcatgc gtttctgacc 780tttgcaacca gcaacccgat gcagtgggca gcagcagtgg
cactgcgtgc accggatagc 840tattataccg aactgaaacg tgattatatg gcgaaacgta
gcattctggt ggaaggcctg 900aaagcggtgg gctttaaagt gtttccgagc agcggcacct
attttgtggt ggtggatcat 960accccgtttg gccatgaaaa cgatattgcg ttttgcgaat
atctggtgaa agaagtgggc 1020gtggtggcga ttccgaccag cgtgttttat ctgaacccgg
aagaaggcaa aaacctggtg 1080cgttttacct tttgcaaaga tgaaggcacc ctgcgtgcgg
cggtggatcg tatgaaagaa 1140aaactgcgta aagtcgacaa gcttgcggcc gcactcgagc
accaccacca ccaccactga 120017399PRTArtificial Sequencesynthetic Danio
rerio (zebra fish) glutamine phenylpyruvate transaminase (GPT) 5'
and 3' sequences from vector/cloning and His tag on C-terminus
17Met Ser Val Ala Lys Arg Leu Glu Lys Phe Lys Thr Thr Ile Phe Thr1
5 10 15 Gln Met Ser Met
Leu Ala Ile Lys His Gly Ala Ile Asn Leu Gly Gln 20
25 30 Gly Phe Pro Asn Phe Asp Gly Pro Asp
Phe Val Lys Glu Ala Ala Ile 35 40
45 Gln Ala Ile Arg Asp Gly Asn Asn Gln Tyr Ala Arg Gly Tyr
Gly Val 50 55 60
Pro Asp Leu Asn Ile Ala Ile Ser Glu Arg Tyr Lys Lys Asp Thr Gly65
70 75 80 Leu Ala Val Asp Pro
Glu Lys Glu Ile Thr Val Thr Ser Gly Cys Thr 85
90 95 Glu Ala Ile Ala Ala Thr Val Leu Gly Leu
Ile Asn Pro Gly Asp Glu 100 105
110 Val Ile Val Phe Ala Pro Phe Tyr Asp Ser Tyr Glu Ala Thr Leu
Ser 115 120 125 Met
Ala Gly Ala Lys Val Lys Gly Ile Thr Leu Arg Pro Pro Asp Phe 130
135 140 Ala Leu Pro Ile Glu Glu
Leu Lys Ser Thr Ile Ser Lys Asn Thr Arg145 150
155 160 Ala Ile Leu Leu Asn Thr Pro His Asn Pro Thr
Gly Lys Met Phe Thr 165 170
175 Pro Glu Glu Leu Asn Thr Ile Ala Ser Leu Cys Ile Glu Asn Asp Val
180 185 190 Leu Val Phe
Ser Asp Glu Val Tyr Asp Lys Leu Ala Phe Asp Met Glu 195
200 205 His Ile Ser Ile Ala Ser Leu Pro
Gly Met Phe Glu Arg Thr Val Thr 210 215
220 Met Asn Ser Leu Gly Lys Thr Phe Ser Leu Thr Gly Trp
Lys Ile Gly225 230 235
240 Trp Ala Ile Ala Pro Pro His Leu Thr Trp Gly Val Arg Gln Ala His
245 250 255 Ala Phe Leu Thr
Phe Ala Thr Ser Asn Pro Met Gln Trp Ala Ala Ala 260
265 270 Val Ala Leu Arg Ala Pro Asp Ser Tyr
Tyr Thr Glu Leu Lys Arg Asp 275 280
285 Tyr Met Ala Lys Arg Ser Ile Leu Val Glu Gly Leu Lys Ala
Val Gly 290 295 300
Phe Lys Val Phe Pro Ser Ser Gly Thr Tyr Phe Val Val Val Asp His305
310 315 320 Thr Pro Phe Gly His
Glu Asn Asp Ile Ala Phe Cys Glu Tyr Leu Val 325
330 335 Lys Glu Val Gly Val Val Ala Ile Pro Thr
Ser Val Phe Tyr Leu Asn 340 345
350 Pro Glu Glu Gly Lys Asn Leu Val Arg Phe Thr Phe Cys Lys Asp
Glu 355 360 365 Gly
Thr Leu Arg Ala Ala Val Asp Arg Met Lys Glu Lys Leu Arg Lys 370
375 380 Val Asp Lys Leu Ala Ala
Ala Leu Glu His His His His His His385 390
395 181236DNAArtificial Sequencesynthetic Arabidopsis
truncated glutamine phenylpyruvate transaminase (GPT) -30
construct, 30 amino acids removed from the targeting sequence
18atggccaaaa tccatcgtcc tatcggagcc accatgacca cagtttcgac tcagaacgag
60tctactcaaa aacccgtcca ggtggcgaag agattagaga agttcaagac tactattttc
120actcaaatga gcatattggc agttaaacat ggagcgatca atttaggcca aggctttccc
180aatttcgacg gtcctgattt tgttaaagaa gctgcgatcc aagctattaa agatggtaaa
240aaccagtatg ctcgtggata cggcattcct cagctcaact ctgctatagc tgcgcggttt
300cgtgaagata cgggtcttgt tgttgatcct gagaaagaag ttactgttac atctggttgc
360acagaagcca tagctgcagc tatgttgggt ttaataaacc ctggtgatga agtcattctc
420tttgcaccgt tttatgattc ctatgaagca acactctcta tggctggtgc taaagtaaaa
480ggaatcactt tacgtccacc ggacttctcc atccctttgg aagagcttaa agctgcggta
540actaacaaga ctcgagccat ccttatgaac actccgcaca acccgaccgg gaagatgttc
600actagggagg agcttgaaac cattgcatct ctctgcattg aaaacgatgt gcttgtgttc
660tcggatgaag tatacgataa gcttgcgttt gaaatggatc acatttctat agcttctctt
720cccggtatgt atgaaagaac tgtgaccatg aattccctgg gaaagacttt ctctttaacc
780ggatggaaga tcggctgggc gattgcgccg cctcatctga cttggggagt tcgacaagca
840cactcttacc tcacattcgc cacatcaaca ccagcacaat gggcagccgt tgcagctctc
900aaggcaccag agtcttactt caaagagctg aaaagagatt acaatgtgaa aaaggagact
960ctggttaagg gtttgaagga agtcggattt acagtgttcc catcgagcgg gacttacttt
1020gtggttgctg atcacactcc atttggaatg gagaacgatg ttgctttctg tgagtatctt
1080attgaagaag ttggggtcgt tgcgatccca acgagcgtct tttatctgaa tccagaagaa
1140gggaagaatt tggttaggtt tgcgttctgt aaagacgaag agacgttgcg tggtgcaatt
1200gagaggatga agcagaagct taagagaaaa gtctga
123619411PRTArtificial Sequencesynthetic Arabidopsis truncated glutamine
phenylpyruvate transaminase (GPT) -30 construct, 30 amino acids
removed from the targeting sequence 19Met Ala Lys Ile His Arg Pro Ile
Gly Ala Thr Met Thr Thr Val Ser1 5 10
15 Thr Gln Asn Glu Ser Thr Gln Lys Pro Val Gln Val Ala
Lys Arg Leu 20 25 30
Glu Lys Phe Lys Thr Thr Ile Phe Thr Gln Met Ser Ile Leu Ala Val
35 40 45 Lys His Gly Ala
Ile Asn Leu Gly Gln Gly Phe Pro Asn Phe Asp Gly 50 55
60 Pro Asp Phe Val Lys Glu Ala Ala Ile
Gln Ala Ile Lys Asp Gly Lys65 70 75
80 Asn Gln Tyr Ala Arg Gly Tyr Gly Ile Pro Gln Leu Asn Ser
Ala Ile 85 90 95
Ala Ala Arg Phe Arg Glu Asp Thr Gly Leu Val Val Asp Pro Glu Lys
100 105 110 Glu Val Thr Val Thr
Ser Gly Cys Thr Glu Ala Ile Ala Ala Ala Met 115
120 125 Leu Gly Leu Ile Asn Pro Gly Asp Glu
Val Ile Leu Phe Ala Pro Phe 130 135
140 Tyr Asp Ser Tyr Glu Ala Thr Leu Ser Met Ala Gly Ala
Lys Val Lys145 150 155
160 Gly Ile Thr Leu Arg Pro Pro Asp Phe Ser Ile Pro Leu Glu Glu Leu
165 170 175 Lys Ala Ala Val
Thr Asn Lys Thr Arg Ala Ile Leu Met Asn Thr Pro 180
185 190 His Asn Pro Thr Gly Lys Met Phe Thr
Arg Glu Glu Leu Glu Thr Ile 195 200
205 Ala Ser Leu Cys Ile Glu Asn Asp Val Leu Val Phe Ser Asp
Glu Val 210 215 220
Tyr Asp Lys Leu Ala Phe Glu Met Asp His Ile Ser Ile Ala Ser Leu225
230 235 240 Pro Gly Met Tyr Glu
Arg Thr Val Thr Met Asn Ser Leu Gly Lys Thr 245
250 255 Phe Ser Leu Thr Gly Trp Lys Ile Gly Trp
Ala Ile Ala Pro Pro His 260 265
270 Leu Thr Trp Gly Val Arg Gln Ala His Ser Tyr Leu Thr Phe Ala
Thr 275 280 285 Ser
Thr Pro Ala Gln Trp Ala Ala Val Ala Ala Leu Lys Ala Pro Glu 290
295 300 Ser Tyr Phe Lys Glu Leu
Lys Arg Asp Tyr Asn Val Lys Lys Glu Thr305 310
315 320 Leu Val Lys Gly Leu Lys Glu Val Gly Phe Thr
Val Phe Pro Ser Ser 325 330
335 Gly Thr Tyr Phe Val Val Ala Asp His Thr Pro Phe Gly Met Glu Asn
340 345 350 Asp Val Ala
Phe Cys Glu Tyr Leu Ile Glu Glu Val Gly Val Val Ala 355
360 365 Ile Pro Thr Ser Val Phe Tyr Leu
Asn Pro Glu Glu Gly Lys Asn Leu 370 375
380 Val Arg Phe Ala Phe Cys Lys Asp Glu Glu Thr Leu Arg
Gly Ala Ile385 390 395
400 Glu Arg Met Lys Gln Lys Leu Lys Arg Lys Val 405
410 201194DNAArtificial Sequencesynthetic Arabidopsis
truncated glutamine phenylpyruvate transaminase (GPT) -45
construct, 45 amino acids removed from the targeting sequence
20atggcgactc agaacgagtc tactcaaaaa cccgtccagg tggcgaagag attagagaag
60ttcaagacta ctattttcac tcaaatgagc atattggcag ttaaacatgg agcgatcaat
120ttaggccaag gctttcccaa tttcgacggt cctgattttg ttaaagaagc tgcgatccaa
180gctattaaag atggtaaaaa ccagtatgct cgtggatacg gcattcctca gctcaactct
240gctatagctg cgcggtttcg tgaagatacg ggtcttgttg ttgatcctga gaaagaagtt
300actgttacat ctggttgcac agaagccata gctgcagcta tgttgggttt aataaaccct
360ggtgatgaag tcattctctt tgcaccgttt tatgattcct atgaagcaac actctctatg
420gctggtgcta aagtaaaagg aatcacttta cgtccaccgg acttctccat ccctttggaa
480gagcttaaag ctgcggtaac taacaagact cgagccatcc ttatgaacac tccgcacaac
540ccgaccggga agatgttcac tagggaggag cttgaaacca ttgcatctct ctgcattgaa
600aacgatgtgc ttgtgttctc ggatgaagta tacgataagc ttgcgtttga aatggatcac
660atttctatag cttctcttcc cggtatgtat gaaagaactg tgaccatgaa ttccctggga
720aagactttct ctttaaccgg atggaagatc ggctgggcga ttgcgccgcc tcatctgact
780tggggagttc gacaagcaca ctcttacctc acattcgcca catcaacacc agcacaatgg
840gcagccgttg cagctctcaa ggcaccagag tcttacttca aagagctgaa aagagattac
900aatgtgaaaa aggagactct ggttaagggt ttgaaggaag tcggatttac agtgttccca
960tcgagcggga cttactttgt ggttgctgat cacactccat ttggaatgga gaacgatgtt
1020gctttctgtg agtatcttat tgaagaagtt ggggtcgttg cgatcccaac gagcgtcttt
1080tatctgaatc cagaagaagg gaagaatttg gttaggtttg cgttctgtaa agacgaagag
1140acgttgcgtg gtgcaattga gaggatgaag cagaagctta agagaaaagt ctga
119421397PRTArtificial Sequencesynthetic Arabidopsis truncated glutamine
phenylpyruvate transaminase (GPT) -45 construct, 45 amino acids
removed from the targeting sequence 21Met Ala Thr Gln Asn Glu Ser Thr
Gln Lys Pro Val Gln Val Ala Lys1 5 10
15 Arg Leu Glu Lys Phe Lys Thr Thr Ile Phe Thr Gln Met
Ser Ile Leu 20 25 30
Ala Val Lys His Gly Ala Ile Asn Leu Gly Gln Gly Phe Pro Asn Phe
35 40 45 Asp Gly Pro Asp
Phe Val Lys Glu Ala Ala Ile Gln Ala Ile Lys Asp 50 55
60 Gly Lys Asn Gln Tyr Ala Arg Gly Tyr
Gly Ile Pro Gln Leu Asn Ser65 70 75
80 Ala Ile Ala Ala Arg Phe Arg Glu Asp Thr Gly Leu Val Val
Asp Pro 85 90 95
Glu Lys Glu Val Thr Val Thr Ser Gly Cys Thr Glu Ala Ile Ala Ala
100 105 110 Ala Met Leu Gly Leu
Ile Asn Pro Gly Asp Glu Val Ile Leu Phe Ala 115
120 125 Pro Phe Tyr Asp Ser Tyr Glu Ala Thr
Leu Ser Met Ala Gly Ala Lys 130 135
140 Val Lys Gly Ile Thr Leu Arg Pro Pro Asp Phe Ser Ile
Pro Leu Glu145 150 155
160 Glu Leu Lys Ala Ala Val Thr Asn Lys Thr Arg Ala Ile Leu Met Asn
165 170 175 Thr Pro His Asn
Pro Thr Gly Lys Met Phe Thr Arg Glu Glu Leu Glu 180
185 190 Thr Ile Ala Ser Leu Cys Ile Glu Asn
Asp Val Leu Val Phe Ser Asp 195 200
205 Glu Val Tyr Asp Lys Leu Ala Phe Glu Met Asp His Ile Ser
Ile Ala 210 215 220
Ser Leu Pro Gly Met Tyr Glu Arg Thr Val Thr Met Asn Ser Leu Gly225
230 235 240 Lys Thr Phe Ser Leu
Thr Gly Trp Lys Ile Gly Trp Ala Ile Ala Pro 245
250 255 Pro His Leu Thr Trp Gly Val Arg Gln Ala
His Ser Tyr Leu Thr Phe 260 265
270 Ala Thr Ser Thr Pro Ala Gln Trp Ala Ala Val Ala Ala Leu Lys
Ala 275 280 285 Pro
Glu Ser Tyr Phe Lys Glu Leu Lys Arg Asp Tyr Asn Val Lys Lys 290
295 300 Glu Thr Leu Val Lys Gly
Leu Lys Glu Val Gly Phe Thr Val Phe Pro305 310
315 320 Ser Ser Gly Thr Tyr Phe Val Val Ala Asp His
Thr Pro Phe Gly Met 325 330
335 Glu Asn Asp Val Ala Phe Cys Glu Tyr Leu Ile Glu Glu Val Gly Val
340 345 350 Val Ala Ile
Pro Thr Ser Val Phe Tyr Leu Asn Pro Glu Glu Gly Lys 355
360 365 Asn Leu Val Arg Phe Ala Phe Cys
Lys Asp Glu Glu Thr Leu Arg Gly 370 375
380 Ala Ile Glu Arg Met Lys Gln Lys Leu Lys Arg Lys
Val385 390 395
221680DNALycopersicon esculentumtomato ribulose bisphosphate carboxylase
(RUBISCO) promoter, TOMATO RuBisCo rbcS3C promoter 22ggtaccgttt
gaatcctcct taaagttttt ctctggagaa actgtagtaa ttttactttg 60ttgtgttccc
ttcatctttt gaattaatgg catttgtttt aatactaatc tgcttctgaa 120acttgtaatg
tatgtatatc agtttcttat aatttatcca agtaatatct tccattctct 180atgcaattgc
ctgcataagc tcgacaaaag agtacatcaa cccctcctcc tctggactac 240tctagctaaa
cttgaatttc cccttaagat tatgaaattg atatatcctt aacaaacgac 300tccttctgtt
ggaaaatgta gtacttgtct ttcttctttt gggtatatat agtttatata 360caccatacta
tgtacaacat ccaagtagag tgaaatggat acatgtacaa gacttatttg 420attgattgat
gacttgagtt gccttaggag taacaaattc ttaggtcaat aaatcgttga 480tttgaaatta
atctctctgt cttagacaga taggaattat gacttccaat ggtccagaaa 540gcaaagttcg
cactgagggt atacttggaa ttgagacttg cacaggtcca gaaaccaaag 600ttcccatcga
gctctaaaat cacatctttg gaatgaaatt caattagaga taagttgctt 660catagcatag
gtaaaatgga agatgtgaag taacctgcaa taatcagtga aatgacatta 720atacactaaa
tacttcatat gtaattatcc tttccaggtt aacaatactc tataaagtaa 780gaattatcag
aaatgggctc atcaaacttt tgtactatgt atttcatata aggaagtata 840actatacata
agtgtataca caactttatt cctattttgt aaaggtggag agactgtttt 900cgatggatct
aaagcaatat gtctataaaa tgcattgata taataattat ctgagaaaat 960ccagaattgg
cgttggatta tttcagccaa atagaagttt gtaccatact tgttgattcc 1020ttctaagtta
aggtgaagta tcattcataa acagttttcc ccaaagtact actcaccaag 1080tttccctttg
tagaattaac agttcaaata tatggcgcag aaattactct atgcccaaaa 1140ccaaacgaga
aagaaacaaa atacaggggt tgcagacttt attttcgtgt tagggtgtgt 1200tttttcatgt
aattaatcaa aaaatattat gacaaaaaca tttatacata tttttactca 1260acactctggg
tatcagggtg ggttgtgttc gacaatcaat atggaaagga agtattttcc 1320ttattttttt
agttaatatt ttcagttata ccaaacatac cttgtgatat tatttttaaa 1380aatgaaaaac
tcgtcagaaa gaaaaagcaa aagcaacaaa aaaattgcaa gtatttttta 1440aaaaagaaaa
aaaaaacata tcttgtttgt cagtatggga agtttgagat aaggacgagt 1500gaggggttaa
aattcagtgg ccattgattt tgtaatgcca agaaccacaa aatccaatgg 1560ttaccattcc
tgtaagatga ggtttgctaa ctctttttgt ccgttagata ggaagcctta 1620tcactatata
tacaaggcgt cctaataacc tcttagtaac caattatttc agcaccatgg
1680231230DNAPhyllostachys bambusoidesbamboo glutamine phenylpyruvate
transaminase (GPT) 23atggcctccg cggccgtctc caccgtcgcc accgccgccg
acggcgtcgc gaagccgacg 60gagaagcagc cggtacaggt cgcaaagcgt ttggaaaagt
ttaagacaac aattttcaca 120cagatgagca tgcttgccat caagcatgga gcaataaacc
tcggccaggg ctttccgaat 180tttgatggcc ctgactttgt gaaagaagct gctattcaag
ctatcaatgc tgggaagaat 240cagtatgcaa gaggatatgg tgtgcctgaa ctgaactcgg
ctgttgctga aaggttcctg 300aaggacagtg gcttgcaagt cgatcccgag aaggaagtta
ctgtcacatc tgggtgcacg 360gaagcgatag ctgcaacgat attgggtctt atcaaccctg
gcgatgaagt gatcttgttt 420gctccattct atgattcata cgaggctacg ctgtcgatgg
ctggtgccaa tgtaaaagcc 480attactctcc gtcctccaga ttttgcagtc cctcttgagg
agctaaaggc cacagtctct 540aagaacacca gagcgataat gataaacaca ccacacaatc
ctactgggaa aatgttttct 600agggaagaac ttgaattcat tgctactctc tgcaagaaaa
atgatgtgtt gctttttgct 660gatgaggtct atgacaagtt ggcatttgag gcagatcata
tatcaatggc ttctattcct 720ggcatgtatg agaggactgt gactatgaac tctctgggga
agacattctc tctaacagga 780tggaagatcg gttgggcaat agcaccacca cacctgacat
ggggtgtaag gcaggcacac 840tcattcctca catttgccac ctgcacacca atgcaatcgg
cggcggcggc ggctcttaga 900gcaccagata gctactatgg ggagctgaag agggattacg
gtgcaaagaa agcgatacta 960gtcgacggac tcaaggctgc aggttttatt gtttaccctt
caagtggaac atactttgtc 1020atggtcgatc acaccccgtt tggtttcgac aatgatattg
agttctgcga gtatttgatc 1080cgcgaagtcg gtgttgtcgc cataccacca agcgtatttt
atctcaaccc tgaggatggg 1140aagaacttgg tgaggttcac cttctgcaag gatgatgata
cgctgagagc cgcagttgag 1200aggatgaaga caaagctcag gaaaaaatga
123024409PRTPhyllostachys bambusoidesbamboo
glutamine phenylpyruvate transaminase (GPT) 24Met Ala Ser Ala Ala
Val Ser Thr Val Ala Thr Ala Ala Asp Gly Val1 5
10 15 Ala Lys Pro Thr Glu Lys Gln Pro Val Gln
Val Ala Lys Arg Leu Glu 20 25
30 Lys Phe Lys Thr Thr Ile Phe Thr Gln Met Ser Met Leu Ala Ile
Lys 35 40 45 His
Gly Ala Ile Asn Leu Gly Gln Gly Phe Pro Asn Phe Asp Gly Pro 50
55 60 Asp Phe Val Lys Glu Ala
Ala Ile Gln Ala Ile Asn Ala Gly Lys Asn65 70
75 80 Gln Tyr Ala Arg Gly Tyr Gly Val Pro Glu Leu
Asn Ser Ala Val Ala 85 90
95 Glu Arg Phe Leu Lys Asp Ser Gly Leu Gln Val Asp Pro Glu Lys Glu
100 105 110 Val Thr Val
Thr Ser Gly Cys Thr Glu Ala Ile Ala Ala Thr Ile Leu 115
120 125 Gly Leu Ile Asn Pro Gly Asp Glu
Val Ile Leu Phe Ala Pro Phe Tyr 130 135
140 Asp Ser Tyr Glu Ala Thr Leu Ser Met Ala Gly Ala Asn
Val Lys Ala145 150 155
160 Ile Thr Leu Arg Pro Pro Asp Phe Ala Val Pro Leu Glu Glu Leu Lys
165 170 175 Ala Thr Val Ser
Lys Asn Thr Arg Ala Ile Met Ile Asn Thr Pro His 180
185 190 Asn Pro Thr Gly Lys Met Phe Ser Arg
Glu Glu Leu Glu Phe Ile Ala 195 200
205 Thr Leu Cys Lys Lys Asn Asp Val Leu Leu Phe Ala Asp Glu
Val Tyr 210 215 220
Asp Lys Leu Ala Phe Glu Ala Asp His Ile Ser Met Ala Ser Ile Pro225
230 235 240 Gly Met Tyr Glu Arg
Thr Val Thr Met Asn Ser Leu Gly Lys Thr Phe 245
250 255 Ser Leu Thr Gly Trp Lys Ile Gly Trp Ala
Ile Ala Pro Pro His Leu 260 265
270 Thr Trp Gly Val Arg Gln Ala His Ser Phe Leu Thr Phe Ala Thr
Cys 275 280 285 Thr
Pro Met Gln Ser Ala Ala Ala Ala Ala Leu Arg Ala Pro Asp Ser 290
295 300 Tyr Tyr Gly Glu Leu Lys
Arg Asp Tyr Gly Ala Lys Lys Ala Ile Leu305 310
315 320 Val Asp Gly Leu Lys Ala Ala Gly Phe Ile Val
Tyr Pro Ser Ser Gly 325 330
335 Thr Tyr Phe Val Met Val Asp His Thr Pro Phe Gly Phe Asp Asn Asp
340 345 350 Ile Glu Phe
Cys Glu Tyr Leu Ile Arg Glu Val Gly Val Val Ala Ile 355
360 365 Pro Pro Ser Val Phe Tyr Leu Asn
Pro Glu Asp Gly Lys Asn Leu Val 370 375
380 Arg Phe Thr Phe Cys Lys Asp Asp Asp Thr Leu Arg Ala
Ala Val Glu385 390 395
400 Arg Met Lys Thr Lys Leu Arg Lys Lys 405
251858DNAArtificial Sequencesynthetic Cambia 1305.1 with (3' end of)
rbcS3C+rice glutamine phenylpyruvate transaminase (GPT), Cambia
1305.1+rbcS3C promoter+catl intron with rice GPT 25aaaaaagaaa aaaaaaacat
atcttgtttg tcagtatggg aagtttgaga taaggacgag 60tgaggggtta aaattcagtg
gccattgatt ttgtaatgcc aagaaccaca aaatccaatg 120gttaccattc ctgtaagatg
aggtttgcta actctttttg tccgttagat aggaagcctt 180atcactatat atacaaggcg
tcctaataac ctcttagtaa ccaattattt cagcaccatg 240gtagatctga gggtaaattt
ctagtttttc tccttcattt tcttggttag gacccttttc 300tctttttatt tttttgagct
ttgatctttc tttaaactga tctatttttt aattgattgg 360ttatggtgta aatattacat
agctttaact gataatctga ttactttatt tcgtgtgtct 420atgatgatga tgatagttac
agaaccgacg aactagtatg aatctggccg gctttctcgc 480cacgcccgcg accgcgaccg
cgacgcggca tgagatgccg ttaaatccct cctcctccgc 540ctccttcctc ctctcctcgc
tccgccgctc gctcgtcgcg tcgctccgga aggcctcgcc 600ggcggcggcc gcggcgctct
cccccatggc ctccgcgtcc accgtcgccg ccgagaacgg 660cgccgccaag gcggcggcgg
agaagcagca gcagcagcct gtgcaggttg caaagcggtt 720ggaaaagttt aagacgacca
ttttcacaca gatgagtatg cttgccatca agcatggagc 780aataaacctt ggccagggtt
ttccgaattt cgatggccct gactttgtaa aagaggctgc 840tattcaagct atcaatgctg
ggaagaatca gtacgcaaga ggatatggtg tgcctgaact 900gaactcagct attgctgaaa
gattcctgaa ggacagcgga ctgcaagtcg atccggagaa 960ggaagttact gtcacatctg
gatgcacaga agctatagct gcaacaattt taggtctaat 1020taatccaggc gatgaagtga
tattgtttgc tccattctat gattcatatg aggctaccct 1080gtcaatggct ggtgccaacg
taaaagccat tactctccgt cctccagatt tttcagtccc 1140tcttgaagag ctaaaggctg
cagtctcgaa gaacaccaga gctattatga taaacacccc 1200gcacaatcct actgggaaaa
tgtttacaag ggaagaactt gagtttattg ccactctctg 1260caaggaaaat gatgtgctgc
tttttgctga tgaggtctac gacaagttag cttttgaggc 1320agatcatata tcaatggctt
ctattcctgg catgtatgag aggaccgtga ccatgaactc 1380tcttgggaag acattctctc
ttacaggatg gaagatcggt tgggcaatcg caccgccaca 1440cctgacatgg ggtgtaaggc
aggcacactc attcctcacg tttgcgacct gcacaccaat 1500gcaagcagct gcagctgcag
ctctgagagc accagatagc tactatgagg aactgaggag 1560ggattatgga gctaagaagg
cattgctagt caacggactc aaggatgcag gtttcattgt 1620ctatccttca agtggaacat
acttcgtcat ggtcgaccac accccatttg gtttcgacaa 1680tgatattgag ttctgcgagt
atttgattcg cgaagtcggt gttgtcgcca taccacctag 1740tgtattttat ctcaaccctg
aggatgggaa gaacttggtg aggttcacct tttgcaagga 1800tgatgagacg ctgagagccg
cggttgagag gatgaagaca aagctcagga aaaaatga 1858261724DNAArtificial
Sequencesynthetic Hordeum vulgare (barley) glutamine phenylpyruvate
transaminase (GPT) in vector Cambia 1305.1 with (3' end of)
rbcS3C+hordeum (IDI4) + catl intron 26aaaaaagaaa aaaaaaacat atcttgtttg
tcagtatggg aagtttgaga taaggacgag 60tgaggggtta aaattcagtg gccattgatt
ttgtaatgcc aagaaccaca aaatccaatg 120gttaccattc ctgtaagatg aggtttgcta
actctttttg tccgttagat aggaagcctt 180atcactatat atacaaggcg tcctaataac
ctcttagtaa ccaattattt cagcaccatg 240gtagatctga gggtaaattt ctagtttttc
tccttcattt tcttggttag gacccttttc 300tctttttatt tttttgagct ttgatctttc
tttaaactga tctatttttt aattgattgg 360ttatggtgta aatattacat agctttaact
gataatctga ttactttatt tcgtgtgtct 420atgatgatga tgatagttac agaaccgacg
aactagtatg gcatccgccc ccgcctccgc 480ctccgcggcc ctctccaccg ccgcccccgc
cgacaacggg gccgccaagc ccacggagca 540gcggccggta caggtggcta agcgattgga
gaagttcaaa acaacaattt tcacacagat 600gagcatgctc gcagtgaagc atggagcaat
aaaccttgga caggggtttc ccaattttga 660tggccctgac tttgtcaaag atgctgctat
tgaggctatc aaagctggaa agaatcagta 720tgcaagagga tatggtgtgc ctgaattgaa
ctcagctgtt gctgagagat ttctcaagga 780cagtggattg cacatcgatc ctgataagga
agttactgtt acatctgggt gcacagaagc 840aatagctgca acgatattgg gtctgatcaa
ccctggggat gaagtcatac tgtttgctcc 900attctatgat tcttatgagg ctacactgtc
catggctggt gcgaatgtca aagccattac 960actccgccct ccggactttg cagtccctct
tgaagagcta aaggctgcag tctcgaagaa 1020taccagagca ataatgatta atacacctca
caaccctacc gggaaaatgt tcacaaggga 1080ggaacttgag ttcattgctg atctctgcaa
ggaaaatgac gtgttgctct ttgccgatga 1140ggtctacgac aagctggcgt ttgaggcgga
tcacatatca atggcttcta ttcctggcat 1200gtatgagagg accgtcacta tgaactccct
ggggaagacg ttctccttga ccggatggaa 1260gatcggctgg gcgatagcac caccgcacct
gacatggggc gtaaggcagg cacactcctt 1320cctcacattc gccacctcca cgccgatgca
atcagcagcg gcggcggccc tgagagcacc 1380ggacagctac tttgaggagc tgaagaggga
ctacggcgca aagaaagcgc tgctggtgga 1440cgggctcaag gcggcgggct tcatcgtcta
cccttcgagc ggaacctact tcatcatggt 1500cgaccacacc ccgttcgggt tcgacaacga
cgtcgagttc tgcgagtact tgatccgcga 1560ggtcggcgtc gtggccatcc cgccaagcgt
gttctacctg aacccggagg acgggaagaa 1620cctggtgagg ttcaccttct gcaaggacga
cgacacgcta agggcggcgg tggacaggat 1680gaaggccaag ctcaggaaga aatgattgag
gggcgcacgt gtga 1724271868DNAArtificial
Sequencesynthetic Arabidopsis glutamine phenylpyruvate transaminase
(GPT) expression cassette under control of CMV 35S promoter
(promoter from Cambia 1201) 27catggagtca aagattcaaa tagaggacct aacagaactc
gccgtaaaga ctggcgaaca 60gttcatacag agtctcttac gactcaatga caagaagaaa
atcttcgtca acatggtgga 120gcacgacaca cttgtctact ccaaaaatat caaagataca
gtctcagaag accaaagggc 180aattgagact tttcaacaaa gggtaatatc cggaaacctc
ctcggattcc attgcccagc 240tatctgtcac tttattgtga agatagtgga aaaggaaggt
ggctcctaca aatgccatca 300ttgcgataaa ggaaaggcca tcgttgaaga tgcctctgcc
gacagtggtc ccaaagatgg 360acccccaccc acgaggagca tcgtggaaaa agaagacgtt
ccaaccacgt cttcaaagca 420agtggattga tgtgatatct ccactgacgt aagggatgac
gcacaatccc actatccttc 480gcaagaccct tcctctatat aaggaagttc atttcatttg
gagagaacac gggggactct 540tgaccatgta cctggacata aatggtgtga tgatcaaaca
gtttagcttc aaagcctctc 600ttctcccatt ctcttctaat ttccgacaaa gctccgccaa
aatccatcgt cctatcggag 660ccaccatgac cacagtttcg actcagaacg agtctactca
aaaacccgtc caggtggcga 720agagattaga gaagttcaag actactattt tcactcaaat
gagcatattg gcagttaaac 780atggagcgat caatttaggc caaggctttc ccaatttcga
cggtcctgat tttgttaaag 840aagctgcgat ccaagctatt aaagatggta aaaaccagta
tgctcgtgga tacggcattc 900ctcagctcaa ctctgctata gctgcgcggt ttcgtgaaga
tacgggtctt gttgttgatc 960ctgagaaaga agttactgtt acatctggtt gcacagaagc
catagctgca gctatgttgg 1020gtttaataaa ccctggtgat gaagtcattc tctttgcacc
gttttatgat tcctatgaag 1080caacactctc tatggctggt gctaaagtaa aaggaatcac
tttacgtcca ccggacttct 1140ccatcccttt ggaagagctt aaagctgcgg taactaacaa
gactcgagcc atccttatga 1200acactccgca caacccgacc gggaagatgt tcactaggga
ggagcttgaa accattgcat 1260ctctctgcat tgaaaacgat gtgcttgtgt tctcggatga
agtatacgat aagcttgcgt 1320ttgaaatgga tcacatttct atagcttctc ttcccggtat
gtatgaaaga actgtgacca 1380tgaattccct gggaaagact ttctctttaa ccggatggaa
gatcggctgg gcgattgcgc 1440cgcctcatct gacttgggga gttcgacaag cacactctta
cctcacattc gccacatcaa 1500caccagcaca atgggcagcc gttgcagctc tcaaggcacc
agagtcttac ttcaaagagc 1560tgaaaagaga ttacaatgtg aaaaaggaga ctctggttaa
gggtttgaag gaagtcggat 1620ttacagtgtt cccatcgagc gggacttact ttgtggttgc
tgatcacact ccatttggaa 1680tggagaacga tgttgctttc tgtgagtatc ttattgaaga
agttggggtc gttgcgatcc 1740caacgagcgt cttttatctg aatccagaag aagggaagaa
tttggttagg tttgcgttct 1800gtaaagacga agagacgttg cgtggtgcaa ttgagaggat
gaagcagaag cttaagagaa 1860aagtctga
1868281780DNAArtificial Sequencesynthetic Cambia
p1305.1 with (3' end of) rbcS3C+Arabidopsis glutamine
phenylpyruvate transaminase (GPT) + catl intron 28aaaaaagaaa
aaaaaaacat atcttgtttg tcagtatggg aagtttgaga taaggacgag 60tgaggggtta
aaattcagtg gccattgatt ttgtaatgcc aagaaccaca aaatccaatg 120gttaccattc
ctgtaagatg aggtttgcta actctttttg tccgttagat aggaagcctt 180atcactatat
atacaaggcg tcctaataac ctcttagtaa ccaattattt cagcaccatg 240gtagatctga
gggtaaattt ctagtttttc tccttcattt tcttggttag gacccttttc 300tctttttatt
tttttgagct ttgatctttc tttaaactga tctatttttt aattgattgg 360ttatggtgta
aatattacat agctttaact gataatctga ttactttatt tcgtgtgtct 420atgatgatga
tgatagttac agaaccgacg aactagtatg tacctggaca taaatggtgt 480gatgatcaaa
cagtttagct tcaaagcctc tcttctccca ttctcttcta atttccgaca 540aagctccgcc
aaaatccatc gtcctatcgg agccaccatg accacagttt cgactcagaa 600cgagtctact
caaaaacccg tccaggtggc gaagagatta gagaagttca agactactat 660tttcactcaa
atgagcatat tggcagttaa acatggagcg atcaatttag gccaaggctt 720tcccaatttc
gacggtcctg attttgttaa agaagctgcg atccaagcta ttaaagatgg 780taaaaaccag
tatgctcgtg gatacggcat tcctcagctc aactctgcta tagctgcgcg 840gtttcgtgaa
gatacgggtc ttgttgttga tcctgagaaa gaagttactg ttacatctgg 900ttgcacagaa
gccatagctg cagctatgtt gggtttaata aaccctggtg atgaagtcat 960tctctttgca
ccgttttatg attcctatga agcaacactc tctatggctg gtgctaaagt 1020aaaaggaatc
actttacgtc caccggactt ctccatccct ttggaagagc ttaaagctgc 1080ggtaactaac
aagactcgag ccatccttat gaacactccg cacaacccga ccgggaagat 1140gttcactagg
gaggagcttg aaaccattgc atctctctgc attgaaaacg atgtgcttgt 1200gttctcggat
gaagtatacg ataagcttgc gtttgaaatg gatcacattt ctatagcttc 1260tcttgccggt
atgtatgaaa gaactgtgac catgaattcc ctgggaaaga ctttctcttt 1320aaccggatgg
aagatcggct gggcgattgc gccgcctcat ctgacttggg gagttcgaca 1380agcacactct
tacctcacat tcgccacatc aacaccagca caatgggcag ccgttgcagc 1440tctcaaggca
ccagagtctt acttcaaaga gctgaaaaga gattacaatg tgaaaaagga 1500gactctggtt
aagggtttga aggaagtcgg atttacagtg ttcccatcga gcgggactta 1560ctttgtggtt
gctgatcaca ctccatttgg aatggagaac gatgttgctt tctgtgagta 1620tcttattgaa
gaagttgggg tcgttgcgat cccaacgagc gtcttttatc tgaatccaga 1680agaagggaag
aatttggtta ggtttgcgtt ctgtaaagac gaagagacgt tgcgtggtgc 1740aattgagagg
atgaagcaga agcttaagag aaaagtctga
1780291155DNAArabidopsis thalianaArabidopsis glutamine phenylpyruvate
transaminase (GPT) (mature protein, no targeting sequence) 29gtggcgaaga
gattagagaa gttcaagact actattttca ctcaaatgag catattggca 60gttaaacatg
gagcgatcaa tttaggccaa ggctttccca atttcgacgg tcctgatttt 120gttaaagaag
ctgcgatcca agctattaaa gatggtaaaa accagtatgc tcgtggatac 180ggcattcctc
agctcaactc tgctatagct gcgcggtttc gtgaagatac gggtcttgtt 240gttgatcctg
agaaagaagt tactgttaca tctggttgca cagaagccat agctgcagct 300atgttgggtt
taataaaccc tggtgatgaa gtcattctct ttgcaccgtt ttatgattcc 360tatgaagcaa
cactctctat ggctggtgct aaagtaaaag gaatcacttt acgtccaccg 420gacttctcca
tccctttgga agagcttaaa gctgcggtaa ctaacaagac tcgagccatc 480cttatgaaca
ctccgcacaa cccgaccggg aagatgttca ctagggagga gcttgaaacc 540attgcatctc
tctgcattga aaacgatgtg cttgtgttct cggatgaagt atacgataag 600cttgcgtttg
aaatggatca catttctata gcttctcttc ccggtatgta tgaaagaact 660gtgaccatga
attccctggg aaagactttc tctttaaccg gatggaagat cggctgggcg 720attgcgccgc
ctcatctgac ttggggagtt cgacaagcac actcttacct cacattcgcc 780acatcaacac
cagcacaatg ggcagccgtt gcagctctca aggcaccaga gtcttacttc 840aaagagctga
aaagagatta caatgtgaaa aaggagactc tggttaaggg tttgaaggaa 900gtcggattta
cagtgttccc atcgagcggg acttactttg tggttgctga tcacactcca 960tttggaatgg
agaacgatgt tgctttctgt gagtatctta ttgaagaagt tggggtcgtt 1020gcgatcccaa
cgagcgtctt ttatctgaat ccagaagaag ggaagaattt ggttaggttt 1080gcgttctgta
aagacgaaga gacgttgcgt ggtgcaattg agaggatgaa gcagaagctt 1140aagagaaaag
tctga
115530384PRTArabidopsis thalianaArabidopsis glutamine phenylpyruvate
transaminase (GPT) (mature protein, no targeting sequence) 30Val Ala Lys
Arg Leu Glu Lys Phe Lys Thr Thr Ile Phe Thr Gln Met1 5
10 15 Ser Ile Leu Ala Val Lys His Gly
Ala Ile Asn Leu Gly Gln Gly Phe 20 25
30 Pro Asn Phe Asp Gly Pro Asp Phe Val Lys Glu Ala Ala
Ile Gln Ala 35 40 45
Ile Lys Asp Gly Lys Asn Gln Tyr Ala Arg Gly Tyr Gly Ile Pro Gln 50
55 60 Leu Asn Ser Ala Ile
Ala Ala Arg Phe Arg Glu Asp Thr Gly Leu Val65 70
75 80 Val Asp Pro Glu Lys Glu Val Thr Val Thr
Ser Gly Cys Thr Glu Ala 85 90
95 Ile Ala Ala Ala Met Leu Gly Leu Ile Asn Pro Gly Asp Glu Val
Ile 100 105 110 Leu
Phe Ala Pro Phe Tyr Asp Ser Tyr Glu Ala Thr Leu Ser Met Ala 115
120 125 Gly Ala Lys Val Lys Gly
Ile Thr Leu Arg Pro Pro Asp Phe Ser Ile 130 135
140 Pro Leu Glu Glu Leu Lys Ala Ala Val Thr Asn
Lys Thr Arg Ala Ile145 150 155
160 Leu Met Asn Thr Pro His Asn Pro Thr Gly Lys Met Phe Thr Arg Glu
165 170 175 Glu Leu Glu
Thr Ile Ala Ser Leu Cys Ile Glu Asn Asp Val Leu Val 180
185 190 Phe Ser Asp Glu Val Tyr Asp Lys
Leu Ala Phe Glu Met Asp His Ile 195 200
205 Ser Ile Ala Ser Leu Pro Gly Met Tyr Glu Arg Thr Val
Thr Met Asn 210 215 220
Ser Leu Gly Lys Thr Phe Ser Leu Thr Gly Trp Lys Ile Gly Trp Ala225
230 235 240 Ile Ala Pro Pro His
Leu Thr Trp Gly Val Arg Gln Ala His Ser Tyr 245
250 255 Leu Thr Phe Ala Thr Ser Thr Pro Ala Gln
Trp Ala Ala Val Ala Ala 260 265
270 Leu Lys Ala Pro Glu Ser Tyr Phe Lys Glu Leu Lys Arg Asp Tyr
Asn 275 280 285 Val
Lys Lys Glu Thr Leu Val Lys Gly Leu Lys Glu Val Gly Phe Thr 290
295 300 Val Phe Pro Ser Ser Gly
Thr Tyr Phe Val Val Ala Asp His Thr Pro305 310
315 320 Phe Gly Met Glu Asn Asp Val Ala Phe Cys Glu
Tyr Leu Ile Glu Glu 325 330
335 Val Gly Val Val Ala Ile Pro Thr Ser Val Phe Tyr Leu Asn Pro Glu
340 345 350 Glu Gly Lys
Asn Leu Val Arg Phe Ala Phe Cys Lys Asp Glu Glu Thr 355
360 365 Leu Arg Gly Ala Ile Glu Arg Met
Lys Gln Lys Leu Lys Arg Lys Val 370 375
380 31384PRTVitis viniferagrape glutamine phenylpyruvate
transaminase (GPT) (mature protein, no targeting sequence) 31Val
Ala Lys Arg Leu Glu Lys Phe Lys Thr Thr Ile Phe Thr Gln Met1
5 10 15 Ser Met Leu Ala Ile Lys
His Gly Ala Ile Asn Leu Gly Gln Gly Phe 20 25
30 Pro Asn Phe Asp Gly Pro Glu Phe Val Lys Glu
Ala Ala Ile Gln Ala 35 40 45
Ile Lys Asp Gly Lys Asn Gln Tyr Ala Arg Gly Tyr Gly Val Pro Asp
50 55 60 Leu Asn Ser
Ala Val Ala Asp Arg Phe Lys Lys Asp Thr Gly Leu Val65 70
75 80 Val Asp Pro Glu Lys Glu Val Thr
Val Thr Ser Gly Cys Thr Glu Ala 85 90
95 Ile Ala Ala Thr Met Leu Gly Leu Ile Asn Pro Gly Asp
Glu Val Ile 100 105 110
Leu Phe Ala Pro Phe Tyr Asp Ser Tyr Glu Ala Thr Leu Ser Met Ala
115 120 125 Gly Ala Gln Ile
Lys Ser Ile Thr Leu Arg Pro Pro Asp Phe Ala Val 130
135 140 Pro Met Asp Glu Leu Lys Ser Ala
Ile Ser Lys Asn Thr Arg Ala Ile145 150
155 160 Leu Ile Asn Thr Pro His Asn Pro Thr Gly Lys Met
Phe Thr Arg Glu 165 170
175 Glu Leu Asn Val Ile Ala Ser Leu Cys Ile Glu Asn Asp Val Leu Val
180 185 190 Phe Thr Asp
Glu Val Tyr Asp Lys Leu Ala Phe Glu Met Asp His Ile 195
200 205 Ser Met Ala Ser Leu Pro Gly Met
Tyr Glu Arg Thr Val Thr Met Asn 210 215
220 Ser Leu Gly Lys Thr Phe Ser Leu Thr Gly Trp Lys Ile
Gly Trp Thr225 230 235
240 Val Ala Pro Pro His Leu Thr Trp Gly Val Arg Gln Ala His Ser Phe
245 250 255 Leu Thr Phe Ala
Thr Cys Thr Pro Met Gln Trp Ala Ala Ala Thr Ala 260
265 270 Leu Arg Ala Pro Asp Ser Tyr Tyr Glu
Glu Leu Lys Arg Asp Tyr Ser 275 280
285 Ala Lys Lys Ala Ile Leu Val Glu Gly Leu Lys Ala Val Gly
Phe Arg 290 295 300
Val Tyr Pro Ser Ser Gly Thr Tyr Phe Val Val Val Asp His Thr Pro305
310 315 320 Phe Gly Leu Lys Asp
Asp Ile Ala Phe Cys Glu Tyr Leu Ile Lys Glu 325
330 335 Val Gly Val Val Ala Ile Pro Thr Ser Val
Phe Tyr Leu His Pro Glu 340 345
350 Asp Gly Lys Asn Leu Val Arg Phe Thr Phe Cys Lys Asp Glu Gly
Thr 355 360 365 Leu
Arg Ala Ala Val Glu Arg Met Lys Glu Lys Leu Lys Pro Lys Gln 370
375 380 32383PRTOryza sativarice
glutamine phenylpyruvate transaminase (GPT) (mature protein, no
targeting sequence) 32Val Ala Lys Arg Leu Glu Lys Phe Lys Thr Thr Ile Phe
Thr Gln Met1 5 10 15
Ser Met Leu Ala Ile Lys His Gly Ala Ile Asn Leu Gly Gln Gly Phe
20 25 30 Pro Asn Phe Asp Gly
Pro Asp Phe Val Lys Glu Ala Ala Ile Gln Ala 35 40
45 Ile Asn Ala Gly Lys Asn Gln Tyr Ala Arg
Gly Tyr Gly Val Pro Glu 50 55 60
Leu Asn Ser Ala Ile Ala Glu Arg Phe Leu Lys Asp Ser Gly Leu
Gln65 70 75 80 Val
Asp Pro Glu Lys Glu Val Thr Val Thr Ser Gly Cys Thr Glu Ala
85 90 95 Ile Ala Ala Thr Ile Leu
Gly Leu Ile Asn Pro Gly Asp Glu Val Ile 100
105 110 Leu Phe Ala Pro Phe Tyr Asp Ser Tyr Glu
Ala Thr Leu Ser Met Ala 115 120
125 Gly Ala Asn Val Lys Ala Ile Thr Leu Arg Pro Pro Asp Phe
Ser Val 130 135 140
Pro Leu Glu Glu Leu Lys Ala Ala Val Ser Lys Asn Thr Arg Ala Ile145
150 155 160 Met Ile Asn Thr Pro
His Asn Pro Thr Gly Lys Met Phe Thr Arg Glu 165
170 175 Glu Leu Glu Phe Ile Ala Thr Leu Cys Lys
Glu Asn Asp Val Leu Leu 180 185
190 Phe Ala Asp Glu Val Tyr Asp Lys Leu Ala Phe Glu Ala Asp His
Ile 195 200 205 Ser
Met Ala Ser Ile Pro Gly Met Tyr Glu Arg Thr Val Thr Met Asn 210
215 220 Ser Leu Gly Lys Thr Phe
Ser Leu Thr Gly Trp Lys Ile Gly Trp Ala225 230
235 240 Ile Ala Pro Pro His Leu Thr Trp Gly Val Arg
Gln Ala His Ser Phe 245 250
255 Leu Thr Phe Ala Thr Cys Thr Pro Met Gln Ala Ala Ala Ala Ala Ala
260 265 270 Leu Arg Ala
Pro Asp Ser Tyr Tyr Glu Glu Leu Arg Arg Asp Tyr Gly 275
280 285 Ala Lys Lys Ala Leu Leu Val Asn
Gly Leu Lys Asp Ala Gly Phe Ile 290 295
300 Val Tyr Pro Ser Ser Gly Thr Tyr Phe Val Met Val Asp
His Thr Pro305 310 315
320 Phe Gly Phe Asp Asn Asp Ile Glu Phe Cys Glu Tyr Leu Ile Arg Glu
325 330 335 Val Gly Val Val
Ala Ile Pro Pro Ser Val Phe Tyr Leu Asn Pro Glu 340
345 350 Asp Gly Lys Asn Leu Val Arg Phe Thr
Phe Cys Lys Asp Asp Glu Thr 355 360
365 Leu Arg Ala Ala Val Glu Arg Met Lys Thr Lys Leu Arg Lys
Lys 370 375 380
33383PRTGlycine maxsoybean glutamine phenylpyruvate transaminase
(GPT) (mature protein, no targeting sequence) 33Ala Lys Arg Leu Glu Lys
Phe Gln Thr Thr Ile Phe Thr Gln Met Ser1 5
10 15 Leu Leu Ala Ile Lys His Gly Ala Ile Asn Leu
Gly Gln Gly Phe Pro 20 25 30
Asn Phe Asp Gly Pro Glu Phe Val Lys Glu Ala Ala Ile Gln Ala Ile
35 40 45 Arg Asp Gly
Lys Asn Gln Tyr Ala Arg Gly Tyr Gly Val Pro Asp Leu 50
55 60 Asn Ile Ala Ile Ala Glu Arg Phe
Lys Lys Asp Thr Gly Leu Val Val65 70 75
80 Asp Pro Glu Lys Glu Ile Thr Val Thr Ser Gly Cys Thr
Glu Ala Ile 85 90 95
Ala Ala Thr Met Ile Gly Leu Ile Asn Pro Gly Asp Glu Val Ile Met
100 105 110 Phe Ala Pro Phe Tyr
Asp Ser Tyr Glu Ala Thr Leu Ser Met Ala Gly 115
120 125 Ala Lys Val Lys Gly Ile Thr Leu Arg
Pro Pro Asp Phe Ala Val Pro 130 135
140 Leu Glu Glu Leu Lys Ser Thr Ile Ser Lys Asn Thr Arg
Ala Ile Leu145 150 155
160 Ile Asn Thr Pro His Asn Pro Thr Gly Lys Met Phe Thr Arg Glu Glu
165 170 175 Leu Asn Cys Ile
Ala Ser Leu Cys Ile Glu Asn Asp Val Leu Val Phe 180
185 190 Thr Asp Glu Val Tyr Asp Lys Leu Ala
Phe Asp Met Glu His Ile Ser 195 200
205 Met Ala Ser Leu Pro Gly Met Phe Glu Arg Thr Val Thr Leu
Asn Ser 210 215 220
Leu Gly Lys Thr Phe Ser Leu Thr Gly Trp Lys Ile Gly Trp Ala Ile225
230 235 240 Ala Pro Pro His Leu
Ser Trp Gly Val Arg Gln Ala His Ala Phe Leu 245
250 255 Thr Phe Ala Thr Ala His Pro Phe Gln Cys
Ala Ala Ala Ala Ala Leu 260 265
270 Arg Ala Pro Asp Ser Tyr Tyr Val Glu Leu Lys Arg Asp Tyr Met
Ala 275 280 285 Lys
Arg Ala Ile Leu Ile Glu Gly Leu Lys Ala Val Gly Phe Lys Val 290
295 300 Phe Pro Ser Ser Gly Thr
Tyr Phe Val Val Val Asp His Thr Pro Phe305 310
315 320 Gly Leu Glu Asn Asp Val Ala Phe Cys Glu Tyr
Leu Val Lys Glu Val 325 330
335 Gly Val Val Ala Ile Pro Thr Ser Val Phe Tyr Leu Asn Pro Glu Glu
340 345 350 Gly Lys Asn
Leu Val Arg Phe Thr Phe Cys Lys Asp Glu Glu Thr Ile 355
360 365 Arg Ser Ala Val Glu Arg Met Lys
Ala Lys Leu Arg Lys Val Asp 370 375
380 34383PRTHordeum vulgarebarley glutamine phenylpyruvate
transaminase (GPT) (mature protein, no targeting sequence) 34Val
Ala Lys Arg Leu Glu Lys Phe Lys Thr Thr Ile Phe Thr Gln Met1
5 10 15 Ser Met Leu Ala Val Lys
His Gly Ala Ile Asn Leu Gly Gln Gly Phe 20 25
30 Pro Asn Phe Asp Gly Pro Asp Phe Val Lys Asp
Ala Ala Ile Glu Ala 35 40 45
Ile Lys Ala Gly Lys Asn Gln Tyr Ala Arg Gly Tyr Gly Val Pro Glu
50 55 60 Leu Asn Ser
Ala Val Ala Glu Arg Phe Leu Lys Asp Ser Gly Leu His65 70
75 80 Ile Asp Pro Asp Lys Glu Val Thr
Val Thr Ser Gly Cys Thr Glu Ala 85 90
95 Ile Ala Ala Thr Ile Leu Gly Leu Ile Asn Pro Gly Asp
Glu Val Ile 100 105 110
Leu Phe Ala Pro Phe Tyr Asp Ser Tyr Glu Ala Thr Leu Ser Met Ala
115 120 125 Gly Ala Asn Val
Lys Ala Ile Thr Leu Arg Pro Pro Asp Phe Ala Val 130
135 140 Pro Leu Glu Glu Leu Lys Ala Ala
Val Ser Lys Asn Thr Arg Ala Ile145 150
155 160 Met Ile Asn Thr Pro His Asn Pro Thr Gly Lys Met
Phe Thr Arg Glu 165 170
175 Glu Leu Glu Phe Ile Ala Asp Leu Cys Lys Glu Asn Asp Val Leu Leu
180 185 190 Phe Ala Asp
Glu Val Tyr Asp Lys Leu Ala Phe Glu Ala Asp His Ile 195
200 205 Ser Met Ala Ser Ile Pro Gly Met
Tyr Glu Arg Thr Val Thr Met Asn 210 215
220 Ser Leu Gly Lys Thr Phe Ser Leu Thr Gly Trp Lys Ile
Gly Trp Ala225 230 235
240 Ile Ala Pro Pro His Leu Thr Trp Gly Val Arg Gln Ala His Ser Phe
245 250 255 Leu Thr Phe Ala
Thr Ser Thr Pro Met Gln Ser Ala Ala Ala Ala Ala 260
265 270 Leu Arg Ala Pro Asp Ser Tyr Phe Glu
Glu Leu Lys Arg Asp Tyr Gly 275 280
285 Ala Lys Lys Ala Leu Leu Val Asp Gly Leu Lys Ala Ala Gly
Phe Ile 290 295 300
Val Tyr Pro Ser Ser Gly Thr Tyr Phe Ile Met Val Asp His Thr Pro305
310 315 320 Phe Gly Phe Asp Asn
Asp Val Glu Phe Cys Glu Tyr Leu Ile Arg Glu 325
330 335 Val Gly Val Val Ala Ile Pro Pro Ser Val
Phe Tyr Leu Asn Pro Glu 340 345
350 Asp Gly Lys Asn Leu Val Arg Phe Thr Phe Cys Lys Asp Asp Asp
Thr 355 360 365 Leu
Arg Ala Ala Val Asp Arg Met Lys Ala Lys Leu Arg Lys Lys 370
375 380 35382PRTDanio reriozebra fish
glutamine phenylpyruvate transaminase (GPT) (mature protein, no
targeting sequence) 35Val Ala Lys Arg Leu Glu Lys Phe Lys Thr Thr Ile Phe
Thr Gln Met1 5 10 15
Ser Met Leu Ala Ile Lys His Gly Ala Ile Asn Leu Gly Gln Gly Phe
20 25 30 Pro Asn Phe Asp Gly
Pro Asp Phe Val Lys Glu Ala Ala Ile Gln Ala 35 40
45 Ile Arg Asp Gly Asn Asn Gln Tyr Ala Arg
Gly Tyr Gly Val Pro Asp 50 55 60
Leu Asn Ile Ala Ile Ser Glu Arg Tyr Lys Lys Asp Thr Gly Leu
Ala65 70 75 80 Val
Asp Pro Glu Lys Glu Ile Thr Val Thr Ser Gly Cys Thr Glu Ala
85 90 95 Ile Ala Ala Thr Val Leu
Gly Leu Ile Asn Pro Gly Asp Glu Val Ile 100
105 110 Val Phe Ala Pro Phe Tyr Asp Ser Tyr Glu
Ala Thr Leu Ser Met Ala 115 120
125 Gly Ala Lys Val Lys Gly Ile Thr Leu Arg Pro Pro Asp Phe
Ala Leu 130 135 140
Pro Ile Glu Glu Leu Lys Ser Thr Ile Ser Lys Asn Thr Arg Ala Ile145
150 155 160 Leu Leu Asn Thr Pro
His Asn Pro Thr Gly Lys Met Phe Thr Pro Glu 165
170 175 Glu Leu Asn Thr Ile Ala Ser Leu Cys Ile
Glu Asn Asp Val Leu Val 180 185
190 Phe Ser Asp Glu Val Tyr Asp Lys Leu Ala Phe Asp Met Glu His
Ile 195 200 205 Ser
Ile Ala Ser Leu Pro Gly Met Phe Glu Arg Thr Val Thr Met Asn 210
215 220 Ser Leu Gly Lys Thr Phe
Ser Leu Thr Gly Trp Lys Ile Gly Trp Ala225 230
235 240 Ile Ala Pro Pro His Leu Thr Trp Gly Val Arg
Gln Ala His Ala Phe 245 250
255 Leu Thr Phe Ala Thr Ser Asn Pro Met Gln Trp Ala Ala Ala Val Ala
260 265 270 Leu Arg Ala
Pro Asp Ser Tyr Tyr Thr Glu Leu Lys Arg Asp Tyr Met 275
280 285 Ala Lys Arg Ser Ile Leu Val Glu
Gly Leu Lys Ala Val Gly Phe Lys 290 295
300 Val Phe Pro Ser Ser Gly Thr Tyr Phe Val Val Val Asp
His Thr Pro305 310 315
320 Phe Gly His Glu Asn Asp Ile Ala Phe Cys Glu Tyr Leu Val Lys Glu
325 330 335 Val Gly Val Val
Ala Ile Pro Thr Ser Val Phe Tyr Leu Asn Pro Glu 340
345 350 Glu Gly Lys Asn Leu Val Arg Phe Thr
Phe Cys Lys Asp Glu Gly Thr 355 360
365 Leu Arg Ala Ala Val Asp Arg Met Lys Glu Lys Leu Arg Lys
370 375 380
36383PRTPhyllostachys bambusoidesbamboo glutamine phenylpyruvate
transaminase (GPT) (mature protein, no targeting sequence) 36Val
Ala Lys Arg Leu Glu Lys Phe Lys Thr Thr Ile Phe Thr Gln Met1
5 10 15 Ser Met Leu Ala Ile Lys
His Gly Ala Ile Asn Leu Gly Gln Gly Phe 20 25
30 Pro Asn Phe Asp Gly Pro Asp Phe Val Lys Glu
Ala Ala Ile Gln Ala 35 40 45
Ile Asn Ala Gly Lys Asn Gln Tyr Ala Arg Gly Tyr Gly Val Pro Glu
50 55 60 Leu Asn Ser
Ala Val Ala Glu Arg Phe Leu Lys Asp Ser Gly Leu Gln65 70
75 80 Val Asp Pro Glu Lys Glu Val Thr
Val Thr Ser Gly Cys Thr Glu Ala 85 90
95 Ile Ala Ala Thr Ile Leu Gly Leu Ile Asn Pro Gly Asp
Glu Val Ile 100 105 110
Leu Phe Ala Pro Phe Tyr Asp Ser Tyr Glu Ala Thr Leu Ser Met Ala
115 120 125 Gly Ala Asn Val
Lys Ala Ile Thr Leu Arg Pro Pro Asp Phe Ala Val 130
135 140 Pro Leu Glu Glu Leu Lys Ala Thr
Val Ser Lys Asn Thr Arg Ala Ile145 150
155 160 Met Ile Asn Thr Pro His Asn Pro Thr Gly Lys Met
Phe Ser Arg Glu 165 170
175 Glu Leu Glu Phe Ile Ala Thr Leu Cys Lys Lys Asn Asp Val Leu Leu
180 185 190 Phe Ala Asp
Glu Val Tyr Asp Lys Leu Ala Phe Glu Ala Asp His Ile 195
200 205 Ser Met Ala Ser Ile Pro Gly Met
Tyr Glu Arg Thr Val Thr Met Asn 210 215
220 Ser Leu Gly Lys Thr Phe Ser Leu Thr Gly Trp Lys Ile
Gly Trp Ala225 230 235
240 Ile Ala Pro Pro His Leu Thr Trp Gly Val Arg Gln Ala His Ser Phe
245 250 255 Leu Thr Phe Ala
Thr Cys Thr Pro Met Gln Ser Ala Ala Ala Ala Ala 260
265 270 Leu Arg Ala Pro Asp Ser Tyr Tyr Gly
Glu Leu Lys Arg Asp Tyr Gly 275 280
285 Ala Lys Lys Ala Ile Leu Val Asp Gly Leu Lys Ala Ala Gly
Phe Ile 290 295 300
Val Tyr Pro Ser Ser Gly Thr Tyr Phe Val Met Val Asp His Thr Pro305
310 315 320 Phe Gly Phe Asp Asn
Asp Ile Glu Phe Cys Glu Tyr Leu Ile Arg Glu 325
330 335 Val Gly Val Val Ala Ile Pro Pro Ser Val
Phe Tyr Leu Asn Pro Glu 340 345
350 Asp Gly Lys Asn Leu Val Arg Phe Thr Phe Cys Lys Asp Asp Asp
Thr 355 360 365 Leu
Arg Ala Ala Val Glu Arg Met Lys Thr Lys Leu Arg Lys Lys 370
375 380 372992DNAArtificial
Sequencesynthetic Oryza sativa (rice) ribulose bisphosphate
carboxylase (RUBISCO) promoter construct with catl intron and part
of Gus plus protein from Cambia 1305.1 vector, expression cassette
37ctgcagcaaa gaaacgttat tagttggtgc ttttggtggt aggaatgtag ttttctgaca
60aagtcaatta ctgaatataa aaaaaatctg cacagctctg cgtcaacagt tgtccaaggg
120atgcctcaaa aatctgtgca gattatcagt cgtcacgcag aagcagaaca tcatggtgtg
180ctaggtcagc ttcttgcatt gggccatgaa tccggttggt tgttaatctc tcctctctta
240ttctcttata ttaagatgca taactctttt atgtagtcta aaaaaaaatc cagtggatcg
300gatagtagta cgtcatggtg ccattaggta ccgttgaacc taacagatat ttatgcatgt
360gtatatatat agctatatag acaaaattga tgccgattat agacccaaaa gcaataggta
420tatataatat aatacagacc acaccaccaa actaagaatc gatcaaatag acaaggcatg
480tctccaaatt gtcttaaact atttccgtag gttcagccgt tcaggagtcg aatcagcctc
540tgccggcgtt ttctttgcac gtacgacgga cacacatggg cataccatat agctggtcca
600tgacattagg agagagaacg tacgtgttga cctgtagctg agatataaca aggttgatta
660taatatcacc aaacatgaaa tcatccaagg atgacccata actatcacta ctatagtact
720gcatctggta aaagaaattg tatagactct atttcgagca ctaccacata acgcctgcaa
780tgtgacaccc tacctattca ctaatgtgcc tcttcccaca cgctttccac ccgtactgct
840cacagcttta agaaccagaa caaatgagta atattagtgt cggttcatgg ctaaaaccag
900cactgatgta catgaccaca tatgtcaaat gctgcttcta ggcatgaccc gctcttacta
960atacctactc atcgctagaa gaattttcgg ctgataaatt ttcaatttaa gcaagagtta
1020tctgcgttgg ttcataactc aaactgatgg ccccaaccat attagtgcaa atttcacata
1080tgatcataac cttttcatat gaaatcggat cgagatgaac tttatataaa cattgtagct
1140gtcgatgata cctacaattt tatagttcac aaccttttta tttcaagtca tttaaatgcc
1200caaataggtg tttcaaatct cagatagaaa tgttcaaaag taaaaaaggt ccctatcata
1260acataattga tatgtaagtg agttggaaaa agataagtac gtgtgagaga gatcggggat
1320caaattctgg tgtaataatg tatgtatttc agtcataaaa attggtagca gtagttgggg
1380ctctgtatat ataccggtaa ggatgggatg gtagtagaat aattcttttt ttgtttttag
1440ttttttctgg tccaaaattt caaatttgga tcccttactt gtaccaacta atattaatga
1500gtgttgaggg tagtagaggt gcaactttac cataatccct ctgtttcagg ttataagacg
1560ttttgacttt aaatttgacc aagtttatgc gcaaatatag taatatttat aatactatat
1620tagtttcatt aaataaataa ttgaatatat tttcataata aatttgtgtt gagttcaaaa
1680tattattaat tttttctaca aacttggtca aacttgaagc agtttgactt tgaccaaagt
1740caaaacgtct tataacttga aacggatgga ttactttttt tgtggggaca agtttacaat
1800gtttaataaa gcacaatcca tcttaatgtt ttcaagctga atattgtaaa attcatggat
1860aaaccagctt ctaaatgttt aaccgggaaa atgtcgaacg acaaattaat atttttaagt
1920gatggggagt attaattaag gagtgacaac tcaactttca atatcgtact aaactgtggg
1980atttattttc taaaatttta taccctgcca attcacgtgt tgtagatctt tttttttcac
2040taaccgacac caggtatatc aattttattg aatatagcag caaaaagaat gtgttgtact
2100tgtaaacaaa aagcaaactg tacataaaaa aaaatgcact cctatataat taagctcata
2160aagatgcttt gcttcgtgag ggcccaagtt ttgatgacct tttgcttgat ctcgaaatta
2220aaatttaagt actgttaagg gagttcacac caccatcaat tttcagcctg aagaaacagt
2280taaacaacga ccccgatgac cagtctactg ctctccacat actagctgca ttattgatca
2340caaaacaaaa caaaacgaaa taaaaatcag cagcgagagt gtgcagagag agacaaaggt
2400gatctggcgt ggatatctcc ccatccatcc tcacccgcgc tgcccatcac tcgccgccgc
2460atactccatc atgtggagag aggaagacga ggaccacagc cagagcccgg gtcgagatgc
2520caccacggcc acaacccacg agcccggcgc gacaccaccg cgcgcgcgtg agccagccac
2580aaacgcccgc ggataggcgc gcgcacgccg gccaatccta ccacatcccc ggcctccgcg
2640gctcgcgagc gccgctgcca tccgatccgc tgagttttgg ctatttatac gtaccgcggg
2700agcctgtgtg cagagcagtg catctcaaga agtactcgag caaagaagga gagagcttgg
2760tgagctgcag ccatggtaga tctgagggta aatttctagt ttttctcctt cattttcttg
2820gttaggaccc ttttctcttt ttattttttt gagctttgat ctttctttaa actgatctat
2880tttttaattg attggttatg gtgtaaatat tacatagctt taactgataa tctgattact
2940ttatttcgtg tgtctatgat gatgatgata gttacagaac cgacgaacta gt
2992381281DNAHordeum vulgarebarley glutamine synthetase (GS1)
38gcgcaggcgg ttgtgcaggc gatgcagtgc caggtggggg tgaggggcag gacggccgtc
60ccggcgaggc agcccgcggg cagggtgtgg ggcgtcagga gggccgcccg cgccacctcc
120gggttcaagg tgctggcgct cggcccggag accaccgggg tcatccagag gatgcagcag
180ctgctcgaca tggacaccac gcccttcacc gacaagatca tcgccgagta catctgggtt
240ggaggatctg gaattgacct cagaagcaaa tcaaggacga tttcgaagcc agtggaggac
300ccgtcagagc tgccgaaatg gaactacgac ggatcgagca cggggcaggc tcctggggaa
360gacagtgaag tcatcctata cccacaggcc atattcaagg acccattccg aggaggcaac
420aacatactgg ttatctgtga cacctacaca ccacaggggg aacccatccc tactaacaaa
480cgccacatgg ctgcacaaat cttcagtgac cccaaggtca cttcacaagt gccatggttc
540ggaatcgaac aggagtacac tctgatgcag agggatgtga actggcctct tggctggcct
600gttggagggt accctggccc ccagggtcca tactactgcg ccgtaggatc agacaagtca
660tttggccgtg acatatcaga tgctcactac aaggcgtgcc tttacgctgg aattgaaatc
720agtggaacaa acggggaggt catgcctggt cagtgggagt accaggttgg acccagcgtt
780ggtattgatg caggagacca catatgggct tccagataca ttctcgagag aatcacggag
840caagctggtg tggtgctcac ccttgaccca aaaccaatcc agggtgactg gaacggagct
900ggctgccaca caaactacag cacattgagc atgcgcgagg atggaggttt cgacgtgatc
960aagaaggcaa tcctgaacct ttcacttcgc catgacttgc acatagccgc atatggtgaa
1020ggaaacgagc ggaggttgac agggctacac gagacagcta gcatatcaga cttctcatgg
1080ggtgtggcga accgtggctg ctctattcgt gtggggcgag acaccgaggc gaagggcaaa
1140ggatacctgg aggaccgtcg cccggcctcc aacatggacc cgtacaccgt gacggcgctg
1200ctggccgaga ccacgatcct gtgggagccg accctcgagg cggaggccct cgctgccaag
1260aagctggcgc tgaaggtatg a
128139426PRTHordeum vulgarebarley glutamine synthetase (GS1) 39Ala Gln
Ala Val Val Gln Ala Met Gln Cys Gln Val Gly Val Arg Gly1 5
10 15 Arg Thr Ala Val Pro Ala Arg
Gln Pro Ala Gly Arg Val Trp Gly Val 20 25
30 Arg Arg Ala Ala Arg Ala Thr Ser Gly Phe Lys Val
Leu Ala Leu Gly 35 40 45
Pro Glu Thr Thr Gly Val Ile Gln Arg Met Gln Gln Leu Leu Asp Met
50 55 60 Asp Thr Thr
Pro Phe Thr Asp Lys Ile Ile Ala Glu Tyr Ile Trp Val65 70
75 80 Gly Gly Ser Gly Ile Asp Leu Arg
Ser Lys Ser Arg Thr Ile Ser Lys 85 90
95 Pro Val Glu Asp Pro Ser Glu Leu Pro Lys Trp Asn Tyr
Asp Gly Ser 100 105 110
Ser Thr Gly Gln Ala Pro Gly Glu Asp Ser Glu Val Ile Leu Tyr Pro
115 120 125 Gln Ala Ile Phe
Lys Asp Pro Phe Arg Gly Gly Asn Asn Ile Leu Val 130
135 140 Ile Cys Asp Thr Tyr Thr Pro Gln
Gly Glu Pro Ile Pro Thr Asn Lys145 150
155 160 Arg His Met Ala Ala Gln Ile Phe Ser Asp Pro Lys
Val Thr Ser Gln 165 170
175 Val Pro Trp Phe Gly Ile Glu Gln Glu Tyr Thr Leu Met Gln Arg Asp
180 185 190 Val Asn Trp
Pro Leu Gly Trp Pro Val Gly Gly Tyr Pro Gly Pro Gln 195
200 205 Gly Pro Tyr Tyr Cys Ala Val Gly
Ser Asp Lys Ser Phe Gly Arg Asp 210 215
220 Ile Ser Asp Ala His Tyr Lys Ala Cys Leu Tyr Ala Gly
Ile Glu Ile225 230 235
240 Ser Gly Thr Asn Gly Glu Val Met Pro Gly Gln Trp Glu Tyr Gln Val
245 250 255 Gly Pro Ser Val
Gly Ile Asp Ala Gly Asp His Ile Trp Ala Ser Arg 260
265 270 Tyr Ile Leu Glu Arg Ile Thr Glu Gln
Ala Gly Val Val Leu Thr Leu 275 280
285 Asp Pro Lys Pro Ile Gln Gly Asp Trp Asn Gly Ala Gly Cys
His Thr 290 295 300
Asn Tyr Ser Thr Leu Ser Met Arg Glu Asp Gly Gly Phe Asp Val Ile305
310 315 320 Lys Lys Ala Ile Leu
Asn Leu Ser Leu Arg His Asp Leu His Ile Ala 325
330 335 Ala Tyr Gly Glu Gly Asn Glu Arg Arg Leu
Thr Gly Leu His Glu Thr 340 345
350 Ala Ser Ile Ser Asp Phe Ser Trp Gly Val Ala Asn Arg Gly Cys
Ser 355 360 365 Ile
Arg Val Gly Arg Asp Thr Glu Ala Lys Gly Lys Gly Tyr Leu Glu 370
375 380 Asp Arg Arg Pro Ala Ser
Asn Met Asp Pro Tyr Thr Val Thr Ala Leu385 390
395 400 Leu Ala Glu Thr Thr Ile Leu Trp Glu Pro Thr
Leu Glu Ala Glu Ala 405 410
415 Leu Ala Ala Lys Lys Leu Ala Leu Lys Val 420
425 404273DNAArtificial Sequencesynthetic Hordeum vulgare
(barley) glutamine synthetase (GS1) expression cassette with Oryza
sativa (rice) ribulose bisphosphate carboxylase (RUBISCO) promoter,
catl intron and part of Gus plus protein 40ctgcagcaaa gaaacgttat
tagttggtgc ttttggtggt aggaatgtag ttttctgaca 60aagtcaatta ctgaatataa
aaaaaatctg cacagctctg cgtcaacagt tgtccaaggg 120atgcctcaaa aatctgtgca
gattatcagt cgtcacgcag aagcagaaca tcatggtgtg 180ctaggtcagc ttcttgcatt
gggccatgaa tccggttggt tgttaatctc tcctctctta 240ttctcttata ttaagatgca
taactctttt atgtagtcta aaaaaaaatc cagtggatcg 300gatagtagta cgtcatggtg
ccattaggta ccgttgaacc taacagatat ttatgcatgt 360gtatatatat agctatatag
acaaaattga tgccgattat agacccaaaa gcaataggta 420tatataatat aatacagacc
acaccaccaa actaagaatc gatcaaatag acaaggcatg 480tctccaaatt gtcttaaact
atttccgtag gttcagccgt tcaggagtcg aatcagcctc 540tgccggcgtt ttctttgcac
gtacgacgga cacacatggg cataccatat agctggtcca 600tgacattagg agagagaacg
tacgtgttga cctgtagctg agatataaca aggttgatta 660taatatcacc aaacatgaaa
tcatccaagg atgacccata actatcacta ctatagtact 720gcatctggta aaagaaattg
tatagactct atttcgagca ctaccacata acgcctgcaa 780tgtgacaccc tacctattca
ctaatgtgcc tcttcccaca cgctttccac ccgtactgct 840cacagcttta agaaccagaa
caaatgagta atattagtgt cggttcatgg ctaaaaccag 900cactgatgta catgaccaca
tatgtcaaat gctgcttcta ggcatgaccc gctcttacta 960atacctactc atcgctagaa
gaattttcgg ctgataaatt ttcaatttaa gcaagagtta 1020tctgcgttgg ttcataactc
aaactgatgg ccccaaccat attagtgcaa atttcacata 1080tgatcataac cttttcatat
gaaatcggat cgagatgaac tttatataaa cattgtagct 1140gtcgatgata cctacaattt
tatagttcac aaccttttta tttcaagtca tttaaatgcc 1200caaataggtg tttcaaatct
cagatagaaa tgttcaaaag taaaaaaggt ccctatcata 1260acataattga tatgtaagtg
agttggaaaa agataagtac gtgtgagaga gatcggggat 1320caaattctgg tgtaataatg
tatgtatttc agtcataaaa attggtagca gtagttgggg 1380ctctgtatat ataccggtaa
ggatgggatg gtagtagaat aattcttttt ttgtttttag 1440ttttttctgg tccaaaattt
caaatttgga tcccttactt gtaccaacta atattaatga 1500gtgttgaggg tagtagaggt
gcaactttac cataatccct ctgtttcagg ttataagacg 1560ttttgacttt aaatttgacc
aagtttatgc gcaaatatag taatatttat aatactatat 1620tagtttcatt aaataaataa
ttgaatatat tttcataata aatttgtgtt gagttcaaaa 1680tattattaat tttttctaca
aacttggtca aacttgaagc agtttgactt tgaccaaagt 1740caaaacgtct tataacttga
aacggatgga ttactttttt tgtggggaca agtttacaat 1800gtttaataaa gcacaatcca
tcttaatgtt ttcaagctga atattgtaaa attcatggat 1860aaaccagctt ctaaatgttt
aaccgggaaa atgtcgaacg acaaattaat atttttaagt 1920gatggggagt attaattaag
gagtgacaac tcaactttca atatcgtact aaactgtggg 1980atttattttc taaaatttta
taccctgcca attcacgtgt tgtagatctt tttttttcac 2040taaccgacac caggtatatc
aattttattg aatatagcag caaaaagaat gtgttgtact 2100tgtaaacaaa aagcaaactg
tacataaaaa aaaatgcact cctatataat taagctcata 2160aagatgcttt gcttcgtgag
ggcccaagtt ttgatgacct tttgcttgat ctcgaaatta 2220aaatttaagt actgttaagg
gagttcacac caccatcaat tttcagcctg aagaaacagt 2280taaacaacga ccccgatgac
cagtctactg ctctccacat actagctgca ttattgatca 2340caaaacaaaa caaaacgaaa
taaaaatcag cagcgagagt gtgcagagag agacaaaggt 2400gatctggcgt ggatatctcc
ccatccatcc tcacccgcgc tgcccatcac tcgccgccgc 2460atactccatc atgtggagag
aggaagacga ggaccacagc cagagcccgg gtcgagatgc 2520caccacggcc acaacccacg
agcccggcgc gacaccaccg cgcgcgcgtg agccagccac 2580aaacgcccgc ggataggcgc
gcgcacgccg gccaatccta ccacatcccc ggcctccgcg 2640gctcgcgagc gccgctgcca
tccgatccgc tgagttttgg ctatttatac gtaccgcggg 2700agcctgtgtg cagagcagtg
catctcaaga agtactcgag caaagaagga gagagcttgg 2760tgagctgcag ccatggtaga
tctgagggta aatttctagt ttttctcctt cattttcttg 2820gttaggaccc ttttctcttt
ttattttttt gagctttgat ctttctttaa actgatctat 2880tttttaattg attggttatg
gtgtaaatat tacatagctt taactgataa tctgattact 2940ttatttcgtg tgtctatgat
gatgatgata gttacagaac cgacgaacta gtgcgcaggc 3000ggttgtgcag gcgatgcagt
gccaggtggg ggtgaggggc aggacggccg tcccggcgag 3060gcagcccgcg ggcagggtgt
ggggcgtcag gagggccgcc cgcgccacct ccgggttcaa 3120ggtgctggcg ctcggcccgg
agaccaccgg ggtcatccag aggatgcagc agctgctcga 3180catggacacc acgcccttca
ccgacaagat catcgccgag tacatctggg ttggaggatc 3240tggaattgac ctcagaagca
aatcaaggac gatttcgaag ccagtggagg acccgtcaga 3300gctgccgaaa tggaactacg
acggatcgag cacggggcag gctcctgggg aagacagtga 3360agtcatccta tacccacagg
ccatattcaa ggacccattc cgaggaggca acaacatact 3420ggttatctgt gacacctaca
caccacaggg ggaacccatc cctactaaca aacgccacat 3480ggctgcacaa atcttcagtg
accccaaggt cacttcacaa gtgccatggt tcggaatcga 3540acaggagtac actctgatgc
agagggatgt gaactggcct cttggctggc ctgttggagg 3600gtaccctggc ccccagggtc
catactactg cgccgtagga tcagacaagt catttggccg 3660tgacatatca gatgctcact
acaaggcgtg cctttacgct ggaattgaaa tcagtggaac 3720aaacggggag gtcatgcctg
gtcagtggga gtaccaggtt ggacccagcg ttggtattga 3780tgcaggagac cacatatggg
cttccagata cattctcgag agaatcacgg agcaagctgg 3840tgtggtgctc acccttgacc
caaaaccaat ccagggtgac tggaacggag ctggctgcca 3900cacaaactac agcacattga
gcatgcgcga ggatggaggt ttcgacgtga tcaagaaggc 3960aatcctgaac ctttcacttc
gccatgactt gcacatagcc gcatatggtg aaggaaacga 4020gcggaggttg acagggctac
acgagacagc tagcatatca gacttctcat ggggtgtggc 4080gaaccgtggc tgctctattc
gtgtggggcg agacaccgag gcgaagggca aaggatacct 4140ggaggaccgt cgcccggcct
ccaacatgga cccgtacacc gtgacggcgc tgctggccga 4200gaccacgatc ctgtgggagc
cgaccctcga ggcggaggcc ctcgctgcca agaagctggc 4260gctgaaggta tga
427341436PRTArtificial
Sequencesynthetic Hordeum vulgare (barley) glutamine synthetase
(GS1) expression cassette translation product 41Met Val Asp Leu Arg Asn
Arg Arg Thr Ser Ala Gln Ala Val Val Gln1 5
10 15 Ala Met Gln Cys Gln Val Gly Val Arg Gly Arg
Thr Ala Val Pro Ala 20 25 30
Arg Gln Pro Ala Gly Arg Val Trp Gly Val Arg Arg Ala Ala Arg Ala
35 40 45 Thr Ser Gly
Phe Lys Val Leu Ala Leu Gly Pro Glu Thr Thr Gly Val 50
55 60 Ile Gln Arg Met Gln Gln Leu Leu
Asp Met Asp Thr Thr Pro Phe Thr65 70 75
80 Asp Lys Ile Ile Ala Glu Tyr Ile Trp Val Gly Gly Ser
Gly Ile Asp 85 90 95
Leu Arg Ser Lys Ser Arg Thr Ile Ser Lys Pro Val Glu Asp Pro Ser
100 105 110 Glu Leu Pro Lys Trp
Asn Tyr Asp Gly Ser Ser Thr Gly Gln Ala Pro 115
120 125 Gly Glu Asp Ser Glu Val Ile Leu Tyr
Pro Gln Ala Ile Phe Lys Asp 130 135
140 Pro Phe Arg Gly Gly Asn Asn Ile Leu Val Ile Cys Asp
Thr Tyr Thr145 150 155
160 Pro Gln Gly Glu Pro Ile Pro Thr Asn Lys Arg His Met Ala Ala Gln
165 170 175 Ile Phe Ser Asp
Pro Lys Val Thr Ser Gln Val Pro Trp Phe Gly Ile 180
185 190 Glu Gln Glu Tyr Thr Leu Met Gln Arg
Asp Val Asn Trp Pro Leu Gly 195 200
205 Trp Pro Val Gly Gly Tyr Pro Gly Pro Gln Gly Pro Tyr Tyr
Cys Ala 210 215 220
Val Gly Ser Asp Lys Ser Phe Gly Arg Asp Ile Ser Asp Ala His Tyr225
230 235 240 Lys Ala Cys Leu Tyr
Ala Gly Ile Glu Ile Ser Gly Thr Asn Gly Glu 245
250 255 Val Met Pro Gly Gln Trp Glu Tyr Gln Val
Gly Pro Ser Val Gly Ile 260 265
270 Asp Ala Gly Asp His Ile Trp Ala Ser Arg Tyr Ile Leu Glu Arg
Ile 275 280 285 Thr
Glu Gln Ala Gly Val Val Leu Thr Leu Asp Pro Lys Pro Ile Gln 290
295 300 Gly Asp Trp Asn Gly Ala
Gly Cys His Thr Asn Tyr Ser Thr Leu Ser305 310
315 320 Met Arg Glu Asp Gly Gly Phe Asp Val Ile Lys
Lys Ala Ile Leu Asn 325 330
335 Leu Ser Leu Arg His Asp Leu His Ile Ala Ala Tyr Gly Glu Gly Asn
340 345 350 Glu Arg Arg
Leu Thr Gly Leu His Glu Thr Ala Ser Ile Ser Asp Phe 355
360 365 Ser Trp Gly Val Ala Asn Arg Gly
Cys Ser Ile Arg Val Gly Arg Asp 370 375
380 Thr Glu Ala Lys Gly Lys Gly Tyr Leu Glu Asp Arg Arg
Pro Ala Ser385 390 395
400 Asn Met Asp Pro Tyr Thr Val Thr Ala Leu Leu Ala Glu Thr Thr Ile
405 410 415 Leu Trp Glu Pro
Thr Leu Glu Ala Glu Ala Leu Ala Ala Lys Lys Leu 420
425 430 Ala Leu Lys Val 435
421992DNAZea maysmaize ubil promoter with 5'UTR intron 42ctgcagtgca
gcgtgacccg gtcgtgcccc tctctagaga taatgagcat tgcatgtcta 60agttataaaa
aattaccaca tatttttttt gtcacacttg tttgaagtgc agtttatcta 120tctttataca
tatatttaaa ctttactcta cgaataatat aatctatagt actacaataa 180tatcagtgtt
ttagagaatc atataaatga acagttagac atggtctaaa ggacaattga 240gtattttgac
aacaggactc tacagtttta tctttttagt gtgcatgtgt tctccttttt 300ttttgcaaat
agcttcacct atataatact tcatccattt tattagtaca tccatttagg 360gtttagggtt
aatggttttt atagactaat ttttttagta catctatttt attctatttt 420agcctctaaa
ttaagaaaac taaaactcta ttttagtttt tttatttaat aatttagata 480taaaatagaa
taaaataaag tgactaaaaa ttaaacaaat accctttaag aaattaaaaa 540aactaaggaa
acatttttct tgtttcgagt agataatgcc agcctgttaa acgccgtcga 600cgagtctaac
ggacaccaac cagcgaacca gcagcgtcgc gtcgggccaa gcgaagcaga 660cggcacggca
tctctgtcgc tgcctctgga cccctctcga gagttccgct ccaccgttgg 720acttgctccg
ctgtcggcat ccagaaattg cgtggcggag cggcagacgt gagccggcac 780ggcaggcggc
ctcctcctcc tctcacggca cggcagctac gggggattcc tttcccaccg 840ctccttcgct
ttcccttcct cgcccgccgt aataaataga caccccctcc acaccctctt 900tccccaacct
cgtgttgttc ggagcgcaca cacacacaac cagatctccc ccaaatccac 960ccgtcggcac
ctccgcttca aggtacgccg ctcgtcctcc cccccccccc ctctctacct 1020tctctagatc
ggcgttccgg tccatggtta gggcccggta gttctacttc tgttcatgtt 1080tgtgttagat
ccgtgtttgt gttagatccg tgctgctagc gttcgtacac ggatgcgacc 1140tgtacgtcag
acacgttctg attgctaact tgccagtgtt tctctttggg gaatcctggg 1200atggctctag
ccgttccgca gacgggatcg atttcatgat tttttttgtt tcgttgcata 1260gggtttggtt
tgcccttttc ctttatttca atatatgccg tgcacttgtt tgtcgggtca 1320tcttttcatg
cttttttttg tcttggttgt gatgatgtgg tctggttggg cggtcgttct 1380agatcggagt
agaattctgt ttcaaactac ctggtggatt tattaatttt ggatctgtat 1440gtgtgtgcca
tacatattca tagttacgaa ttgaagatga tggatggaaa tatcgatcta 1500ggataggtat
acatgttgat gcgggtttta ctgatgcata tacagagatg ctttttgttc 1560gcttggttgt
gatgatgtgg tgtggttggg cggtcgttca ttcgttctag atcggagtag 1620aatactgttt
caaactacct ggtgtattta ttaattttgg aactgtatgt gtgtgtcata 1680catcttcata
gttacgagtt taagatggat ggaaatatcg atctaggata ggtatacatg 1740ttgatgtggg
ttttactgat gcatatacat gatggcatat gcagcatcta ttcatatgct 1800ctaaccttga
gtacctatct attataataa acaagtatgt tttataatta ttttgatctt 1860gatatacttg
gatgatggca tatgcagcag ctatatgtgg atttttttag ccctgccttc 1920atacgctatt
tatttgcttg gtactgtttc ttttgtcgat gctcaccctg ttgtttggtg 1980ttacttctgc
ag
1992431248DNAHordeum vulgarebarley glutamine phenylpyruvate transaminase
(GPT) including targeting sequence coding domain 43atggcatccg
cccccgcctc cgcctccgcg gccctctcca ccgccgcccc cgccgacaac 60ggggccgcca
agcccacgga gcagcggccg gtacaggtgg ctaagcgatt ggagaagttc 120aaaacaacaa
ttttcacaca gatgagcatg ctcgcagtga agcatggagc aataaacctt 180ggacaggggt
ttcccaattt tgatggccct gactttgtca aagatgctgc tattgaggct 240atcaaagctg
gaaagaatca gtatgcaaga ggatatggtg tgcctgaatt gaactcagct 300gttgctgaga
gatttctcaa ggacagtgga ttgcacatcg atcctgataa ggaagttact 360gttacatctg
ggtgcacaga agcaatagct gcaacgatat tgggtctgat caaccctggg 420gatgaagtca
tactgtttgc tccattctat gattcttatg aggctacact gtccatggct 480ggtgcgaatg
tcaaagccat tacactccgc cctccggact ttgcagtccc tcttgaagag 540ctaaaggctg
cagtctcgaa gaataccaga gcaataatga ttaatacacc tcacaaccct 600accgggaaaa
tgttcacaag ggaggaactt gagttcattg ctgatctctg caaggaaaat 660gacgtgttgc
tctttgccga tgaggtctac gacaagctgg cgtttgaggc ggatcacata 720tcaatggctt
ctattcctgg catgtatgag aggaccgtca ctatgaactc cctggggaag 780acgttctcct
tgaccggatg gaagatcggc tgggcgatag caccaccgca cctgacatgg 840ggcgtaaggc
aggcacactc cttcctcaca ttcgccacct ccacgccgat gcaatcagca 900gcggcggcgg
ccctgagagc accggacagc tactttgagg agctgaagag ggactacggc 960gcaaagaaag
cgctgctggt ggacgggctc aaggcggcgg gcttcatcgt ctacccttcg 1020agcggaacct
acttcatcat ggtcgaccac accccgttcg ggttcgacaa cgacgtcgag 1080ttctgcgagt
acttgatccg cgaggtcggc gtcgtggcca tcccgccaag cgtgttctac 1140ctgaacccgg
aggacgggaa gaacctggtg aggttcacct tctgcaagga cgacgacacg 1200ctaagggcgg
cggtggacag gatgaaggcc aagctcagga agaaatga
124844415PRTHordeum vulgarebarley glutamine phenylpyruvate transaminase
(GPT) including targeting sequence 44Met Ala Ser Ala Pro Ala Ser Ala
Ser Ala Ala Leu Ser Thr Ala Ala1 5 10
15 Pro Ala Asp Asn Gly Ala Ala Lys Pro Thr Glu Gln Arg
Pro Val Gln 20 25 30
Val Ala Lys Arg Leu Glu Lys Phe Lys Thr Thr Ile Phe Thr Gln Met
35 40 45 Ser Met Leu Ala
Val Lys His Gly Ala Ile Asn Leu Gly Gln Gly Phe 50 55
60 Pro Asn Phe Asp Gly Pro Asp Phe Val
Lys Asp Ala Ala Ile Glu Ala65 70 75
80 Ile Lys Ala Gly Lys Asn Gln Tyr Ala Arg Gly Tyr Gly Val
Pro Glu 85 90 95
Leu Asn Ser Ala Val Ala Glu Arg Phe Leu Lys Asp Ser Gly Leu His
100 105 110 Ile Asp Pro Asp Lys
Glu Val Thr Val Thr Ser Gly Cys Thr Glu Ala 115
120 125 Ile Ala Ala Thr Ile Leu Gly Leu Ile
Asn Pro Gly Asp Glu Val Ile 130 135
140 Leu Phe Ala Pro Phe Tyr Asp Ser Tyr Glu Ala Thr Leu
Ser Met Ala145 150 155
160 Gly Ala Asn Val Lys Ala Ile Thr Leu Arg Pro Pro Asp Phe Ala Val
165 170 175 Pro Leu Glu Glu
Leu Lys Ala Ala Val Ser Lys Asn Thr Arg Ala Ile 180
185 190 Met Ile Asn Thr Pro His Asn Pro Thr
Gly Lys Met Phe Thr Arg Glu 195 200
205 Glu Leu Glu Phe Ile Ala Asp Leu Cys Lys Glu Asn Asp Val
Leu Leu 210 215 220
Phe Ala Asp Glu Val Tyr Asp Lys Leu Ala Phe Glu Ala Asp His Ile225
230 235 240 Ser Met Ala Ser Ile
Pro Gly Met Tyr Glu Arg Thr Val Thr Met Asn 245
250 255 Ser Leu Gly Lys Thr Phe Ser Leu Thr Gly
Trp Lys Ile Gly Trp Ala 260 265
270 Ile Ala Pro Pro His Leu Thr Trp Gly Val Arg Gln Ala His Ser
Phe 275 280 285 Leu
Thr Phe Ala Thr Ser Thr Pro Met Gln Ser Ala Ala Ala Ala Ala 290
295 300 Leu Arg Ala Pro Asp Ser
Tyr Phe Glu Glu Leu Lys Arg Asp Tyr Gly305 310
315 320 Ala Lys Lys Ala Leu Leu Val Asp Gly Leu Lys
Ala Ala Gly Phe Ile 325 330
335 Val Tyr Pro Ser Ser Gly Thr Tyr Phe Ile Met Val Asp His Thr Pro
340 345 350 Phe Gly Phe
Asp Asn Asp Val Glu Phe Cys Glu Tyr Leu Ile Arg Glu 355
360 365 Val Gly Val Val Ala Ile Pro Pro
Ser Val Phe Tyr Leu Asn Pro Glu 370 375
380 Asp Gly Lys Asn Leu Val Arg Phe Thr Phe Cys Lys Asp
Asp Asp Thr385 390 395
400 Leu Arg Ala Ala Val Asp Arg Met Lys Ala Lys Leu Arg Lys Lys
405 410 415 452735DNAArtificial
Sequencesynthetic tomato ribulose bisphosphate carboxylase
(RUBISCO) small subunit (rbcS3C) promoter + Arabidopsis glutamine
synthetase (GS1), expression cassette 45gtttgaatcc tccttaaagt ttttctctgg
agaaactgta gtaattttac tttgttgtgt 60tcccttcatc ttttgaatta atggcatttg
ttttaatact aatctgcttc tgaaacttgt 120aatgtatgta tatcagtttc ttataattta
tccaagtaat atcttccatt ctctatgcaa 180ttgcctgcat aagctcgaca aaagagtaca
tcaacccctc ctcctctgga ctactctagc 240taaacttgaa tttcccctta agattatgaa
attgatatat ccttaacaaa cgactccttc 300tgttggaaaa tgtagtactt gtctttcttc
ttttgggtat atatagttta tatacaccat 360actatgtaca acatccaagt agagtgaaat
ggatacatgt acaagactta tttgattgat 420tgatgacttg agttgcctta ggagtaacaa
attcttaggt caataaatcg ttgatttgaa 480attaatctct ctgtcttaga cagataggaa
ttatgacttc caatggtcca gaaagcaaag 540ttcgcactga gggtatactt ggaattgaga
cttgcacagg tccagaaacc aaagttccca 600tcgagctcta aaatcacatc tttggaatga
aattcaatta gagataagtt gcttcatagc 660ataggtaaaa tggaagatgt gaagtaacct
gcaataatca gtgaaatgac attaatacac 720taaatacttc atatgtaatt atcctttcca
ggttaacaat actctataaa gtaagaatta 780tcagaaatgg gctcatcaaa cttttgtact
atgtatttca tataaggaag tataactata 840cataagtgta tacacaactt tattcctatt
ttgtaaaggt ggagagactg ttttcgatgg 900atctaaagca atatgtctat aaaatgcatt
gatataataa ttatctgaga aaatccagaa 960ttggcgttgg attatttcag ccaaatagaa
gtttgtacca tacttgttga ttccttctaa 1020gttaaggtga agtatcattc ataaacagtt
ttccccaaag tactactcac caagtttccc 1080tttgtagaat taacagttca aatatatggc
gcagaaatta ctctatgccc aaaaccaaac 1140gagaaagaaa caaaatacag gggttgcaga
ctttattttc gtgttagggt gtgttttttc 1200atgtaattaa tcaaaaaata ttatgacaaa
aacatttata catattttta ctcaacactc 1260tgggtatcag ggtgggttgt gttcgacaat
caatatggaa aggaagtatt ttccttattt 1320ttttagttaa tattttcagt tataccaaac
ataccttgtg atattatttt taaaaatgaa 1380aaactcgtca gaaagaaaaa gcaaaagcaa
caaaaaaatt gcaagtattt tttaaaaaag 1440aaaaaaaaaa catatcttgt ttgtcagtat
gggaagtttg agataaggac gagtgagggg 1500ttaaaattca gtggccattg attttgtaat
gccaagaacc acaaaatcca atggttacca 1560ttcctgtaag atgaggtttg ctaactcttt
ttgtccgtta gataggaagc cttatcacta 1620tatatacaag gcgtcctaat aacctcttag
taaccaatta tttcagcacc atgtctctgc 1680tctcagatct cgttaacctc aacctcaccg
atgccaccgg gaaaatcatc gccgaataca 1740tatggatcgg tggatctgga atggatatca
gaagcaaagc caggacacta ccaggaccag 1800tgactgatcc atcaaagctt cccaagtgga
actacgacgg atccagcacc ggtcaggctg 1860ctggagaaga cagtgaagtc attctatacc
ctcaggcaat attcaaggat cccttcagga 1920aaggcaacaa catcctggtg atgtgtgatg
cttacacacc agctggtgat cctattccaa 1980ccaacaagag gcacaacgct gctaagatct
tcagccaccc cgacgttgcc aaggaggagc 2040cttggtatgg gattgagcaa gaatacactt
tgatgcaaaa ggatgtgaac tggccaattg 2100gttggcctgt tggtggctac cctggccctc
agggacctta ctactgtggt gtgggagctg 2160acaaagccat tggtcgtgac attgtggatg
ctcactacaa ggcctgtctt tacgccggta 2220ttggtatttc tggtatcaat ggagaagtca
tgccaggcca gtgggagttc caagtcggcc 2280ctgttgaggg tattagttct ggtgatcaag
tctgggttgc tcgatacctt ctcgagagga 2340tcactgagat ctctggtgta attgtcagct
tcgacccgaa accagtcccg ggtgactgga 2400atggagctgg agctcactgc aactacagca
ctaagacaat gagaaacgat ggaggattag 2460aagtgatcaa gaaagcgata gggaagcttc
agctgaaaca caaagaacac attgctgctt 2520acggtgaagg aaacgagcgt cgtctcactg
gaaagcacga aaccgcagac atcaacacat 2580tctcttgggg agtcgcgaac cgtggagcgt
cagtgagagt gggacgtgac acagagaagg 2640aaggtaaagg gtacttcgaa gacagaaggc
cagcttctaa catggatcct tacgttgtca 2700cctccatgat cgctgagacg accatactcg
gttga 2735463271DNAArtificial
Sequencesynthetic tomato ribulose bisphosphate carboxylase
(RUBISCO) small subunit (rbcS3C) promoter + Zea mays AAT (glutamine
phenylpyruvate transaminase (GPT)), expression cassette
46ggtaccgttt gaatcctcct taaagttttt ctctggagaa actgtagtaa ttttactttg
60ttgtgttccc ttcatctttt gaattaatgg catttgtttt aatactaatc tgcttctgaa
120acttgtaatg tatgtatatc agtttcttat aatttatcca agtaatatct tccattctct
180atgcaattgc ctgcataagc tcgacaaaag agtacatcaa cccctcctcc tctggactac
240tctagctaaa cttgaatttc cccttaagat tatgaaattg atatatcctt aacaaacgac
300tccttctgtt ggaaaatgta gtacttgtct ttcttctttt gggtatatat agtttatata
360caccatacta tgtacaacat ccaagtagag tgaaatggat acatgtacaa gacttatttg
420attgattgat gacttgagtt gccttaggag taacaaattc ttaggtcaat aaatcgttga
480tttgaaatta atctctctgt cttagacaga taggaattat gacttccaat ggtccagaaa
540gcaaagttcg cactgagggt atacttggaa ttgagacttg cacaggtcca gaaaccaaag
600ttcccatcga gctctaaaat cacatctttg gaatgaaatt caattagaga taagttgctt
660catagcatag gtaaaatgga agatgtgaag taacctgcaa taatcagtga aatgacatta
720atacactaaa tacttcatat gtaattatcc tttccaggtt aacaatactc tataaagtaa
780gaattatcag aaatgggctc atcaaacttt tgtactatgt atttcatata aggaagtata
840actatacata agtgtataca caactttatt cctattttgt aaaggtggag agactgtttt
900cgatggatct aaagcaatat gtctataaaa tgcattgata taataattat ctgagaaaat
960ccagaattgg cgttggatta tttcagccaa atagaagttt gtaccatact tgttgattcc
1020ttctaagtta aggtgaagta tcattcataa acagttttcc ccaaagtact actcaccaag
1080tttccctttg tagaattaac agttcaaata tatggcgcag aaattactct atgcccaaaa
1140ccaaacgaga aagaaacaaa atacaggggt tgcagacttt attttcgtgt tagggtgtgt
1200tttttcatgt aattaatcaa aaaatattat gacaaaaaca tttatacata tttttactca
1260acactctggg tatcagggtg ggttgtgttc gacaatcaat atggaaagga agtattttcc
1320ttattttttt agttaatatt ttcagttata ccaaacatac cttgtgatat tatttttaaa
1380aatgaaaaac tcgtcagaaa gaaaaagcaa aagcaacaaa aaaattgcaa gtatttttta
1440aaaaagaaaa aaaaaaacat atcttgtttg tcagtatggg aagtttgaga taaggacgag
1500tgaggggtta aaattcagtg gccattgatt ttgtaatgcc aagaaccaca aaatccaatg
1560gttaccattc ctgtaagatg aggtttgcta actctttttg tccgttagat aggaagcctt
1620atcactatat atacaaggcg tcctaataac ctcttagtaa ccaattattt cagcaccatg
1680gtagatctga gggtaaattt ctagtttttc tccttcattt tcttggttag gacccttttc
1740tctttttatt tttttgagct ttgatctttc tttaaactga tctatttttt aattgattgg
1800ttatggtgta aatattacat agctttaact gataatctga ttactttatt tcgtgtgtct
1860atgatgatga tgatagttac agaaccgacg aactagtatg aatctggccg ccttttcctc
1920cacccttgcc acgctcccct ggtatgagat gccatcaata aattcctccg caactttctc
1980gtcctcactg ctccgccgct cgctctgcgc gtcgctccgg acgatctccc acatggcctc
2040cgccgccgcc cccacctccg cgcccgtcgc caccaccgag aacggcgccg cgaaggcgat
2100agagcagcgg cccgtgcagg tcgcagagcg gctggaaaag ttcaagacaa caattttcac
2160tcagatgagc atgcttgcca tcaagcatgg agcaataaac cttggccagg gctttccgaa
2220ttttgatggc ccagactttg tgaaagaggc cgcaattcaa gctatcaatg ctgggaagaa
2280tcagtacgca agagggtttg gtgtgcctga actgaactcg gctatcgctg aaaggttcct
2340gaaggacagt ggattgcaag ttgaccctga caaggaagtc actgttacat ctggatgcac
2400tgaggcaata gctgcaacca tactaggtct gatcaatcct ggcgacgagg tgatactgtt
2460cgccccattc tacgattcat acgaggctac actgtcgatg gccggtgcca acgtgaaggc
2520cattaccctc cgcgctccag atttcgcggt cccgcttgag gagctggagg ctgcagtctc
2580caaggacacg aaagcgataa tgataaacac gccgcacaac ccaaccggga aaatgttcac
2640cagggaggag ctcgaatcca tcgccgccct ctgcaaggaa aacgacgttt tgctgttctc
2700agatgaggtc tatgacaagc tggtgtttga ggctgaccac atatccatgg cttctatccc
2760gggcatgtac gagaggacgg tgaccatgaa ctctctgggg aagacgttct ctcttacagg
2820atggaagatc gggtgggcaa tcgcgccgcc gcacctgaca tggggcctca ggcaggcgca
2880ctcgttcctg acgttcgcca cctgcacacc gatgcaggcg gcggccgcgg cggctctgag
2940ggcaccggac agctactacg acgagctgaa gagggactac agcgcgaaga aggctatcct
3000gctggaagga ctcgaagccg cagggttcat cgtctaccca tcgagtggga catactacat
3060catggtcgac cacaccccgt tcggtttcga cagcgacgta gagttctgcg agtacttgat
3120ccgcgaagtc ggcgtctgcg ctataccgcc cagcgtgttc tacctcgacc ccgaagaggg
3180aaagaaattg gtgaggttca ccttcagcaa ggacgaaggc acgctgcggg ccgcggtcga
3240gaggttgaag gcgaagctca ggaggaaatg a
327147467PRTArtificial Sequencesynthetic Zea mays AAT (glutamine
phenylpyruvate transaminase (GPT)) expression cassette translation
product with N-terminal Gus plus sequence 47Met Val Asp Leu Arg Asn Arg
Arg Thr Ser Met Asn Leu Ala Ala Phe1 5 10
15 Ser Ser Thr Leu Ala Thr Leu Pro Trp Tyr Glu Met
Pro Ser Ile Asn 20 25 30
Ser Ser Ala Thr Phe Ser Ser Ser Leu Leu Arg Arg Ser Leu Cys Ala
35 40 45 Ser Leu Arg Thr
Ile Ser His Met Ala Ser Ala Ala Ala Pro Thr Ser 50 55
60 Ala Pro Val Ala Thr Thr Glu Asn Gly
Ala Ala Lys Ala Ile Glu Gln65 70 75
80 Arg Pro Val Gln Val Ala Glu Arg Leu Glu Lys Phe Lys Thr
Thr Ile 85 90 95
Phe Thr Gln Met Ser Met Leu Ala Ile Lys His Gly Ala Ile Asn Leu
100 105 110 Gly Gln Gly Phe Pro
Asn Phe Asp Gly Pro Asp Phe Val Lys Glu Ala 115
120 125 Ala Ile Gln Ala Ile Asn Ala Gly Lys
Asn Gln Tyr Ala Arg Gly Phe 130 135
140 Gly Val Pro Glu Leu Asn Ser Ala Ile Ala Glu Arg Phe
Leu Lys Asp145 150 155
160 Ser Gly Leu Gln Val Asp Pro Asp Lys Glu Val Thr Val Thr Ser Gly
165 170 175 Cys Thr Glu Ala
Ile Ala Ala Thr Ile Leu Gly Leu Ile Asn Pro Gly 180
185 190 Asp Glu Val Ile Leu Phe Ala Pro Phe
Tyr Asp Ser Tyr Glu Ala Thr 195 200
205 Leu Ser Met Ala Gly Ala Asn Val Lys Ala Ile Thr Leu Arg
Ala Pro 210 215 220
Asp Phe Ala Val Pro Leu Glu Glu Leu Glu Ala Ala Val Ser Lys Asp225
230 235 240 Thr Lys Ala Ile Met
Ile Asn Thr Pro His Asn Pro Thr Gly Lys Met 245
250 255 Phe Thr Arg Glu Glu Leu Glu Ser Ile Ala
Ala Leu Cys Lys Glu Asn 260 265
270 Asp Val Leu Leu Phe Ser Asp Glu Val Tyr Asp Lys Leu Val Phe
Glu 275 280 285 Ala
Asp His Ile Ser Met Ala Ser Ile Pro Gly Met Tyr Glu Arg Thr 290
295 300 Val Thr Met Asn Ser Leu
Gly Lys Thr Phe Ser Leu Thr Gly Trp Lys305 310
315 320 Ile Gly Trp Ala Ile Ala Pro Pro His Leu Thr
Trp Gly Leu Arg Gln 325 330
335 Ala His Ser Phe Leu Thr Phe Ala Thr Cys Thr Pro Met Gln Ala Ala
340 345 350 Ala Ala Ala
Ala Leu Arg Ala Pro Asp Ser Tyr Tyr Asp Glu Leu Lys 355
360 365 Arg Asp Tyr Ser Ala Lys Lys Ala
Ile Leu Leu Glu Gly Leu Glu Ala 370 375
380 Ala Gly Phe Ile Val Tyr Pro Ser Ser Gly Thr Tyr Tyr
Ile Met Val385 390 395
400 Asp His Thr Pro Phe Gly Phe Asp Ser Asp Val Glu Phe Cys Glu Tyr
405 410 415 Leu Ile Arg Glu
Val Gly Val Cys Ala Ile Pro Pro Ser Val Phe Tyr 420
425 430 Leu Asp Pro Glu Glu Gly Lys Lys Leu
Val Arg Phe Thr Phe Ser Lys 435 440
445 Asp Glu Gly Thr Leu Arg Ala Ala Val Glu Arg Leu Lys Ala
Lys Leu 450 455 460
Arg Arg Lys465 48392PRTChlorella vulgarisChlorella glutamine
phenylpyruvate transaminase (GPT) 48Met Ala Ala Ala Ala Ala Gly Gly
Asp Gly Pro Ser Ala Ala Arg Arg1 5 10
15 Phe Asn Ser Thr Phe Ser Ser Leu Pro Thr Thr Ile Phe
Glu Gln Met 20 25 30
Ser Leu Leu Ala Ala Lys His Gln Ser Thr Asn Leu Gly Gln Gly Phe
35 40 45 Pro Asp Asn Glu
Leu Glu Gly Pro Glu Ser Met Lys Lys Val Met Ile 50 55
60 Ser Leu Tyr Glu His Ser Asn Gln Tyr
Pro Pro Leu Met Gly Leu Pro65 70 75
80 Glu Leu Arg Gln Ala Val Ala Ala His Ser Ala Arg His Ala
Gly Ile 85 90 95 Pro
Val Asp Trp Gln Ala Glu Thr Leu Val Thr Val Gly Ala Thr Glu
100 105 110 Ala Leu Ala Ala Ala
Phe Leu Gly Leu Leu Asp Ala Gly Asp Glu Val 115
120 125 Ile Phe Phe Glu Pro Leu Tyr Asp Ser
Tyr Val Pro Met Ala Arg Arg 130 135
140 Ala Gly Ala Ile Pro Arg Ile Val Gln Leu Tyr Pro Pro
Ala Trp Ser145 150 155
160 Ile Asp Ala Ala Glu Leu Glu Ala Ala Phe Ser Pro Gln Thr Lys Leu
165 170 175 Leu Val Leu Asn
Thr Pro His Asn Pro Thr Gly Lys Val Phe Gly Ala 180
185 190 Glu Glu Leu Gln Leu Ile Ala Asp Leu
Cys Gln Lys His Asp Cys Leu 195 200
205 Cys Leu Leu Asp Glu Val Tyr Glu His Leu Val Phe Pro Gly
Thr Arg 210 215 220
His Thr Ser Leu Gln Ser Leu Pro Gly Met Arg Glu Arg Cys Leu Arg225
230 235 240 Val Gly Trp Leu Ser
Gly Pro His Asp Leu Leu Ala Ala Val Thr Lys 245
250 255 Ala His Gln Phe Leu Ile Phe Thr Val Pro
Ser Ala Leu Gln Arg Ala 260 265
270 Val Ala Tyr Gly Leu Glu Gln Glu Glu Ala Phe Cys Cys Gly Leu
Gly 275 280 285 Ala
Ala Leu Ser Lys Lys Arg Gln Leu Leu Glu Gly Gln Leu Ala Glu 290
295 300 Ile Gly Phe Ala Val Leu
Pro Ala Gln Gly Thr Tyr Phe Leu Val Ala305 310
315 320 Asp Phe Ala Gly Leu Leu Pro Ala Gly Ser Ser
Glu Asp Asp Val Gln 325 330
335 Phe Cys His Arg Leu Thr Val Glu Ala Gly Val Thr Leu Ile Pro Val
340 345 350 Ser Ala Phe
Tyr Ala Asp Arg Ala Ala Thr Pro Arg Thr Leu Val Arg 355
360 365 Phe Val Phe Cys Lys Thr Asp Glu
Lys Leu Asn Thr Ala Cys Gly Lys 370 375
380 Leu Arg Thr Tyr Phe Gly Arg Gln385
390
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