NZ716717B2

NZ716717B2 - An expression construct and process for enhancing the carbon, nitrogen, biomass and yield of plants

Info

Publication number: NZ716717B2
Application number: NZ716717A
Authority: NZ
Inventors: Paramvir Singh Ahuja; Anish Kaachra; Sanjay Kumar; Surender Kumar Vats
Original assignee: Council Of Scientific & Industrial Research
Priority date: 2011-04-19
Filing date: 2012-04-19
Publication date: 2017-07-04

Abstract

Disclosed is an expression construct comprising SEQ ID NO. 7 for co-expression of the genes AspAT, GS and PEPCase comprising nucleotide sequences SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, linked to at least one control sequence and a transcription terminator sequence, useful for enhancing the carbon, nitrogen, biomass and yield of plants as compared to wild type or untransformed plant. bon, nitrogen, biomass and yield of plants as compared to wild type or untransformed plant.

Description

[Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by m [Annotation] m Unmarked set by amandam AN EXPRESSION CONSTRUCT AND PROCESS FOR ENHANCING THE CARBON, NITROGEN, BIOMASS AND YIELD OF PLANTS The following specification particularly describes the invention and the manner in which it is to be med: FIELD OF THE INVENTION The present invention relates to an expression construct for enhancing the carbon (C), nitrogen (N), biomass and yield of plants.

Further, the present invention provides the process for enhancement of C and N levels and subsequent improvement in the biomass and yield of plant by using the aforesaid expression construct which utilizes co-overexpression of genes from enzymes phosphoenolpyruvate carboxylase (hereinafter, referred as ”PEPCase”), glutamine synthetase (hereinafter, referred as ”GS”) and aspartate aminotransferase nafter, referred as ”AspAT”). In particular, the present invention is directed to transgenic plants where nucleic acid sequences ng the said proteins are sed in plant cells.

More particularly, the t invention relates to the transformation of a plant with genetic uct involving rexpression of three genes wherein one gene PEPCase encodes enzyme responsible to capture C02 and the other two encode for s (AspAT and GS) involved in N assimilation wherein the N assimilation requires C skeleton which is met by PEPCase, under the control of constitutive promoter comprising plant Arabidopsis thaliana transformed with AspAT+ G5 + PEPCase gene and expression of this gene in plants, thereby enhancing the status of C and N, biomass and yield of plant.

BACKGROUND OF THE ION AND PRIOR ART The present invention relates to a transformed plant with co-overexpression of three genes, viz.

AspAT, G5 and PEPCase, leading to enhanced C, N content, biomass, and yield component.

PEPCase (EC. 31) is a ubiquitous enzyme in plants that catalyses the B-carboxylation of yieldphosphgolpyruvate (hereinafter, referred as ”PEP”) in the presence of HC03_and Mg2+ to0 acetate (hereinafter, referred as ”OAA”) and inorganic phosphate (hereinafter, [Annotation] amandam None set by amandam [Annotation] amandam ionNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam ation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam ed as ”Pi”), and it primarily has an anaplerotic function of replenishing the tricarboxylic acid cycle with intermediates. In higher plants, there are several ms of PEPCase of different organ specificities and they are involved in a variety of functions including stomata opening, fruit ripening and seed maturation. The leaves of C4 and CAM plants n high levels of PEPCase, which catalyze the initial C02 fixation of photosynthesis. The much lower levels of PEPCase seen in the leaves of C3 plants contribute to an anaplerotic function and play a role in tion ofthe cellular pH.

GS (EC 6.3.1.2) catalyses the ATP-dependent condensation of ammonia (hereinafter, referred as ”NH3”) with glutamate (hereinafter, referred as ”Glu”) to produce glutamine (hereinafter, referred as ”Gln”). Subsequently, glutamate synthase ) ers the amide group of Gln to CL —ketoglutarate ing two molecules of Glu. Both Gin and Glu are the primary source of organic N for proteins, nucleic acid and chlorophyll.

AspAT (EC 2.6.1.1) catalyzes the reversible transfer of the amino group of te (hereinafter, referred as ”Asp”) to OL-ketoglutarate to form OAA and Glu. In plants, AspAT has been proposed to play several metabolic roles including: recycling of C skeletons during NH3+ lation in roots, providing amide precursors for biosynthesis of major nitrogen transport molecules such as asparagines (hereinafter, referred as ”Asn”) and ureides, recruiting Asn nitrogen during seed g and ipating in intracellular C shuttles in C4 plants providing precursors for the biosynthesis of the Asp family of amino acids.

Plant performance in terms of biomass production, yield or harvest index depends upon number of internal and environmental factors. Among all these factors, plant C and N level is one of the important factors governing plant productivity. The ng details of C and N assimilation suggest that a regulatory system coordinates the uptake and distribution of these nutrients in se to both metabolic and environmental cues. Plants sense changes in their C and N status and relay this ation to the nucleus where changes in gene expression are brought about. Since plant growth and crop yield are largely influenced by the assimilated C and N, many attempts have been made in the past to engineer efficient C and N assimilation. However, there is no report yet which show significant improvement in the st of C, N, biomass and yield in plants.

[Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam Table 1 illustrates the status of information available on the various strategies to improve C and/or N and biomass in different plants.

Table 1: Functions Transformation Results Reference System adopted NAD kinase2 Arabidopsis NADK2 NADK2 overexpressors shi, H., (NADK2) overexpressor and were characterized by Takahara, K., nade mutant were increase in calvin cycle Hashida, S., Catalyzes the studied to igate intermediates and Hirabayashi,T., synthesis of the impact of altering amino acid like Glu Fujimori,T., NADP from NAD NADP level on plant and Gln. However, Yamada,M.K., in chloroplasts metabolism. there is no clear Yamaya,T., evidence on role of Yanagisawa, S.

NADK2 influencing C and Uchimiy, H. and N lism. 2009. Plant Physiol. 151: 100- 113.

Dof 1 Maize Dofl cDNA was Dofl overexpression Yanagisawa, S., overexpressed in in Arabidopsis has led Akiyama, A., Dofl is a Arabidopsis plants to co-operative Kisa ka, H., transcription under tive of modification of plant C Uchimiya, H. and activator for the 355 promoter and N content, with Miwa, T. 2004. le gene designated as ed growth Proc. Natl. Acad. expressions 35$C4PPDK. under low N Sci. USA. 101: associated with conditions. r, 7833—7838 the organic acid effect of CN lism, alteration on plant including biomass or yield was PEPCase. not discussed.

GS i.) A soybean cytosolic Over expression of Vincent, R., GS gene (G515) fused lic GS Fraisier, V., GS ses the with the constitutive accelerated plant Chaillou, 5., ATP- ent CaMV 35$ promoter in development, g Limami, M. A., condensation of order to direct its over- to early senescence Deleens, E., NH3 with (Glu) to expression in Lotus and premature Phillipson, B., produce (Gln). corniculatus L. plants. ing when grown Douat, C., NH4+ rich medium. , J.-P. and Limitation of C Hirel, B. skeleton and energy 1997. Planta. for enhanced NH4+ 201: 424-433.

D assimilation were anticipated. ation] m None set by m [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by m [Annotation] amandam Unmarked set by amandam ii.) A pea cytosolic GS Overexpression of Oliveira, |.., gene was lic GS in relation , T., overexpressed in to N, light and Knight, T., Clark, tobacco plants photorespiration A. and Coruzzi, suggested an G. 2002, Plant alternative route to Physiol. chloroplastic GS for 129:1170-1180 assimilation of photorespiratory ammonium. iii.) Full-length cDNAs An increased metabolic Cai, H., Zhou, Y., encoding rice cytosolic level in GS- Xiao, J., Li, X., GS genes ;1 overexpressed plants Zhang, Q. and and 2) along was obtained which , Lian, X. 2009, with E. coli GS gene showed higher total GS Plant Cell Rep. (glnA) were activities and soluble 28: 527-537 overexpressed in the protein concentrations rice plant under in leaves and higher constitutive CaMV 35$ total amino acids and promoter. total N content in the whole plant. However, decrease in both grain yield production and total amino acids were observed in seeds of GS-overexpressed plants compared with wild-type plants. iv) cDNA encoding alfa Transgenic plants Fuentes, S., Allen, alfa cytosolic GS over grew better under N D., Ortiz-Lopez, A. expressed in tobacco starvation by and Hernandez, plants maintaining G. 2001. J. Exp. photosynthesis at rate Bot. 1- able to those 1081. of plants under high N, while photosynthesis in control plants was inhibited by 40-50%.

These results further reflect the need for cooperative modification of CN metabolism for developing plants with better agronomic traits.

[Annotation] amandam None set by amandam ation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam ation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam PEPCase i) The intact maize Transgenic plants Agarie, S., Miura, gene encoding C4- exhibited higher A., Sumikura, R., PEPCase catalyses specific e used PEPCase activity with Tsukamoto, S., the [3- for transformation of reduced 02 inhibition Nose, A., Arima, S., carboxylation of rice plants of photosynthesis. It Matsuoka, M. and PEP in the was found that the Tokutomi, MM. presence of HC03 reduced 02 inhibition 2002. Plant Sci. ‘and Mg2+to yield photosynthesis was 162: 257-265.

OAA and Pi. ily due to reduction of Pi rather than se in the partial direct ﬁxation of atmospheric C02 via the enhanced maize PEPCase.

However, no report on biomass accumulation or yield as a consequence of PEPCase overexpression was reported. ii) Maize PEPCase Higher levels of maize Hudspeth, introduced in to PEPCase transcript of R.L.,Grula, o plants under the correct size were J.W.,Dai, Z., the l maize obtained using tobacco Edwards, G.E. and e and tobacco (chlorophyll a/b Ku, M.S.B. 1992. chlorophyll a/b binding n gene Plant Physiol. 98: binding protein gene promoter. With two 458-464 promoter. fold incerase in PEPCase activities in leaf, transgenic plants had significantly elevated levels of titratable yand malic acid. However, these biochemical differences did not e any significant physiological changes with respect to photosynthetic rate or C02 compensation point.

AspAT i) m miliaceum mAspAT- or cAspAT- Sentoku, N., L. mitochondrial and transformed plants Taniguchi , M., AspAp cytosolic AspAT had about threefold or Sugiyama, T., [Annotation] amandam None set by amandam ation] m ionNone set by amandam [Annotation] amandam Unmarked set by m [Annotation] amandam None set by m [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam catalyzes (mAspAT and cAspAT, 3.5-fold higher AspAT ru, K., the reversible respectively) genes activity in , R., transfer of the were expressed in the leafthan non- Takaiwa, F. and amino group of tobacco plants under transformed plants, Toki, S. 2000.

(Asp) to a- CaMV 35$ promoter. respectively. Plant Cell Rep. ketogluta rate to Leaves of both 19598 - 603. form OAA and transformed plants Glu had increased levels of PEPCase and transformed plants with cAspAT also had increased levels of mAspAT in the leaf.

These results further ted interaction between C and N metabolism. ii) Three AspAT genes Compared with Zhou. Y., Cai ,H., from rice (OsAAT3) control Xiao, J. and one AspAT gene plants, the Li, X., Zhang, Q. from E. coli (EcAAT) transformants showed and Lian, X. 2009. were over expressed significantly increased Theor Appl under CaMV 35$ leaf AspAT activity and Genet. 11821381- er in rice greater seed amino 1390 plants . acid and n contents. However, influence of CN level on biomass or yield was not discussed.

Higher activity of PEPCase shall facilate C02 capturing and makes the carbon backbone available for routing of en in to organic form through joint activity of AspAT and GS. As a result, the inventors have found that object of the present invention can be attained by itant increase in expression of genes encoding AspAT, GS and PEPCase to establish the present invention.

Below is given a state of the art knowledge in relation to the present invention and the attempts previously made to enhance either carbon and / or nitrogen levels in the plant.

Reference may be made to article by Hudspeth, R.L., Grula, J.W., Dai, 2., Edwards, G.E. and Ku, M.S.B., entitled ”Expression of miaze phosphoenolpyruvate carboxylase in transgenic tobacocnl992, Plant Physiology 98: , 458-464), wherein PEPCase from maize was expressed under a tobacco(Nicotiana plumbaginifolia) chlorophyll a/b binding protein gene [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] m None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam ed set by amandam promoter in tobacco plants. Up to two fold higher activity of PEPCase was observed in the transgenic leaves as compared to non-transformants with elevated levels of titratable acidity and malic acid. However, these biochemical differences did not produce any significant physiological changes with respect to photosynthetic rate or C02 sation point.

Reference may be made to article by Lebouteiller, B., Dupont, A.G., Pierre, J.N., Bleton , J., Tchapla, A., Maucourt, M. and Moing, A., Rolin, D., and Vidal, J. entitled ”Physiological impacts of modulating phosphoenolpyruvate carboxylase levels in leaves and seeds of Arabidopsis thaliana” (2007, Plant Science, 172:256-272,), wherein the PEPCase of sorghum was expressed under CaMV 35$ promoter in Arabidopsis plant. The leaves of the primary transformants showed up to ten-fold increase in PEPCase activity and up to 30% increase in the dry weight and total protein content of seeds. However, the transformants (primary and progeny) did not show any ed growth phenotype or modiﬁcation in seed production per plant nce may be made to yet another e by Chen, L.M., Li, K.Z. Miwa, T. and Izui, K. ed ”Overexpression of a acterial phosphoenol pyruvate ylase with diminished ivity to feedback inhibition in Arabidopsis changes amino acid metabolism” (2004, Planta, 219: 440-419.), wherein the cyanobacterial Synechococcus us phosphoenolpyruvate carboxylase (SvPEPCase) with diminished sensitivity to feed back tion, was over sed under the control of CaMV 35$ promoter in Arabidopsis plant. One third of the T1 transformants showed severe phenotypes as bleached leaves and were infertile when grown on soil. However, no such phenotype was observed with Arabidopsis transformed with maize PEPCase (ZmPEPC) for C4 photosynthesis, which is normally sensitive to a feedback inhibitor, L-malate. The growth inhibition of SvPEPC ormed T2 plants was presumed to be primarily due to a decreased availability of phosphoenolpyruvate (PEP), one of the precursors for the shikimate pathway for the synthesis of aromatic amino acids and phenylpropanoids.

Referernmay be made to yet another article by ma, H., Hatch, M.D., Tamai, T., Tsuchida, H., Sudoh, S., Furbank, RT and Miyao, M., entitled ”Activity regulation and [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by m [Annotation] amandam Unmarked set by amandam ation] amandam None set by amandam [Annotation] m MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam physiological impacts of maize C (4)-specific phosphoenolpyruvate carboxylase overproduced in transgenic rice plants” (2003, Photosynthesis Research, 77: 227-239), n the intact maize PEPCase gene was overexpressed in the leaves of rice plants.

Introduced PEPCase in transgenic rice leaves underwent activity regulation through protein phosphorylation in manner similar to nous rice e but contrary to that occurring in maize leaves, being downregulated in the light and upregulated in the dark.

Compared with untransformed rice, the level of PEP was slightly lower and the product (OAA) was slightly higher in transgenic rice, suggesting that maize PEPCase was functioning even though it remained dephosphorylated and less active in the light. 14C02 labeling experiments ted that maize PEPCase did not contribute signiﬁcantly to the photosynthetic C02 ﬁxation of transgenic rice plants. Rather, it slightly lowered the C02 lation rate. This effect was ascribable to the stimulation of ation in the light, which was more marked at lower 02 concentrations. It was concluded that overproduction of e does not directly affect photosynthesis signiﬁcantly but it suppresses photosynthesis indirectly by stimulating respiration in the light.

Reference may be made to yet another article by Vincent, R., er, V., Chaillou, S., Limami, M.A., Deleens, E., Phillipson, B., Douat, C., Boutin, J.P. and Hirel, B., entitled ”Overexpression of a soybean gene encoding cytosolic glutamine synthetase in shoots of transgenic Lotus corniculatus L. plants triggers changes in ammonium assimilation and plant development” (1997, Planta. 201:424-433), wherein a soyabean cytosolic GS gene 6515 was fused with CaMV 35$ promoter to achieve constitutive expression in the lotus corniculatus L. plants. On growing the transgenic plants under different N regimes an increase in free amino acids and ammonium was observed anied by a decrease in soluble carbohydrates in the transgenic plants cultivated with 12 mM NH4+ in comparison to the wild type grown under the same conditions. ing experiments revealed that both ammonium uptake in the roots and the subsequent translocation of amino acids to the shoots was lower in plants over expressing GS. However the early floral development in the ormed plants suggested the role of GS in the early senescence and premature ing when plants were grown on an ammonium-rich medium. Limitation of C skeleton and enen for enhanced similation were anticipated.

[Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam ation] amandam ed set by amandam [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam Reference may be made to yet another article by Fuentes, S.|., Allen, D.J., Ortiz-Lopez, A. and Hernandez, G., entitled ”Overexpression of cytosolic glutamine synthetase increases photosynthesis and growth at low nitrogen conditions” (2001, Journal of Experimental , 52:1071-1081), wherein the alfa alfa GS driven by constitutive CaMV 35$ er uced into tobacco plants. Leaf GS activity in the transgenic plants increased up to six times of untrasformed plants. Under N starvation GS transgenic grew better by maintenance of photosynthesis at rates indistinguishable from plants under high N, while photosynthesis in the control plants was inhibited by 40-50 % by N deprivation. However, under optimum N ization conditions, no effect of GS overexpression on photosynthesis or growth was observed.

Reference may be made to yet another article by Oliveira, |.., Brears, T., Knight, T., Clark, A. and Coruzzi, G., ed ”Overexpression of cytosolic glutamine synthetase. Relation to nitrogen, light, and photorespiration” (2002, Plant Physiology, 129: 1170-1180), n the overexpression of pea cytosolic GS was d in relation to nitrogen, light and photorespiration. o plants, which cally overexpress cytosolic GSl in leaves, display a light-dependent improved growth phenotype under N-limiting and N-non-limiting ions as evident by se in fresh weight, dry weight, and leaf soluble protein. The cytosolic GSl transgenic plants also exhibit an increase in the C02 photorespiratory burst and an increase in levels of espiratory intermediates, suggesting changes in photorespiration. However, the effect of stimulation of photorespiration by GS overexression on plant productivity was not discussed.

Reference may be made to yet r article by Cai, H., Zhou, Y., Xiao, J., Li, X., Zhang, Q. and Lian, X., entitled ”Overexpressed glutamine synthetase gene modifies nitrogen metabolism and abiotic stress response in rice” (2009, Plant Cell Reports. 28: 7), wherein the full-length cDNAs encoding rice (Oryza sativa) cytosolic GS genes (OsGSl;1 and OsGSl;2) along with E. coli GS gene (glnA) were pressed in the rice plant under constitutive CaMV 35$ promoter. An increased metabolic level in GS-overexpressed plants was obtained which showed higher total GS activities and soluble protein concentrations in leaves and higher total amino acids and total N content in the whole plant. However, ation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam ed set by amandam [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam ation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam decrease in both grain yield production and total amino acids were observed in seeds of GS- overexpressed plants compared with wild-type plants.

Reference may be made to yet another e by Sentoku, N., Taniguchi, M., Sugiyama, T., Ishimaru, K., Ohsugi, R., Takaiwa, F. and Toki, S., entitled ” Analysis of the transgenic o plants expressing Panicum miliaceum aspartate aminotransferase genes" (2000, Plant Cell Reports, 19: 598-603), n the effects of the overexpression of Panicum mitochondrial and cytoplasmic AspAT (mAspAT and cAspAT respectively) under the control of CaMV 35$ promoter were evaluated on transgenic tobacco plants. The mAspAT- or cAspAT-transformed plants had about old or 3.5-fold higher AspAT activity in the leaf than ansformed plants, respectively. Interestingly, the leaves of both transformed plants had sed levels of PEPCase and transformed plants with cAspAT also had increased levels of mAspAT in the leaf. These results suggest that the increased expression of Panicum cAspAT in transgenic tobacco enhances the expression of its endogenous mAspAT and PEPCase, and the increased expression of Panicum mAspAT enhances the expression of its endogenous PEPCase.

However, there is no account on effect of AspAT overexpression on plant growth and productivity.

Reference may be made to yet another article by Zhou, Y., Cai, H., Xiao, J., Li, X., Zhang, Q. and Lian, X., entitled ”Over-expression of aspartate aminotransferase genes in rice resulted in altered nitrogen metabolism and increased amino acid t in seeds” (2009, tical and Applied Genetics, 118:1381—1390 ), wherein three AspAT genes from rice (OsAAT1-3) encoding chloroplastic, cytoplasmic, and mitochondrial AspAT isoenzymes, respectively and one AspAT gene from E. coli (EcAAT) were overexpressed in rice plant under the l of CaMV 35$ promoter. The OsAAT 1, OsAATZ, and EcAATtransformants showed significantly increased leaf AspAT activity and greater seed amino acid and protein contents. r no significant changes were found in leaf AspAT activity, seed amino acid content or protein content in OsAAT3 over-expressed plants.

Reference may be made to yet another article by Murooka, Y., Mori, Y. and Hayashi, M., entitled ” ion of the amino acid content of Arabidopsis seeds by expressing soyabean aspartate aminotransferase gene” (2009, Journal of Bioscience and Bioengineering, 94: 225- 230), Wain AspAT5 encoding the chloroplast AspAT from Soyabean was linked to CaMV [Annotation] amandam None set by m [Annotation] amandam MigrationNone set by amandam [Annotation] amandam ed set by amandam [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam [Annotation] m MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam 355 promoter for achieving its overexpression in the Arabidopsis plant. Expression of AspAT5 in transformants caused 3-, 4-, 23-, and d increases in the contents of free glycine, alanine, asparagine, and Glu, respectively, in the T3 seeds. However, a decrease in the contents of valine, ne, isoleucine, leucine, and phenylalanine by several folds was also observed. Further, there is no report on effect of overexpression of AspAt on plant growth and productivity.

Reference may be made to yet another article by Yanagisawa, S., Akiyama, A., Kawaka, H., Uchimiya, H. and Miwa, T. entitled ”Metabolic engineering with Dofl transcription factor in : Improved nitrogen assimilation and growth under low-nitrogen conditions" (2004, Proceedings of the National Academy of Sceinces (USA), 101:7833-7838), wherein over- expression of Dofl transcription factor from maize improves N assimilation in transgenic Arabidopsis plants. Dofl expressing plants showed up-regulation of genes encoding enzymes for C on production, a marked increase of amino acid contents, and a reduction of the glucose level. The results suggest cooperative modification of C and N metabolisms on the basis of their intimate link. Elementary analysis revealed that the N content increased in the Dofl transgenic plants (230%), indicating promotion of net N assimilation. However, effect of C N alteration on plant biomass or yield was not discussed.

Reference may be made to still another e by shi, H., Takahara, K., Hashida, S., Hirabayashi, T., Fujimori, T., Kawai-Yamada, M., , T., Yanagisawa, S. and Hirofumi Uchimiya, H., entitled ”Pleiotropic Modulation of carbon and nitrogen metabolism in Arabidopsis plants overexpressing the NAD 2 gene” by (2009, Plant logy. 151:100-113), wherein enic Arabidopsis plants with over expression of NAD kinase2 (NADK2) along with NADK2 mutants were raised to investigate the impacts of altering NADP level on plant metabolism. Metabolite proﬁling revealed that NADP (H) concentrations were tional to NADK activity in NADK2 overexpressors and in the NADK2 mutant. l metabolites associated with the calvin cycle were also higher in the overexpressors, accompanied by an increase in overall Rubisco activity. Furthermore, enhanced NADP (H) production due to NADK2 overexpression increased N assimilation. Gin and Glu concennons, as well as some other amino acids, were higher in the overexpressors.

However, there is no clear evidence on role of NADK2 influencing C and N metabolism.

[Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam [Annotation] amandam ionNone set by amandam ation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam ation] amandam Unmarked set by amandam The improvement in the C and N status of plants is a major concern to improve productivity.

However, there is no report yet which show enhancement of C and N levels and subsequent improvement in the biomass and yield of plant. r, no attempt has been made to co-over express three genes, viz. AspAT, GS and PEPCase, leading to enhanced status of C and N, s, and yield.

OBJECTIVES OF THE INVENTION The main objective of the present invention is to provide an expression construct for enhancing the carbon, nitrogen, biomass and yield of plants which obviates the drawbacks ofthe hitherto known prior art as detailed above. r objective of the present invention is to provide an expression construct for co- overexpression of AspAT (SEQ ID NO: 1), GS (SEQ ID NO: 2). and PEPcase (SEQ ID NO: 3) wherein PEPCase efficiently captures C02 whereas the other two genes encoding for enzymes (AspAT and GS) have role in N assimilation, using the carbon backbone provided by PEPCase ed reaction resulting in the ement of C and N status with improved biomass and yield of .

Yet another objective of the present invention is to raise transgenic plant exhibiting co- overexpression of genes AspAT, GS and PEPCase.

Still another objective of the present invention is to evaluate the expression of AspAT, GS and PEPCase genes in transgenic plants.

Still another objective of the present invention is to evaluate the enic plants for status of C and N, biomass and yield compared to wild plants.

Y OF THE INVENTION Accordingly, the present ion provides an expression construct represented by SEQ ID NO. 7 for co-expression of the genes AspAT, GS and PEPCase comprising nucleotide sequences represented by SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, wherein SEQ ID NO: 1 ents AspAT genes, SEQ ID NO: 2 represents GS genes and SEQ ID NO: 3 represents PEPCase genes linked to atleast one control sequence and a transcription terminator sequence, useful for enhancing the carbon, nitrogen, biomass and yield of plants as comde to wild type or untransformed plant.

[Annotation] m None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam [Annotation] m MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam In an embodiment of the present invention, the control sequence is preferably ented by SEQ ID NO: 4.

In r embodiment of the present invention, the ription terminator sequence is represented by SEQ ID NO: 5.

In an embodiment, the present ion provides an expression construct prepared from the cytoso|ic AspATgene from soyabean, cytoso|ic GS gene from tobacoo and cytoso|ic PEPCase gene from maize.

In another embodiment of the present invention, the po|ynuc|eotide having SEQ ID No: 7 is overexpressed in plants.

In still another ment of the present invention, the control sequence used is a constitutive promoter selected from the group ting of CaMV 35$ promoter, rubisco promoter, ubiquitin promoter, actin promoter.

In still another embodiment of the present invention, the ator used is preferably selected from the group consisting of Nos terminator and CaMV 3’ UTR.

In still another embodiment of the present invention, a process for preparing the expression construct wherein the process sing the steps of: i) amplifying cDNA sequences encoding genes represented by SEQ ID NO: 1 using primers represented by SEQ ID NO: 10 and SEQ ID NO: 11, SEQ ID NO: 2 using primers represented by SEQ ID NO: 8 and SEQ ID NO: 9 and SEQ ID NO: 3 using primers represented by SEQ ID NO: 12 and SEQ ID NO: 13; ii) cloning independently the amplified product of SEQ ID NO: 1, 2 and 3 as obtained in step (i) into pGEM-T easy vector; iii) digesting independently the plasmid from the positive clones as obtained in step (ii) along with A 1302 and further ligating the digested gene ts and pCAMBIA 1302 and transforming into E.coli DH5 0L cells; iv) sequencing the plasmid from the ve colonies obtained in step (iii) confirming the inframe cloning of AspAT::pCAMBIA1302; GS::pCAMBIA1302 and PEPCase::pCAMBIA 1302. v) amplifying the products ed in step (iv) by using primers represented by D SEQ ID NO: 10 and SEQ ID NO: 16; SEQ ID NO: 14 and SEQ ID NO: 15 and SEQ ID NO: 17 and SEQ ID NO: 18.

[Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by m [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam ation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam vi) cloning, digesting, ligating and sequencing was again performed ndently for the amplified GS coding sequence to form GS+pCAMB|A1302 which was further digested and ligated with the plasmids of positive clones of amplified AspATcoding sequence to form A5pAT+GS+pCAMB|A1302 expression cassette; vii) ligating the digested plasmids of positive clones of amplified e coding sequence with the destination pCAMB|A1302 which was previously cloned with the AspAT+GS+ expression cassette as obtained in step (vi) such that the genes AspA, GS and e were controlled by independent CaMV 35$ promoter and Nos transcriptional terminator to form single plant expression construct AspAT + GS + PEPCase ented by SEQ ID NO: 7.

In still another embodiment of the present invention, a process for enhancing the carbon, nitrogen, biomass and yield of plants using the sion construct, wherein the said process comprising the steps of: a) transforming Agrobacterium tumefacians strain with the expression uct as claimed in claim 1; b) orming the explants with the recombinant Agrobacterium cians strain as obtained in step (a); c) selecting the transformed explants of step (b) to obtain the desired transformed plants having ed level of carbon, nitrogen, biomass and yield of plants as compared to wild type plant.

In still another embodiment of the present invention, a process wherein the ormed plants display an increase of about 45-50% in PEPCase activity, atleast 55% in GS activity and 55-60% in AspAT ty as compared to wild type, resulting in increase in carbon and nitrogen levels in the plant.

In another embodiment of the present invention, the Agrobacterium strain provided is selected from a group consisting of GV3101 with ATCC number Agrobacterium tumefaciens (GV3101 (pMP90RK) (C58 derivative) ATCC® Number: 33970 Reference: Hayashi H, Czaja |, Lubenow H, Schell J n R. 1992.

In yet another embodiment of the present invention, the ormed plants are selected from the group consisting of grain crops, pulses, ble crops, oilseed crop and ornamens.

[Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam [Annotation] m ionNone set by amandam [Annotation] amandam Unmarked set by amandam ation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam In yet another ment, the transformed plants are selected from the group consisting of arabidopsis, tomato, potato, tobacco, maize, wheat, rice, cotton, mustard, pigeon pea, cowpea, pea, sugarcane, soyabean and sorghum.

In still another embodiment, the transformed plants as compared to wild type display increased yield and/or s, indicated by increased seed yield and/or pod yield.

In still another embodiment, the transformed plants display enhanced growth characteristics characterized by sed shoot fresh weight, shoot dry weight, root fresh and dry weight as compared to wild type or untransformed plant.

In yet another embodiment of the present invention, the transformed plant shows enhanced levels of carbon, nitrogen, s and yield as compared to wild plants.

In still another ment of the present invention, the sion and functionality of over expressed enzymes in transgenic plants is evaluated.

In yet r embodiment of the present invention, the selectable marker used is hpt gene (hygromycin phosphotransferase) represented by SEQ ID NO: 6 for hygromycin resistance controlled by duplicated CaMV 35$ promoter and terminated by CaMV 3'UTR (polyA signal).

In another embodiment of the present invention, biochemical assays and RT-PCR were performed to evaluate the expression of introduced genes and the functionality of over expressed enzymes in transgenic plants.

In a further embodiment of the present invention, the enic plants were investigated for different growth and yield ters and compared to wild plants ated under the same conditions.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS Figure 1 represents a schematic view of T- DNA region of plant transformation vector pCAMBIA1302 for co-overexpression of AspAT, GS and PEPCase (a) and amplification of coding sequences for AspAT, GS and PEPCase from respective plant sources (b) as discussed in Examples 1 to 4.

Figure 2 represents DNA analysis (a) and RNA analysis (b) of WT, LI and L2, where WT = wild,' L1 and L2 = two different transgenic lines co-overexpressing AspAT, GS and PEPCase.

Figure Dapresents shoot fresh weight (FW) (a), shoot dry weight (DW) (b), root fresh weight (c) and root dry weight (d) of WT and AspAT+GS+PEPCase transgenic plants at 60 [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] m ed set by amandam [Annotation] amandam None set by m [Annotation] amandam MigrationNone set by amandam [Annotation] amandam ed set by amandam [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam days of sowing. Data is mean of five separate biological replicates with standard deviation marked on each bar.

Figure 4 represents AspAT activity (a) GS activity (b) and PEPCase activity (d) of WT, LI and L2 at 42 days of sowing. Data is mean of three separate biological replicates with rd deviation marked on each bar.

Figure 5 represents Analyses of N (a) and C (b) content from ent plant parts of WT, LI and L2 lines at 65 days of sowing. Data is mean of three separate biological ates with standard deviation marked on each bar.

Figure 6 represents a representative WT and AspAT+GS+PEPCase transgenic plants at 75 days of sowing.

Figure 7 represents pod number (a) and seed yield (b) in WT, LI and L2 at 75 days of sowing.

Data is mean of five separate biological replicated with standard deviation marked on each bar.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to c engineering of C and N metabolism in plants. In particular, the present invention relates to an expression construct for co-overexpression of AspAT, GS and PEPCase for concomitant alteration in the s involved in C and N assimilation or utilization and/or their expression in order to engineer plants with increased C and N levels thereby promoting better growth and biomass production and enhanced yield.

The term "vector" refers to a construct made up of nucleic acids wherein gene from a n source can be ligated and isolated when . The construct is usually a plasmid (i.e. extra chromosomal self replicating nucleic acid) and is propagated, for example bacterial cell of Eco/i. The vector in the present invention was used to transfer the gene from one source to r.

The term "gene" refers to the sequence of nucleic acids that can produce a polypeptide chain.

The term "gene expression" refers to the level/amount of RNA (i.e. sequence of ribonucleic acid) of choice ribed (i.e. the process of sis of RNA by DNA) by DNA (i.e. sequean deoxyribonucleic acid). When the gene was transcribed in higher amounts as compared to the control, it was referred to as "over-expression” of gene.

[Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by m [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by m [Annotation] amandam Unmarked set by amandam ation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam The term ”selectable marker” refers to a gene, which allows a cell to survive in the presence of an otherwise toxic antibiotic The term "transgenic plant" refers to genetically transformed plants with stable integration of introduced gene in to its genome The term ”promoter” refers to the specific DNA sequence, usually located upstream (5') to the DNA sequence involved in transcription, n the enzyme RNA polymerase binds for the process of ription. “Constitutive promoters” direct expression of the gene in all tissues and during all periods regardless of the surrounding environment and development stage of the organism.

The term ssion cassettes” refers to vector comprising of (a) a tutive promoter; (b) all the three genes cloned 3' to the constitutive promoter, (c) a polyadenylation signal located 3' to the coding sequence. and capable of passing genetic information on to successive generations. type" plants are untransformed .

The term "To" refers to the first set of genetically transformed plants that can be identified and selected upon growth in presence ofa selection agent antibiotic, for which the transgenic plant contains the corresponding resistance gene. The term "T1" refers to the generation of plants obtained after self-fertilization of the s of T 0 generation plants, previously selected as being transgenic. "T2” plants are generated from T1 plants, and so on.

The present invention will be illustrated in greater details by the following examples.

EXAMPLES The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention.

Sequences of the primers used in the present invention are listed as follows: Name of the ce Sequence Purpose sequence ID No.

AspAT cDNA atggcttctc acgacagcat ctccgcttct ccaacctccg cttctgattc cgtcttcaat 60 ents 1 cacctcgttc ccga agatcctatc ctcggggtaa ctgtcgctta taacaaagat 120 sequence nucleotide ccaagtccag ttaagctcaa cttgggagtt tacc gaactgagga aggaaaacct 180 cttgttttga atgtagtgag gcgagttgaa cagcaactca acgt gtcacgcaac 240 sequences of aaggaatata ttccgatcgt tgggcttgct aata aattgagtgc taagcttatt 300 AspAT genes for tttggggctg acagccctgc tattcaagac aacagggtta ccactgttca atgcttgtct 360 ggaactggtt ctttaagagt tgaa tttttggcta aacactatca ccaacggact 420 making an atatacttgc caacaccaac ttggggcaat cacccgaagg ttttcaactt agcaggcttg 480 expression tctgtcaaaa cataccgcta tcca gcaacacgag gacttgactt tcaaggactt 540 ctggaagacc ttggttctgc tccatctgga tctattgttt tgctacatgc atgcgcacat 600 actg gtgtggatcc aacccttgag gagc agattaggca gctaataaga 660 tcaaaagctt cttt ctttgacagt gcttatcagg gttttgctag tggaagtcta 720 gatgcagatg cccaacctgt tcgtttgttt gttgctgatg gaggcgaatt gctggtagca 780 caaagctatg caaagaatct gggtctttat ggggaacgtg cctt aagcattgtc 840 tgcaagtcag ctgatgttgc aagcagggtt gagagccagc tgaagctagt gattaggccc 900 atgtactcaa gtcctcccat tcatggtgca tccattgtgg ctgccattct ccgg 960 aatttgttca atgactggac tattgagttg aaggcaatgg gcat tatg 1020 cgccaagaac ttttcgatgc tttatgttcc agaggcacac ctggcgattg gagtcacatt 1080 atcaaacaga tgtt tactttcact ggattgaatg cggaacaagt ttccttcatg 1140 actaaagagt tccatatata catgacatct gatgggagga ttagcatggc tggtctgagt 1200 tccaaaactg tcccacttct ggcggatgcg atacatgcag ctgtaacccg agttgtctaa 1260 GS cDNA atggctcatc tttcagatct cgttaatctc aatctctctg actccactca gaaaattatt 60 Represents 2 gctgaataca tatggattgg tggatcagga atggacgtca ggagcaaagc cagaacactt 120 sequence nucleotide tctggacctg ttgatgatcc ttcaaagctt cccaaatgga attatgatgg ttctagcaca 180 gctc ctggagaaga cagtgaagag atcctatatc ctcaagcaat tttcaaggat 240 sequences of GS agaa ggggcaacaa tatcttggtc atttgtgatt gttacacccc tgaa 300 genes for making cccattccaa caaacaaaag gcacagtgct gccaagattt tcagccaccc tgatgttgtt 360 gttgaggaac cctggtatgg tcttgagcaa gaatacacct aaaa agatatcaat 420 an expression tggcctcttg gatggcctct tggtggtttt cctggaccac agggaccata ctattgcgga 480 construct attggagctg gaaaggtctt tggacgcgat atcgttgact ctcattataa tctc 540 tatgctggga ttaacatcag tggtatcaat ggagaagtga tgcccggaca gtgggaattt 600 caagttggac cttcagttgg catttcagca gctgatgaat tgtgggcagc tcgttacatt 660 cttgagagga ttactgagat tgctggagtt gtggtctcat ttgaccccaa acctattccg 720 ggtgactgga ctgg caca aactacagca caaagtctat gaggaatgaa 780 ggaggctatg aagtcattaa gaaggcaatt gagaaccttg gactgaggca caaggagcat 840 gcat atggtgaagg caacgagcgt cgtctcactg gaagacacga aacagctgac 900 acat tcaaatgggg agttgcgaac cgtggtgcat ctattcgtgt gggaagagac 960 acggagagag aagggaaggg atacttcgag gataggaggc ctgcttcgaa tatggatcca 1020 ttcgtcgtga cttccatgat tgctgagacc ctat ccgagccttg a 1071 PEPCase atggcgtcga ccaaggctcc cggccccggc cacc actccatcga cgcgcagctc 60 Represents 3 cgtcagctgg tcccaggcaa ggtctccgag gacgacaagc tcatcgagta cgatgcgctg 120 cDNA nucleotide ctcgtcgacc gcttcctcaa catcctccag gacctccacg ggcccagcct tcgcgaattt 180 ce gtccaggagt gctacgaggt ctcagccgac tacgagggca aaggagacac gacgaagctg 240 sequences of ggcgagctcg gcgccaagct cacggggctg gcccccgccg acgccatcct cgtggcgagc 300 PEPCase genes for tccatcctgc acatgctcaa cctcgccaac ctggccgagg aggtgcagat cgcgcaccgc 360 cgccgcaaca gcaagctcaa gaaaggtggg ttcgccgacg agggctccgc caccaccgag 420 making an tccgacatcg aggagacgct caagcgcctc gtgtccgagg tcggcaagtc ccccgaggag 480 sion gtgttcgagg cgctcaagaa ccagaccgtc gtct tcaccgcgca tcctacgcag 540 tccgcccgcc gctcgctcct gcaaaaaaat gccaggatcc gaaattgtct gacccagctg 600 uct aatgccaagg acatcactga cgacgacaag caggagctcg atgaggctct gcagagagag 660 atccaagcag ccttcagaac cgatgaaatc aggagggcac cccc cgaa 720 atgcgctatg ggatgagcta catccatgag actgtatgga agggtgtgcc taagttcttg 780 cgccgtgtgg atacagccct gaagaatatc ggcatcaatg agcgccttcc ctacaatgtt 840 tctctcattc ggttctcttc gggt ggtgaccgcg atcc aagagttacc 900 ccggaggtga caagagatgt atgcttgctg gccagaatga tggctgcaaa cttgtacatc 960 gatcagattg aagagctgat gtttgagctc tctatgtggc gctgcaacga tcgt 1020 gttcgtgccg tcca cagttcgtct ggttccaaag ttaccaagta ttacatagaa 1080 ttctggaagc aaattcctcc aaacgagccc taccgggtga tactaggcca tgtaagggac 1140 aagctgtaca acacacgcga gcgtgctcgc catctgctgg gagt ttctgaaatt 1200 tcagcggaat cgtcatttac cagtatcgaa gagttccttg agccacttga gctgtgctac 1260 aaatcactgt gtgactgcgg cgacaaggcc atcgcggacg ggagcctcct ggacctcctg 1320 cgccaggtgt tcacgttcgg gctctccctg gtgaagctgg acatccggca ggagtcggag 1380 cggcacaccg acgtgatcga cgccatcacc acgcacctcg gcatcgggtc gtaccgcgag 1440 tggcccgagg acaagaggca ggagtggctg ctgtcggagc tgcgaggcaa gcgcccgctg 1500 ctgcccccgg ccca gaccgacgag gacg gcgc gttccacgtc 1560 ctcgcggagc tcccgcccga cagcttcggc ccctacatca tctccatggc cccc 1620 tcggacgtgc tcgccgtgga gctcctgcag cgcgagtgcg gcca gccgctgccc 1680 gtggtgccgc tgttcgagag gctggccgac tcgg cgcccgcgtc cgtggagcgc 1740 ctcttctcgg tggactggta catggaccgg atcaagggca agcagcaggt catggtcggc 1800 tactccgact ccggcaagga cgccggccgc ctgtccgcgg cgtggcagct gtacagggcg 1860 caggaggaga tggcgcaggt ggccaagcgc gtca agctcacctt gttccacggc 1920 cgcggaggca ccgtgggcag gggtggcggg cccacgcacc ttgccatcct gtcccagccg 1980 ccggacacca tcaacgggtc catccgtgtg acggtgcagg gcgaggtcat cgagttctgc 2040 ttcggggagg agcacctgtg cttccagact ctgcagcgct tcacggccgc cacgctggag 2100 cacggcatgc acccgccggt caag cccgagtggc gcaagctcat gatg 2160 gcggtcgtgg ccacggagga gtaccgctcc gtca aggaggcgcg cttcgtcgag 2220 tacttcagat cggctacacc ggagaccgag tacgggagga tgaacatcgg cagccggcca 2280 gccaagagga ggcccggcgg cggcatcacg accctgcgcg ccatcccctg gatcttctcg 2340 tggacccaga ccaggttcca cctccccgtg tggctgggag tcggcgccgc attcaagttc 2400 gccatcgaca aggacgtcag gaacttccag gtcctcaaag agatgtacaa cgagtggcca 2460 ttcttcaggg tcaccctgga cctgctggag atggttttcg ccaagggaga ccccggcatt 2520 ttgt atgacgagct gcttgtggcg gaagaactca agccctttgg gctc 2580 agggacaaat acgtggagac acagcagctt caga tcgctgggca caaggatatt 2640 cttgaaggcg atccattcct gaagcagggg ctggtgctgc ccta cacc 2700 ctgaacgtgt tccaggccta cacgctgaag cggataaggg accccaactt caaggtgacg 2760 ccccagccgc cgctgtccaa ggagttcgcc gacgagaaca agcccgccgg actggtcaag 2820 ctgaacccgg cgagcgagta cccgcccggc gaca tcct caccatgaag 2880 ggcatcgccg ccggcatgca gaacactggc tag 2913 CaMV 35S catggagtca caaa tagaggacct aacagaactc gccgtaaaga ctggcgaaca 60 Represents control 4 acag agtctcttac atga gaaa atcttcgtca acatggtgga 120 promoter sequence gcacgacaca cttgtctact ccaaaaatat caaagataca gtctcagaag accaaagggc 180 sequence aattgagact tttcaacaaa gggtaatatc cggaaacctc ctcggattcc attgcccagc 240 tatctgtcac tttattgtga tgga aaaggaaggt taca aatgccatca 300 ttgcgataaa ggaaaggcca tcgttgaaga tgcctctgcc ggtc ccaaagatgg 360 acccccaccc acgaggagca tcgtggaaaa agaagacgtt ccaaccacgt cttcaaagca 420 agtggattga tgtgatatct ccactgacgt aagggatgac gcacaatccc actatccttc 480 gcaagaccct tcctctatat aaggaagttc atttcatttg gagagaacac gggggact 538 nos cgttcaaaca tttggcaata aagtttctta agattgaatc ccgg tcttgcgatg 60 Represents 5 attatcatat aatttctgtt gaattacgtt aagcatgtaa taattaacat gtaatgcatg 120 (nopaline transcription acgttattta tgagatgggt ttttatgatt ccgc aattatacat ttaatacgcg 180 synthase) atagaaaaca aaatatagcg cgcaaactag ttat cgcgcgcggt gtcatctatg 240 terminator 3'UTR TTACTAGATCGGG sequence (polyAsignal) sequence hygromycin ctatttcttt gccctcggac tggg gcgtcggttt ccactatcgg cgagtacttc 60 Represents hpt 6 otransfe tacacagcca tcggtccaga cggccgcgct tctgcgggcg atttgtgtac gcccgacagt 120 gene (hygromycin rase cccggctccg gatcggacga ttgcgtcgca tcgaccctgc gcccaagctg catcatcgaa 180 attgccgtca accaagctct gatagagttg gtcaagacca atgcggagca tatacgcccg 240 phosphotransferas gagtcgtggc gatcctgcaa gctccggatg cctccgctcg aagtagcgcg tctgctgctc 300 e)for hygromycin catacaagcc aaccacggcc tccagaagaa gatgttggcg acctcgtatt gggaatcccc 360 cgcc tcgctccagt caatgaccgc tgttatgcgg ccattgtccg tcaggacatt 420 resistance gttggagccg aaatccgcgt gcacgaggtg ccggacttcg gggcagtcct cggcccaaag 480 catcagctca tcgagagcct gcgcgacgga cgcactgacg gtgtcgtcca tcacagtttg 540 ccagtgatac acatggggat cagcaatcgc gcatatgaaa tcacgccatg attg 600 accgattcct tgcggtccga atgggccgaa cccgctcgtc tggctaagat cggccgcagc 660 gatcgcatcc atagcctccg cgaccggttg tagaacagcg ggcagttcgg tttcaggcag 720 gtcttgcaac gtgacaccct gtgcacggcg gcaa taggtcaggc tctcgctaaa 780 [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by m [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam [Annotation] m MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam ctccccaatg lcaagcacxt ccggaatcgg gagcgcgcc gatgcaaagt gccgataaac 840 atct ttgtagaaac ca tcggcgca gctamacc cgcaggacat atcca cgccc 900 tcctacatcg aagctgaaag cacgagattc ttcgccctcc gagagctgca lcaggtcgga 960 gacgcmtcp, aacttttcga tcagaaactt ctcnacaaac gtcgcggtga gttcaggctt 1020 meat 1026 expression Represents an cassettes for expression construct Asp/\T, GS (or ression of and PEPCasc the genes Asp/W; 65 .C HC';::5 » coding - a atctccacbac.-.._ := L z andPEPCasr.’ Wmecamr-mg Spel l ccs.

CIOHEd cacnacagcatdccncttctccaacctccncttcmattccgtcttcaatcacctcgttcntg ctcccga agarcctatcctcngggt aactgtcgcttataacaaagatccaagtccagttaagctcaacttpgg u n dcr agttgglgcttaccga actgaggaaggaaaacctcttnttttgaatmagtgangcgngttnaacagcaact C0" "0| 0“ ca taaa tgacg tgtca aaggaatatattccgatcgttgggctmctga tttlaataaattgamﬂct aagcttatttttggggctga tgctattca agacaacagggrraccactgttcaatgcttgtctgga ac Camv 3 SS tggttctttaagagttgggggtgaamttggctaaacactatcaccaacggactatatacﬂgccaa caccaa er ctlggngcaatcacccgaamttttcaacttagcapgcttgtctgtca aaacataccgctactatgctccagc ( ) a n d N as aacacgaggacttgactttcaaggacttctggaaga ccttggttctgctcca tctgga tctattgttttgctaca ‘ tgcatgcgcacataaccccactrgmmgatccaacccnnagcaarmnagcagattaggcagcta ata an terminator atcaaa agcmgttacctttctttgaca mgcttatca gggttttgctagtggaagtctagamcagatﬁccca a cctgttcgtttgtttgttgctgatggaggcgaattgctggtagcacaaagcta mcaaa gaatctngntct tt (a) in atggggaacgtgttggcgccttaagcattgtctgcaagtcagctgatgttgcaagcagggttgagagccanc PCAM BIA tgaagcmgtgattaggccca tgtactcaagtcctcccattcatggtgcatcca ttgtggctgccattctca ag 1302 gaccggaamgttcaatnactggactattgagttgaaggcaatggctgatcgcatcatcagtatgcgccaag aacttttcgamcntatgtxccagamcacacctmcnatmgagtcacattatcaaacagattggaatgttt actltcactggattgaatgcﬁgaacaagtttcc‘ttcatgactaaagagttccatatatacatgacatctgatgg gaggattagcatggctggrctgagttccaaaactgtcccacttctggcngamcgatacatgcanctgtaa'or: gagttgtdaagg? Mtgaat:ggtgaccagctcgaamccccgatggguaaaca-mggcaataaagsnmaazanggarc gtgggxaﬁtggtﬁtcgtaxaazmmgmacgggaggajgtaataattaacaszag meal‘zacagtatttaxaawmmpjj.smzaﬁcmsgmmmmzmsm a__caaaa_§a!a c caaacta ﬁaﬁﬁggggggggggmcatctajﬁ .tactagalcgggaatta3%atngcgéatgctagagcagcttgagcttggatcagattgtcgtttccc nccncagtttagcttWWW Wm:mctcatctttca GS coding sequence —" gatncgnaarctca atctctctgactccactcagaaaattattgctga atacatatgganggmgarcapg aatggacgtcaggagcaaagccagaacacmctngaccmttgatgatccttcaaagcltcccaaatggaa Ktatgatggttctagcacaggawagctcctggagaagacagtga agagatcctatatcctcaagcaaltnc aangatccancagaaggggcaacaatatcttggtcatttgtgattgttacaccccagctggtgaacccattc caaca a acaaaa gtgctgcca agattttcagccaccctga tgttgttgttgaggaaccctggtatg mmgagcaanaatacaccltmtgcaaaaagatatcaattggcctcttggatggcctcttggtggttttcct ggaccacaggga cca ta ciattgcgnaattgga gctggaaaggtcmggacgcgatatcgttgactctcatt ataaggcatgtctctatgctgggattaacatcagtggtatcaatggagaaglgatgcccggacagtgggaat ttgga ccttcagttggcatttcagcagctgatgaa ttgtggncagctcgttacattcttgagagga tt actgagattgctpgagttgtggtctcantgaccccaaacctattccgggtgactgnaatggtgctgga gctc acacaaactacagcacaaagtctarga gga atgaaggaggctatgaagtcattaaga aggcaangagaa ccttggactgaggca caagga gcatattgcagcatatggtgaaggcaacgagentcgtctcactgga agac /‘ [Annotation] m None set by amandam ation] amandam MigrationNone set by amandam [Annotation] m Unmarked set by amandam [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] m None set by amandam [Annotation] amandam ionNone set by amandam [Annotation] amandam Unmarked set by amandam ' ‘h'l gacacggagagagaagggaaggga tacttcgagaztaggaggcctgcttcgaatatggatccattcgtcgt gacttccatgattgctgagaccactatcctatccgagccttg- : - as Ianaxcatat aa ca aaca aa cat ac tamasg agataggggggggggaggccggcaattatacamaataggggatagaaaacaagataxa. aggggcaaacta catct acta an: aattaaactatcagt gmgacaggatatattggcﬁigcgcgcaaatggcgaa agcagqtrgagcttggatcagattgtcg -..| ;-.-;v!.‘;-_ aLQ.‘1".','..'-, ".. El--'.:- . a z : - - - ‘ _ . . . -. mmcttgacca PEPCan: cod 3 sequznce ——> tgg-tatggcgtcgaccaaggctcccggccccggcgagaagcaccactccatqgaqzcgcagctc cgtcagctggtcccaggcaaggtctccgaggacgacaagctca(cgagtacgatgcgctgctcgtcgaccgc ttcctcaacatcctccaggacctccacgggcccagccttcgcgaatttgtccaggagtgctacgaggtctcag ‘ ccgacta cgagggcaa aggagacacgacgaagctgggcgagctcggcgccaagctcacggggctggcccc cgccgacgccatcctcgtggcgagctccazcctgcacalgctcaacczcgccaacctggccgaggaggtgca ga tcgcgca ccgccgccgcaacagca agctcaagaaaggtgggttcgccgacgagggctccgcca ccacc gagtccg‘acatcgaggagacgctcaagcgcctcgtgtccgaggtcggcaagtcccccgaggaggtgttcga ggcgctcaagaaccagaccgtcgacctcgtcttcaccgcgcatcctacgcagtccgcccgccgctcgctcctg caaaaaaatgccaggatccgaaattgtctgacccagctgaatgccaaggacatcactgacgacgacaagc tcgatgaggctctgcagagagagatccaagcagccttcagaaccgatgaaatcaggagggcac aacccaccccgca ggccgaaatgcgctatgggatgagctacatccatgagactgtatggaagggtg‘gcct an gttcttgcgccgtgtggatacagccctgaagaatatcggcatcaatgagcgccttccctacaatgtttctct cattcggttctcttcttggatgggtggtgaccgcgaxggaaatccaagagtta ccccggaggtgacaagaga tgtatgcttgctggccagaatgatggctgca aacttgtacatcgatcagangaagagctgatgtttgagent ctatgtggcgctgcaacga(gagcltcgtgttcgtgccgaagagdccacagttcgtctggttccaaagttacc aagtattacatagaattckggaagcaaattcctccaaacgagccctaccgggtgatactaggocacgtaagg gacaagctgtacaacacacgcgagcgtgctcgccatctgctggcttctggagtttctgaaamcagcggaat cgtcamaccagtatcga agagnccttgagccacttgagctgtgctacaaa tcactgtgtgactgcggcga ca aggccatcgcggacgggagcctcctggacctcctgcgccaggtgttcacgttcgggctctccctggtgaa gctggacatccggcaggagtcggagcggcacaccgacgtgatcgacgccatcaccacgcacctcggcatcg ggtcgtaccgcgagtggcccgaggacaagaggcaggagtggctgctgtcggagctgcgaggcaagcgccc gctgctgcccccggaccttccccagaccgacgagatcgccgacgtcatcggcgcgttccacgtéctcgcgga gctcccgcccgacagcttcggcccctacatcatctccatggcgacggccccctcggacgtgctcgccgtggag ctcctg cagcgcgagtgcggcgtgcgccagccgctgcccgtggtgccgctgttcgagasgctggccgacctg cagtcggcgcccgcgtccgtggagcgcctmctcggtggactggtacatggaccggatcaagggca agcag caggtcatggtcggctactccgactccggcaaggacgccggccgcctgtcc gcggcgtggcagctgtacagggcgca ggaggagatggcgcaggtggccaagcgctacggcgtcaagctca ccttgttccacggccgcggaggcaccgtgggcaggggtggcgggcccacgcaccttgccatcctgtcccagc cgccgga cacca ggtccatccgtgtgacggtgcagggcgaggtcatcgagttctgcncggggagg agcacctgtgcttccagactctgcagcgcncacggccgccacgctggagcacggcatgcacccgccggtct ctcccaagcccgagtggcgcaagctcatggacgagalggcggtcgtggccacggaggagtaccgctccmc gtcgtcaaggaggcgcgcttcgtcgagtacttcagatcggctacaccggagaccgagtacgggaggatgaa catcggcagccggccagccaagaggaggcccggcggcggcatcacgaccctgcgcgccatcccctggatct ggacccagaccaggttccacctccccgtgtggctgggagtcggcgccgcancaagncgccatcga ca agga cgtcaggaacttccaggtcctcaa agagatgtacaacgagtggccattcttcagggtcaccctgga cctgctggagatggttttcgccaagggagaccccggcattgccggcttgtatgacgagctgcttgtggcggaa gaaacaagcocmgggaagcagctcagggacaaa‘acgtggagacacagcagcttctcctccagatcgct gggcacaaggatattcttgaaggcgatccattcctgaagcaggggctggtgctgcgcaacccctacatcacc accctgaacgtgttccaggcctacacgctgaagcggata agggaccccaacttca aggtgacgccccagcc gccgctgtccaaggagttcgccgacgagaacaagcccgccggactggtcaagctgaacccgg cgagcgagtacccgcccggcctggaagacacgctcatcctcaccatgaagggcatcgccgccggcatgca g abut! Pmn aacactggctam gaattggtgaccagctcgaatttccccgatgmcmcmigg m:_a_aa.mt__s_maaagpaxcmmmmmmm ca wannaaca aa ca. a ante, mm a w anatamnaatacc - -.-_aaaa_caaaa.t,axac 1:.:-. 1: 1- :_:~: 2: : [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam [Annotation] m MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] m None set by amandam [Annotation] m MigrationNone set by amandam [Annotation] amandam Unmarked set by m momma: Forward primer for ampliﬁcation of ‘ tobacco GS coding 65”,». F S'- TGCCATGGCTCA‘i‘CTITCGGATCTCG'iT -3' B sequence, including restriction site for enzyme Ncol.

Reverse primer for ampliﬁcation of S'- GGGTGACCTCAAGGCTCGGATAGGATAGTG ~3' tobacco GS coding sequence, including restriction site for enzyme BstEli.

Forward primer for ampliﬁcation of S’- CATAGATCTTATGGCTTCTCACGACAGCATCT -3’ Soyabean Asp/ﬂ” coding sequence, including restriction site for enzyme Bglil.

Reverse primer for amplification of Soyabean MpA'i‘ As pAT pm]. R ACGTGTTAGACAACTCGGGTTACAGCTG-B' 11 coding sequence, including restriction site for enzyme PmIi.

Forward primer for amplification of PEPCase 5'- ATAGATCTTATGGCGTCGACCAAGGCTCCG -3‘ maize PEPGase .59“. F coding sequence, ing restriction site for enzyme Bglll. _W—_—_ Reverse primer for ampliﬁcation of PEPCase maize PEPCase ’-AGACTAGTGCCAGTGTTCTGCATGCCGGCGG3’ 13 59:! R . coding sequence, Including ction site for enzyme Spei.

Forward primer for ' ampliﬁcation of 355 5,”. F S‘-GGACTAGTAATGGCGAATGCTAGAGCAGCTTGAG —3' 14 CaMV 355 promoter ‘ sequence, including [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam ed set by amandam [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam [Annotation] m MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam restriction site for enzyme .5"ch Reverse primer for ‘-GC(‘ACGTGTCCTCAGCTGGCGCGCCCGCCA- amplification of N05 Nos'i'Am, A'I‘A'l‘A'lCCI‘G'l’CAAACACTGATAGT -3' terminator sequence, —____..A._.___._.4 ing ction site for enzyme Ascl, 8val,Pm/i w « __- ...

Reverse prirncrio'r'ﬂ‘ amplification of Nos S’—GGAC’I'AGTTTAATTCCCGA'I'Cl‘AGTAACA’l'AGA'l'GJ‘ terminator seq uence. 16 ing restriction site for enzyme Spel.

Forvmw— ”W" ampliﬁcation of (.an 355 promoter '-A'l'C'l'GGCGCGCCAATGGCGAATGCTAGAGCAGC‘TTGAG 3‘ 17 sequence, including restriction site for enzyme Ascl. e primer for qmplifica tion of maize PEPCase PEPCase “Va ’ - GTGCCTCAGCQTAGCCAGTGTTCTGCATGCCGG -3' coding sequence, 18 including restriction site for enzyme Bval. __ '- Forward primer raF'""' ampliflcatlon of hygromycin hpt F S' - GAGGGCGAAGAATCTCG‘I'GC -3’ 19 phosphotransferase for screening transgenic plants.

Reverse primer for ampliﬁcation of ycin ' - GATGTTGGCGACCl'CGTATTGG -3' 20 phosphotransferase for screening transgenic plants.

Forward primer for PEPCase Exp 5' — ACG'i'CAGGAACITCCAGGTC -3‘ maize PEPCaSC, used F for RT-PCR based evaluation of [Annotation] amandam None set by amandam ation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam ation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by m [Annotation] amandam None set by amandam [Annotation] amandam ionNone set by amandam [Annotation] amandam ed set by amandam PEPCase transgene expression.

Reverse primer for maize PEPCase, used PEPCase Exp for RT-PCR based S’ — CTI'GTTCTCGTCGGCGAAC -3' 22 R evaluation of PEPCase transgene expression.

Forward primer for tobacco GS, used for RT-PCR based ' - ACTTTCTGGACCFGTTGAT -3' 23 evaluation of GS tra nsgene expression.

Reverse primer for tobacco GS, used for RT~PCR based ' - ACTGTGCCTT -3' 24 evaluation of GS transgene expression. ﬁorward primer for soyabean AspAl' , used for RT-PCR Asp/\T Exp F 5' - ATGGCTTCTCACGACAGCATC -3‘ 25 based evaluation of GS transgene expression. e primer for soyabean ASpAT, used for RT-PCR ASpAT Exp R 5' - TTGCGTGACACGTCATTTATGAGT -3' 26 based evaluation of GS transgene expression.

Forward primer for ISSrRNA, used as internal control for S'-CACAATGATAGGMGAGCCGAC-3' 265 r- RT-PCR based 27 evaluation of transgene expression.

Reverse primer for 265 R S’WGGGAACGGGCTTGGCAGMTC-S’ ' ZSSrRNA, used as 28 internal control for [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam ation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] m ed set by amandam RT-PCR baséd evaluation of transgene expression MAS!lDSISASPTSASDSVFNHLVRAPEDPILGVTVAYNKDPSPVK|.NI.GVGAYRTEEG Represents Proteins KPLVLNWRRVE of AspAT genes QQLINDVSRNKEYIPNGLADFNKLSAKLIFGADSPAIQDNRVTTVQCLSGTGSLRVGG EFlAKHYHQRT IYLPTPTWGNHEKVFNLAGLSVKTYRYYAPATRGLDFQGLLEDLGSAPSGSIVLLIMCA AspATPr HNPTGVDPTLE QWEOJRQLIRSKALLPFFDSAYQGFASGSLDADAQPVRLFVADGGELLVAQSYAKNLG LYGERVGALSIV CKSADVASRVESQLKLVIRPMYSSPPIHGASIVA/\ILKDRNLFNDWTIELKAMADRIISM RQELFDALG WSHIIKQIGMFTFTGLNAEQVSFMTKEHIIYMTSDGRISMAGLSSKTVPLLA DAIHAAVTRW _‘ _ __ MAIILSDLVNINLSDSTQKIIAEYIWIGGSGMDVRSKARTLSGPVDDPSKLPKWNYDG Represents Proteins SSTGQAPGEDSEE of 65 genes ILYPQAIFKDPFRRGNNILVICDCYTPAGEPIPTNKRIiSMKlFSHPDWVEEPWYGLEQ EYTLLQKDIN WPLGWPIGGFPGPQGPYYCGIGAGKVFGRDIVDSHYKACLYAGINISGINGEVMPGQ GSPr WEFQVGPSVGISA 30 ARYILERITEIAGVVVSF DPKPIPGDWNGAGAI ITNYSTKSMRNEGGYEVI KK AIENLGLRHKEH IAAVGEGNERRLTGRHETADINTFKWGVANRGASI RVGRDTEREGKGYFEDRRPASN MDPFWI'SMIAET TILSEP PGPGEKHHSIDAQLRQLVPGKVSEDDKLIEYDALUEIFLNILQDLI (GPSIRE Represents Proteins FVQEOIEVSAD of PEPCase genes TTKLGELGAKLTGLAPADAILVASSILI lMlN LANLAEEVQIAHRRRNSKLKKG GFADEGSATI'E SDIEETLKRLVSEVG KSPEEVFEALKNQTVDLVFTN(PTQSARRSLLQKNARIRNCLTQL NAKDITDDDK QELDEALQREIQAAFRTDEIRRAQPTPQAEMRYG MSYIHETVWKGVPKFLRRVDTAL ERLPVNV SLIRFSSWMGGDROGNPRVTPEVTRDVCLLARMMAANLYIDQIEELMFELSMWRCN DELRVRAEELHSSS GSKVTKYYIEFWKQIPPNEPYRVILGIWROKLYNTRERARHLLASGVSEISAESSFTSIEE FLEPLELCY KSLCDCGDKAIADGSLLDLLRQVFTFGLSLVKLDIRQESERHTDVIDAIT‘I’HLGIGSYRE PEPCdsePr QEWL LSELRG KRPLLPPDLPQTDE IADVIGAFHVLAELPPDSFG PYIISMATAPSDVLAVELLQR ECGVRQPLP VVPLFERLADLOSAPASVERLFSVDWYM DRIKGKQQVMVGYSDSGKDAGRLSAAW QLYRAQEEMAQVAKR YGVKLTLFHGRGGTVGRGGGPTHLAILSQPPDTINGSIRVTVQGEVI EFCFGEEHLCFQ TLQRFTAATLE HGMHPPVSPKPEWRKLMDEMAWATEEYRSVWKEARFVEYFRSATPETEYGRMNI GSRPAKRRPGGGIT TLRAIPWIFSWTQTRFI ILPVWLGVGAAFKFAIDKDVRNFQVLKEMVNEWPFFRVTLD LLEMVFAKGDPGI AGLYDELLVAEELKPFGKQLR DKYVETQQLLLOJAGHKDILEGDPFLKQGLVLRNPYITI' LNVFQAYTLK RIRDPNFKVTPQPPLSKEFADEN KPAGLVKLNPASEYPPGLEDTLI LTMKGIAAGMQN [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by m EXAMPLE 1 Ampliﬁcation and cloning ofAspA T gene Nucleotide sequence encoding soyabean cytosolic AspAT gene (SEQ ID NO: 1) was obtained from the NCBI database of nucleotide ces (GenBank Accession No. AFO34210.1; (hgpzllwwwncbinlm.nih.gov/nuccore/AF034210.l) RNA from soyabean plant was ed using iRIS Plant RNA Kit (Ghawana et al., US Patent no 0344NF2004/LN). cDNA was synthesized using total RNA preparations (2 pg) in the presence of 1 pg oligo(dT).2.|3 and 400 U of reverse transcriptase Superscript II (Invitrogen) aﬁer digesting with 2 U DNase I (ampliﬁcation grade, ogen, USA) following the manufacturer’s instructions. The full coding region of AspAT was then ed from soyabean cDNA using primers AspAT Es!" F (SEQ ID NO: 10) and AspAT pm“ R (SEQ ID NO: 11) such that restriction sites Bglll (AGATQI) and Pmll (QACGTG) is incorporated in the coding sequence for AspA T. Qiagen High Fidelity Taq rase enzyme was used for the PCR using the following conditions: initial denaturating at 94 °C for 3 minutes , 30 cycles of 94 °C for 30 seconds, annealing at 59 °C for 305econds, extension at 72 °C for 1 minute 20 seconds, with a ﬁnal extension of 72 °C for 7 minutes. The ampliﬁcation product was cloned in to pGEM-T easy vector (Promega, USA). Plasmid from the positive clones and pCAMBIA 1302 plasmid were digested with Bglll and Pmll and digested products isolated from an agarose gel electrophoresis were ligated and ormed in to E. coli DHSa cells which were ed from Takara Bio Company, Japan (Cat. No. 9057). Plasmid from the positive colonies were sequenced to verify the in frame cloning of the AspAT coding sequence placed between CaMV 3SS promoter (SEQ ID NO: 4) and Nos terminator (SEQ ID NO: 5) of pCAMBIAI302 and ing vector was designated as AspATzzpCAMBIAl302. ' EXAMPLE 2 Ampliﬁcation and cloning of GS gene Nucleotide sequence encoding tobacco cytosolic GS gene (SEQ ID NO: 2) was obtained from the NCBI se of nucleotide sequences (GenBank Accession No. X95932.1; ihttp://www.ncbi.nlm.nih.gov/nuccore/X95932.l) .RNA from tobacco plant was isolated using iRIS Plant RNA Kit (Ghawana et al., US Patent no 0344NF2004/lN). cDNA was synthesized using total RNA preparations (2 pg) in the presence of 1 pg dT)lz..3 and 400 U of e riptase Superscript II (Invitrogen) aﬁer digesting with 2 U DNase I ﬁcation grade, Invitrogen, USA) following the manufacturer‘s instructions.

[Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam ed set by amandam [Annotation] amandam None set by m [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam The full coding region of GS was ampliﬁed from tobacco cDNA using primers GSA/m. F with restriction sites NcoI (CCATGG) (SEQ ID NO: 8) and 05mm R with restriction sites for BstEII (GGTGACC) (SEQ ID NO: 9). 68”“. F primers was modiﬁed so as to eliminate the BgIII site by replacement of ‘A’ nucleotide by ‘G ‘ at posiu'on 15.

Qiagen High ty Taq polymerase enzyme was used for the PCR using the following ions: initial denaturating at 94 °C for 3 minutes , 30 cycles of 94 °C for 30 s, annealing at 59 °C for 305econds, extension at 72 °C for e 10 seconds, with a ﬁnal extension of 72 °C for 7 minutes. The cation product was cloned in to pGEM-T easy vector (Promega, USA). Plasmids from the positive colonies and binary vector pCAMBIA 1302 were ed with Neal and BstEII and digested product isolated from an agarose gel electrophoresis were ligated such that GS is placed downstream of CaMV 3SS promoter of pCAMBIA vector. The ligation product was transformed in to E. cali DHSa cells and transformants were sequenced to verify the in frame claning of the GS coding sequence and the resulting vector was designated as GS::pCAMBIAl302.

EXAMPLE 3 Ampliﬁcation and cloning of maize e gene Nucleotide sequence encoding maize PEPCase gene (SEQ ID NO: 3) was obtained from the NCBI database of nucleotide sequences (NCBI Reference Sequence: NM_OOIlll948.I; (http://www.ncbi.nIm.nih.gov/nuccore/NM 001111948.” RNA ﬁ'om maize plant was isolated using iRIS Plant RNA Kit (Ghawana et al., US Patent no 0344NF2004/IN). cDNA was synthesized using total RNA preparations (2 pg) in the presence of 1 ug oligo(dT).2..3 and 400 U of reverse transcriptase Superscript II (Invitrogen) after ing with 2 U DNase I (ampliﬁcation grade, Invitrogen, USA) following the manufacturer’s ctions.

The full coding region of PEPCase was ed ﬁom maize cDNA using primers PEPCase 331" F with restriction sites for BglII (AGATCI ) (SEQ ID NO: 12) and PEPCaSe 5,,“ R with restricition sites for SpeI (ACTAGI) (SEQ ID NO: 13). Qiagen High Fidelity Taq polymerase enzyme supplemented with Q-soiution (facilitating ampliﬁcation of GC-rich templates) was used for PCR using the following conditions: initial rating at 94 °C for 3 minutes, 32 cycles of 94 °C for 30 seconds, annealing at 58 “C for 30 seconds, extension at 72 °C for 3 minute, with a ﬁnal extension of 72 °C for 7 minutes. The cation product was cloned in to pGEM-T easy vector ga, USA). Plasmid from the positive clones and pCAMBIA 1302 plasmids were digested with BgIII and SpeI and ed product isolated [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by m [Annotation] amandam None set by amandam [Annotation] amandam ionNone set by amandam [Annotation] amandam Unmarked set by amandam from an agarose gel ophoresis were ligated and then transformed in to E. coli DHSo cells. Transformants were ced to verify the in frame cloning of the PEPCase coding sequence and resulting vector was ated as PEPCasezzpCAMBlA 1302.

EXAMPLE 4 Assembly of expression cassettes for Asp/1T, GS and e in single pCAMBIA 1302 vector (generous g1]? from “Centre for Application of Molecular Biology to International Agriculture ", A ustralia) A stepwise method for ampliﬁcation and integration of expression tes each for AspA T, GS and e in to single plant transformation vector pCAMBIA 1302 is described as follows: GS expression cassette sing CaMV3SS promoter, downstream cloned GS and nopaline synthase (hereinaﬂer, referred as “Nos”) terminator was ampliﬁed from 08:: pCAMBlA 1302 vector ( Example 2 ), using s 358 5,,“ F( SEQ ID NO: l4) and NosT Am, BM'PMHR (SEQ ID NO: 15). The primers were designed to incorporate the Spel (ACTAGT) in the forward primer and Ascl (GGCGCGCC ), Bva'I (CCTCAGC)and Pmll (CACGTG) in reverse primer to tate the ning of OS expression cas3ette in to SpeI and PmlI sites ofpCAMBlA 1302 vector as well as to create the additional restriction sites ( Ascl 3’ end in , BvaI ) at the vector backbone . Qiagen High Fidelity Taq polymerase enzyme was used for the PCR using the following conditions: initial denaturating at 94 °C for 3 minutes , 30 cycles of 94 °C for 30 seconds, annealing at 59 °C for 30 seconds, extension at 72 °C for 2 minutes, with a ﬁnal extension of 72 °C for 7 minutes. The ampliﬁcation product was cloned in to pGEM-T easy vector (Promega, USA). Plasmids from the positive clones was digested with 81221 and Pmll, and the digested product was then ed from an agarose gel electrophoresis and ligated in to Spel and PmlI sites of pCAMBIA 1302 vector. The ligation t was transformed in to E. coli DHSa cells and transformants were veriﬁed by sequencing of plasmid.

AspAT coding sequence along with 3’Nos terminator sequence was ampliﬁed from AspATz: pCAMBIA I302 vector (Example 1 ) using primers AspAT fig/ll F (SEQ ID NO: 10) and NosT 5-,”. (SEQ ID NO: 16) with restriction sites for BgIII (AGATCT) and SpeI (ACTAGjl) respectively.

Qiagen High Fidelity Taq polymerase enzyme was used for the PCR using the following conditions: initial denaturation at 94 °C for 3 s °C for 30 seconds, , 30 cycles of 94 [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by m [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam ing at 59 °C for 30 seconds, extension at 72 °C for 2 minutes, with a ﬁnal extension of 72 °C for 7 minutes. The ampliﬁcation product was cloned in to pGEM—T easy vector (Promega, USA). Plasmids from the positive clones upon ion with BgIII and SpeI , cloned downstream of CaMV 35$ promoter of destination pCAMBIA 1302 ( previously cloned with GS expression cassette). The ligation product was then transformed in to E. coli DHSa cells and transformants were sequenced to verify the in frame cloning of the AspAT coding sequence.

CM 358 promoter along with the downstream cloned e gene from PEPCaSe:: pCAMBIA 1302 vector (example 3) was ampliﬁed with the primers 358 A“! F (SEQ ID NO: 17) having restriction site for AscI GCC) and PEPCase awe. R (SEQ ID NO: 18) having ction site for Bb VCI (CCTCAGC). ‘ Qiagen High Fidelity Taq polymerase enzyme was used for the PCR using the ing ions: initial denaturation at 94 °C for 3 minutes , 30 cycles of 94 °C for 30 seconds, annealing at 60 °C for 30 seconds, extension at 72 °C for 4 minutes, with a ﬁnal extension of 72 °C for 7 minutes. The cation product was cloned in to pGEM-T easy vector (Promega, USA), plasmid from the positive clones was digested with AscI (GGCGCGCC) and BbVCI (CCTCAGC) and digested product isolated from an agarose gel electrophoresis ligated upstream of Nos terminator sequence of destination A. 1302 previously cloned with GS and AspAT expression cassettes. The ligation product was transformed in to E. coli DHSa cells and transformants ced to verify the in frame cloning of the PEPCase coding sequence. Resultant plant expression vector was designated as AspAT + GS + PEPCase for co-overexpression of AspAT, GS and PEPcase. A hygromycin resistance gene (SEQ ID No.6) was included as a selectable marker for screening transgenic plants.

Schematic m of expression uct is shown in Figurel, represented by SEQ ID NO. 7 for plant transformation such that the transgenic plant produces higher amount of proteins ented by SED ID NO. 29. 30, and 31.

EXAMPLE 5 g of enic Arabidopsis plants co« over expressing genes AspAT. GS and PEPCase Generation of plant expression vector (AspAT + GS + PEPCase) Briefly, the plant expression vector was constructed as follows: cDNA sequences encoding soybean AspAT gene (SEQ ID NO: 1), tobacco cytosolic GS gene (SEQ ID NO: 2) and maize PEPCase gene (SEQ ID NO: 3), were ﬁrst independently cloned in to pCAMBIA 1302 [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam ation] amandam MigrationNone set by amandam ation] amandam Unmarked set by amandam vector. The elements for expression te for AspAT, GS and PEPCase were then ampliﬁed and assembled in to destination pCAMBlAl302 such that genes AspAT, GS and PEPCase were controlled by independent CaMV 35$ promoter and Nos transcriptional terminator.

Agrobacterium ed plant transformation: AspAT + GS + PEPCase were transferred to Agrobacterium tumefaciens strain GV3101 with ATCC number Agrobacterium tumefaciens (GV3101 (pMP90RK) (C58 derivative) ATCC® Number: 33970 Reference: Hayashi H, Czaja I, Lubenow H, Schell J ,Walden R. 1992 using standard triparental mating method.

Brieﬂy, E. coli DHSa cells harboring the recombinant uct AspAT + G8 + e and those harboring helper plasmid pRK2013 were Cultured overnight at 37°C. Agrobaterium strain GV3101 grown at 28°C for 48hrs. All the three cultures were then pelleted, washed, and mixed, followed by plating on YEM (Yeast Extract Mannitol) plates supplemented with the antibiotics kanamycin (SOuyml) and cin (50ug/ml). Antibiotic resistant colonies were veriﬁed by colony PCR to assure the transformation of Agrobacterium with the recombinant construct AspAT + GS + PEPCase.

Arabidopsis seeds of the Columbia ecolype were generous giﬁ by Dr. Christine H Foyer of; IACR-Rothamsted, Harpenden, UK.

Arabidopsis plants were transformed with Agrobacteria harboring ‘ + GS + PEPCase using vaCuum inﬁltration method. Brieﬂy, liquid S-ml cultures were established from single transformed clerium colony and grown in YEM medium supplemented with SOug/ml kanamycin, l rifampicin at 28°C up to the late logarithmic phase. Next, 1 ml of bacterial sion Was diluted with 100 ml of YEB culture medium mented with the same antibiotics. The culture was grown ght until their l density reached 1.2—1.8 at 600 nm. The bacteria were spinned for 20 min at 2000 g at room temperature and suspended in a solution for inﬁltration containing half strength MS (Murashige and Skoog) medium with 2% sucrose, 0.05% MES (Sigma,) and 0.01% of Silwet L-77 (Lehle Seeds, United States). Arabidopsis inﬂorescences were dipped in bacterial suspension and ated under vacuum for 10 minutes. Plants were then transferred to growth chamber and grown under controlled long day conditions (16-h light at 22—23°C and 8-h darkness at 20°C) for seed set.

Selection of Primary transformant To transgenic Arabidopsis plant: Seeds from transformed plants were surface sterilized by ion in 70% (v/v) ethanol for 2 min, [Annotation] amandam None set by amandam [Annotation] m MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam followed by immersion in 10% (v/v) sodium hypochlorite solution. Seeds were then washed four times with sterile distilled water and sown onto 1% agar containing MS medium supplemented with hygromycin B at a cancentration of 20 pg ml'I (Sigma # . Seeds were then ﬁed for 2 days in the dark at 4°C. After stratiﬁcation plates were transferred to a growth r with 16 h light and 8 h dark cycle for ation. After 14.days, hygromycin resistant seedlings were selected as putative primary transformants (To) sand transferred to pots ning vermiculite, perlite and cocopeat mix (1:111) and grown to ty under cantrolled iou of light, temperature and humidity for growth and seed set.

Raising T1 and '1'; generation AspAT + GS + PEPCase transgenic : Seeds harvested from To enic plants were germinated on MS + hygromycin B (at a cancentration of 20ug ml") plates and transgenic lines ting a segregation ratio of 3:1 (scored by their sensitivity to hygromycin B) were ed to raise T1 generation of transgenic plants . Homozygous transgenic plants were obtained in the T2 generation and evaluated for different physiological and biochemical parameters in comparison to wild control plants.

EXAMPLE6 Analysis of the Genomic DNA from Arabidopsis thaliana plants ormed with AspAT + GS + PEPCase opsis plants from two independent transgenic lines transformed with AspAT + GS + PEPCase were selected to verify the insertion of transgenes in to plant genome. The genomic DNA was isolated using DNeasy Plant mini kit (QIAGEN Co.). PCR was carried out by using the isolated DNA as template with primers hpt F (SEQ ID NO: 19) and hp! R (SEQ ID NO: 20) annealing to the hygromycin phosphtransferaes (hp!) gene (SEQ ID NO: 6) (plant selection marker from pCAMBIA 1302 vector).

PCR Cycling conditions deﬁned by initial denaturation at 94 °C for 3 minutes , 28' cycles of 94 °C for 30 seconds, annealing at 58 “C for 30 secOnds, extension at 72 °C for 1 , * with a ﬁnal extensiOn of 72 “C for 7 minutes.

The result is shown in Figure 2A, in which WT represents the wild and L1 and L2 represent two different transgenic lines. The ampliﬁcation of hpt gene was observed only with transgenic conﬁrming insertion of AspAT+GS+PEPCase in to Arabidopsis plants.

[Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] m Unmarked set by amandam [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam ation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam EXAMPLE? Evaluation of AspAT + GS + PEPCase transgenics by reverse riptase - rase chain reaction (RT-PCR) RNA analysis of transformants was done to conﬁrm the expression of Asp/1T, GS and e. Total RNA was isolated from leaf and root of transgenic plants using iRIS Plant RNA Kit (Ghawana er al., US Patent no 0344NF2004/IN). cDNA was synthesized using total RNA preparations (2 pg) in the presence of 1 pg oligo(dT).2.lg and 400 U of reverse transcriptase Superscript II (lnvitrogen) aﬁer digesting with 2 U DNase I (ampliﬁcation grade, lnvitrogen, USA) following the manufacturer’s instructions). Expression of transgenes was evaluated using gene speciﬁc primer for AspAT, GS and PEPCase. designated as PEPCase Exp F (SEQ ID NO: 21), PEPCase Exp R (SEQ ID NO: 22), GS Exp F (SEQ ID NO: 23), GS Exp R (SEQ ID NO: 24), AspAT Exp F (SEQ ID NO: 25) and ASPAT Epr (SEQ ID NO: 26). As a positive l for RT-PCR, 26$ rRNA was ampliﬁed using primers 268 F (SEQ ID NO: 27) and 268 R (SEQ ID NO: 28).

The results of analyses are shown in Figure ZB, in which WT represents wild and L1 and L2 represent two transgenic lines. The ampliﬁcation of RT—PCR products were observed only in trangenics conﬁrming the expression of introduced genes.

Enzymatic assays from wild type and AspAT + GS + PEPCasetransgenic Arabidopsis plants tic assays were med with AspAT + GS + PEPCase transgenic and wild plants as follows: PEPCase Activity Measurement: Frozen leaf samples (200 mg) ground with a mortar and pestle in lml of extraction buffer ning 50 mM Tris-Cl buffer (pH 7.5), 1.0 mM MgCl2, .0 mM DTT, 1.0 mM PMSF, 2% (w/v) PVPP, 10% (v/v) glycerol and 0.1% (v/v) Triton X— 100. The extract was centrifuged at 12,000 g for 10 min at 4 °C and the supernatant was used for the determination of enzyme activity. PEPCase was assayed Spectrophotometrically at 340 nm in the presence of excess MDH and lactate dehydrogenase (Ashton et al. 1990). The reaction mixture contained 50 mM Tris-Cl (pH 8.0), 5 mM MgC12, 5 mM DTT, 1 mM NaHCO;, 5 mM glucose—6-phosphate, 0.2 mM NADH, 2 units MDH, 0.1 units lactate dehydrogenase and crude extract. The on was ted by the addition of 5 mM PEP.

[Annotation] amandam None set by m [Annotation] amandam MigrationNone set by m [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam AspAT Activity Measurement: Extraction buffer for AspAT consisted of 200 mM Tris-Cl buffer (pH 7.5), 2.0 mM EDTA and 20% glycerol.

The enzyme was d in an MDH-coupled reaction essentially as described by Ireland and Joy (1990). Brieﬂy the reaction mixture contained 10 mM 2-oxoglutarate, 2 mM aspartate, 0.2 mM NADH, and 50 mM HEPES buffer (pH 8.0). Reaction was started by addition of 2- oxoglutarate. Assay control was run by excluding the 2-oxoglutarate from the reaction mix.

GS Activity Measurement: GS (glutamine synthetase) was extracted in the ng medium containing 50 mM'Tris-Cl buffer (pH 7.8), 1 mM EDTA, 10 mM MgSO.:, 5 mM sodium glutamate, 10% (v/v) glycerol and ble PVPP (2% w/v). Enzyme assay was performed as described earlier by Lea et a1. (1990) and the activity was ated from the standard curve prepared with y—glutamylhydroxamate.

The results of the analyses are shown in the Figure 5A to SC, an increase of about 45 to 50% in PEPCase activity, 55% in GS activity and 55 to 60% in AspAT ty was observed with two independent AspAT + GS + PEPCase transgenic plants compared to wild plants.

EXAMPLE 9 C and N analyses in wild and AspAT + GS + PEPCase transgenic Arabidopsis plants Seeds of AspAT + GS + PEPCase transformed Arabiopdris thaliana plants and wild control plants were germinated on half strength MS plates supplemented with 20g/l sucrose. 14 days- old ngs were transferred to pots containing mix of vermiculite,- perlite and coco peat in the ratio of 1:1 :1 and grown under long-day conditions comprising 16 hours of light period at 22°C and 8 hours of dark period at 20°C maintained in the Arabidopsis growth chamber.

Different plant parts including rosette leaf, stem, cauline leaf and green pods were ted from 65 —days old plants and dried at 80 °C for 48 hrs. The tative determination of the C and N elements was conducted with Elementar CHNS analyzer using sulfanilamide as standard. The results are shown in Figure 6. The elementary is showed that the total C and N content in AspAT + GS + PEPCase transgenic plant leaves has signiﬁcantly increased by co-overexpression of AspAT, GS and PEPCase compared to wild plants.

EXAMPLE 10 Investigation of growth and yield in wild and GS+PEPCase transgenic plants Wild and AspAT + GS + PEPCase transgenic plants were analyzed for different growth characteristics. Shoot, root fresh and dry weight was recorded for s old plants. Across different parameters evaluated, AspAT + GS + PEPCase plants showed enhanced growth [Annotation] amandam None set by m ation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] m Unmarked set by amandam [Annotation] amandam None set by amandam ation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam characteristics. In particular, the transgenic plants have more number of leaves per rosette having larger area. Transgenic plants exhibited about 70% increase in the shoot fresh weight with 60% increase in the shoot dry weight whereas the increase of about 40% and 30 % was observed in the root fresh and dry weight respectively ( shown in Figure 3).

Total number of pods from 72-days old AspAT + GS + PEPCase transgenic plants was calculated and compared to untransformed wild plants (shown in Figure 7 a). Furthermore total seed yield (total seed weight per plant) was also measured for enic and control plants. Across both the parameters, AlspAT + GS + PEPCase transgenic Arabidopsis plant showed increase in yield compared to wild plants as shown in Figure 7 b.

ADVANTAGES OF THE INVENTION 1. There have have been efforts to enhance carbon and nitrogen status of plants, a step towards food security. 2. The present ion provides an innovative approach wherein overexpression of e provides a carbon skeleton to e nitrogen assimilated through over expression ofAspA Tand GS. 3. The improved capacity of plant for carbon and nitrogen capture was also reﬂected in improved plant productivity both in terms of plant seed and plant biomass production.

Claims

The Claims :

1. An expression construct comprising SEQ ID NO. 7 for co-expression of the genes AspAT, GS and PEPCase comprising nucleotide ces SEQ ID NO: 1, SEQ ID 5 NO: 2 and SEQ ID NO: 3, linked to at least one l sequence and a transcription terminator sequence, useful for ing the carbon, nitrogen, s and yield of plants as compared to wild type or untransformed plant.

2. An expression construct as d in claim 1, wherein the control sequence comprises a sequence as provided in SEQ ID NO: 4 and the transcription terminator sequence 10 comprises a sequence as provided in SEQ ID NO: 5.

3. An expression construct as claimed in claim 1, wherein the said control sequence is a constitutive promoter selected from the group consisting of CaMV 35S promoter, rubisco promoter, ubiquitin promoter, actin promoter.

4. An expression construct as claimed in claim 1, wherein the terminator used is selected 15 from the group consisting of Nos terminator and CaMV 3'UTR.

5. An expression construct as claimed in claim 1, wherein the polynucleotide comprising SEQ ID No: 7 is overexpressed in plants.

6. A process for preparing the expression construct as claimed in claim 1, wherein the process comprising the steps of: 20 i) amplifying cDNA sequences encoding genes comprising SEQ ID NO: 1 using primers comprising SEQ ID NO: 10 and SEQ ID NO: 11, SEQ ID NO: 2 using primers comprising SEQ ID NO: 8 and SEQ ID NO: 9 and SEQ ID NO: 3 using primers comprising SEQ ID NO: 12 and SEQ ID NO: 13; 25 ii) cloning independently the amplified product of SEQ ID NO: 1, 2 and 3 as obtained in step (i) into pGEM-T easy vector; iii) digesting independently the d from the ve clones as obtained in step (ii) along with pCAMBIA 1302 and further ligating the 30 ed gene ts and pCAMBIA 1302 and transforming into E. coli DH5 α cells; iv) sequencing the plasmid from the positive colonies obtained in step (iii) confirming the inframe cloning of AspAT::pCAMBIA1302; GS::pCAMBIA1302 and PEPCase::pCAMBIA 1302; [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam v) amplifying the ts obtained in step (iv) by using primers comprising SEQ ID NO: 10 and SEQ ID NO: 16; SEQ ID NO: 14 and SEQ ID NO: 15 and SEQ ID NO: 17 and SEQ ID NO: 18; vi) cloning, digesting, ligating and sequencing was again performed 5 independently for the amplified GS coding sequence to form GS+pCAMBIA1302 which was further digested and ligated with the plasmids of positive clones of amplified AspAT coding sequence to form AspAT +GS+pCAMBIA1302 expression cassette; vii) ligating the ed plasmids of positive clones of amplified PEPCase 10 coding sequence with the destination pCAMBIA1302 which was previously cloned with the AspAT +GS+ expression te as obtained in step (vi) such that the genes AspA, GS and PEPCase were controlled by independent CaMV 35S promoter and Nos transcriptional ator to form single plant expression construct AspAT + GS + PEPCase 15 represented by SEQ ID NO: 7.

7. A process for enhancing the carbon, nitrogen, biomass and yield of plants using the sion construct as claimed in claim 1, wherein the said process comprising the steps of: 20 a) transforming Agrobacterium tumefacians strain with the expression construct as claimed in claim 1; b) transforming plant explants with the recombinant Agrobacterium cians strain as obtained in step (a); c) selecting the transformed explants of step (b) to obtain the desired transformed 25 plants having enhanced level of carbon, en, biomass and seed yield of plants as compared to wild type plant.

8. A s as claimed in claim 7, wherein the transformed plants is selected from the group sing of arabidopsis, tomato, potato, tobacco, maize, wheat, rice, cotton, 30 mustard, pigeon pea, cowpea, pea, sugarcane, soyabean and sorghum.

9. A process as claimed in claim 7, wherein the ormed plants display an increase of about 45-50% in PEPCase activity, t 55% in GS activity and 55-60% in AspAT activity as compared to wild type, resulting in increase in carbon and nitrogen levels in 35 the plant. [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam

10. A process as claimed in claim 7, wherein the transformed plants as compared to wild type y increased seed yield and/or biomass, indicated by increased seed yield and/or pod yield. 5

11. A process as claimed in claim 7, wherein the ormed plants display enhanced growth characteristics characterized by increased shoot fresh , shoot dry weight, root fresh and dry weight as compared to wild type or untransformed plant.