NZ716717A - An expression construct and process for enhancing the carbon, nitrogen, biomass and yield of plants - Google Patents

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.

Description

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

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 sion construct which utilizes co-overexpression of genes from enzymes phosphoenolpyruvate carboxylase (hereinafter, referred as ”PEPCase”), glutamine synthetase (hereinafter, referred as ”GS”) and aspartate aminotransferase (hereinafter, referred as ”AspAT”). In particular, the present invention is directed to transgenic plants where nucleic acid sequences ng the said proteins are expressed in plant cells.

More particularly, the present invention relates to the transformation of a plant with genetic construct involving rexpression of three genes wherein one gene PEPCase encodes enzyme responsible to capture C02 and the other two encode for enzymes (AspAT and GS) involved in N assimilation wherein the N assimilation requires C skeleton which is met by PEPCase, under the l 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 INVENTION AND PRIOR ART The present ion 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. 4.1.1.31) is a ubiquitous enzyme in plants that catalyses the B-carboxylation of yieldphosphgolpyruvate nafter, 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 MigrationNone set by amandam [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] m Unmarked set by amandam referred as ”Pi”), and it primarily has an anaplerotic function of replenishing the tricarboxylic acid cycle with intermediates. In higher plants, there are several isoforms of PEPCase of different organ specificities and they are involved in a variety of functions including stomata g, fruit ripening and seed maturation. The leaves of C4 and CAM plants contain 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 bute to an anaplerotic function and play a role in regulation ofthe cellular pH.

GS (EC 6.3.1.2) catalyses the ATP-dependent condensation of ammonia (hereinafter, referred as ”NH3”) with glutamate nafter, referred as ”Glu”) to produce ine (hereinafter, referred as ”Gln”). uently, glutamate synthase (GOGAT) transfers the amide group of Gln to CL lutarate producing 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) zes the reversible transfer of the amino group of asparate (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+ assimilation 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 filling and participating in intracellular C shuttles in C4 plants providing precursors for the biosynthesis of the Asp family of amino acids.

Plant mance in terms of s production, yield or t 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 emerging s of C and N assimilation suggest that a regulatory system coordinates the uptake and distribution of these nutrients in response to both metabolic and environmental cues. Plants sense changes in their C and N status and relay this information 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, s and yield in plants.

[Annotation] m None set by amandam [Annotation] amandam MigrationNone set by m [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 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 Takahashi, H., (NADK2) pressor and were characterized by Takahara, K., nade mutant were increase in calvin cycle a, S., Catalyzes the studied to investigate 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 y, H. and N metabolism. 2009. Plant Physiol. 151: 100- 113.

Dof 1 Maize Dofl cDNA was Dofl overexpression Yanagisawa, S., overexpressed in in Arabidopsis has led a, A., Dofl is a Arabidopsis plants to co-operative Kisa ka, H., ription under derivative of cation of plant C Uchimiya, H. and activator for the 355 promoter and N content, with Miwa, T. 2004. multiple gene designated as improved growth Proc. Natl. Acad. expressions 35$C4PPDK. under low N Sci. USA. 101: associated with conditions. However, 7833—7838 the organic acid effect of CN metabolism, 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 cytosolic GS Fraisier, V., GS catalyses the with the constitutive rated plant Chaillou, 5., ATP- dependent CaMV 35$ promoter in development, leading 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. flowering when grown Douat, C., NH4+ rich medium. Boutin, J.-P. and Limitation of C Hirel, B. skeleton and energy 1997. . for enhanced NH4+ 201: 424-433.

D lation were anticipated.

[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] m Unmarked set by amandam [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by m [Annotation] m Unmarked set by amandam ii.) A pea cytosolic GS Overexpression of Oliveira, |.., gene was cytosolic 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 lic Cai, H., Zhou, Y., encoding rice cytosolic level in GS- Xiao, J., Li, X., GS genes (OsGSl;1 overexpressed plants Zhang, Q. and and OsGSl;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 er. 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 . iv) cDNA encoding alfa Transgenic plants Fuentes, S., Allen, alfa cytosolic GS over grew better under N D., Ortiz-Lopez, A. sed in tobacco tion by and Hernandez, plants maintaining G. 2001. J. Exp. photosynthesis at rate Bot. 52:1071- comparable to those 1081. of plants under high N, while photosynthesis in control plants was ted by 40-50%.

These results further reflect the need for cooperative modification of CN metabolism for developing plants with better agronomic traits. ation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam ation] m 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 e i) The intact maize Transgenic plants Agarie, S., Miura, gene encoding C4- exhibited higher A., Sumikura, R., PEPCase catalyses specific PEPCase 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 d 02 inhibition 2002. Plant Sci. ‘and Mg2+to yield photosynthesis was 162: 257-265.

OAA and Pi. primarily due to reduction of Pi rather than se in the partial direct n 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 e Higher levels of maize Hudspeth, introduced in to PEPCase transcript of R.L.,Grula, tobacco plants under the correct size were J.W.,Dai, Z., the l maize obtained using tobacco Edwards, G.E. and PEPCase and tobacco (chlorophyll a/b Ku, M.S.B. 1992. chlorophyll a/b binding protein gene Plant Physiol. 98: binding protein gene promoter. With two 4 promoter. fold incerase in PEPCase activities in leaf, transgenic plants had significantly elevated levels of titratable acidityand malic acid. However, these mical differences did not produce any significant physiological changes with respect to photosynthetic rate or C02 compensation point.

AspAT i) Panicum eum 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 [Annotation] amandam MigrationNone set by m [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam [Annotation] m MigrationNone set by amandam ation] amandam Unmarked set by m [Annotation] amandam None set by amandam ation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam catalyzes (mAspAT and cAspAT, 3.5-fold higher AspAT |shimaru, 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 suggested interaction between C and N metabolism. ii) Three AspAT genes Compared with Zhou. Y., Cai ,H., from rice (OsAAT3) l 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- promoter in rice greater seed amino 1390 plants . acid and protein 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 nitrogen in to c form through joint activity of AspAT and GS. As a result, the inventors have found that object of the present ion can be attained by concomitant increase in expression of genes encoding AspAT, GS and e 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 oenolpyruvate carboxylase in transgenic tobacocnl992, Plant Physiology 98: , 4), n PEPCase from maize was expressed under a tobacco(Nicotiana plumbaginifolia) chlorophyll a/b binding protein gene ation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [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] m None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam promoter in o 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 s 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., rt, M. and Moing, A., Rolin, D., and Vidal, J. entitled ”Physiological impacts of modulating phosphoenolpyruvate ylase 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 e 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 improved growth phenotype or modification in seed tion per plant Reference may be made to yet another article by Chen, L.M., Li, K.Z. Miwa, T. and Izui, K. entitled ”Overexpression of a cyanobacterial phosphoenol pyruvate carboxylase with diminished sensitivity to feedback inhibition in Arabidopsis s amino acid metabolism” (2004, Planta, 219: 440-419.), wherein the cyanobacterial Synechococcus vulcanus phosphoenolpyruvate carboxylase Case) with diminished sensitivity to feed back inhibition, was over expressed 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 transformed T2 plants was presumed to be primarily due to a decreased availability of oenolpyruvate (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 Fukayama, H., Hatch, M.D., Tamai, T., Tsuchida, H., Sudoh, S., Furbank, RT and Miyao, M., entitled ity regulation and [Annotation] amandam None set by m [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 m [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), wherein the intact maize PEPCase gene was overexpressed in the leaves of rice plants.

Introduced e in transgenic rice leaves underwent activity regulation through n phosphorylation in manner similar to endogenous rice PEPCase but contrary to that occurring in maize , 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 indicated that maize PEPCase did not contribute significantly to the ynthetic C02 n of transgenic rice plants. Rather, it ly lowered the C02 assimilation rate. This effect was ascribable to the stimulation of respiration in the light, which was more marked at lower 02 concentrations. It was concluded that overproduction of PEPCase does not directly affect ynthesis significantly but it sses photosynthesis indirectly by stimulating respiration in the light.

Reference may be made to yet another article by Vincent, R., Fraisier, V., Chaillou, S., , 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 ulatus L. plants. On growing the transgenic plants under different N regimes an increase in free amino acids and ammonium was observed accompanied 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. Labelling ments 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 flowering when plants were grown on an ammonium-rich medium. Limitation of C skeleton and enen for enhanced NH4+assimilation were anticipated.

[Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam ation] 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 MigrationNone set by amandam ation] m 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, l of Experimental Botany, 52:1071-1081), wherein the alfa alfa GS driven by constitutive CaMV 35$ promoter 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 fertilization conditions, no effect of GS overexpression on photosynthesis or growth was Reference may be made to yet another article by Oliveira, |.., Brears, T., Knight, T., Clark, A. and Coruzzi, G., entitled ”Overexpression of cytosolic glutamine synthetase. Relation to nitrogen, light, and photorespiration” (2002, Plant logy, 129: 1170-1180), wherein the overexpression of pea cytosolic GS was studied in relation to nitrogen, light and photorespiration. Tobacco plants, which cally overexpress cytosolic GSl in leaves, y a light-dependent improved growth phenotype under N-limiting and limiting ions as evident by increase 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 se in levels of photorespiratory intermediates, suggesting changes in photorespiration. However, the effect of stimulation of photorespiration by GS ression on plant productivity was not discussed.

Reference may be made to yet another e 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: 527-537), 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 overexpressed 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 t in the whole plant. However, ation] 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 ation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] m Unmarked set by amandam decrease in both grain yield production and total amino acids were observed in seeds of GS- overexpressed plants ed with wild-type plants.

Reference may be made to yet another article by Sentoku, N., Taniguchi, M., Sugiyama, T., Ishimaru, K., Ohsugi, R., Takaiwa, F. and Toki, S., entitled ” Analysis of the transgenic tobacco plants expressing Panicum miliaceum aspartate aminotransferase genes" (2000, Plant Cell Reports, 19: 3), n the effects of the overexpression of Panicum mitochondrial and cytoplasmic AspAT T and cAspAT respectively) under the control of CaMV 35$ promoter were ted on transgenic tobacco plants. The mAspAT- or cAspAT-transformed plants had about threefold or 3.5-fold higher AspAT activity in the leaf than non-transformed plants, respectively. stingly, the leaves of both transformed plants had increased 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 d nitrogen metabolism and increased amino acid content in seeds” (2009, Theoretical and d 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 pressed in rice plant under the control of CaMV 35$ promoter. The OsAAT 1, OsAATZ, and EcAATtransformants showed significantly sed leaf AspAT activity and greater seed amino acid and protein contents. However no significant changes were found in leaf AspAT activity, seed amino acid content or protein t in OsAAT3 xpressed plants.

Reference may be made to yet another article by Murooka, Y., Mori, Y. and Hayashi, M., entitled ” Variation 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 amandam [Annotation] amandam ionNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam ation] 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 355 er for achieving its overexpression in the Arabidopsis plant. Expression of AspAT5 in transformants caused 3-, 4-, 23-, and 50-fold increases in the contents of free glycine, alanine, asparagine, and Glu, respectively, in the T3 seeds. However, a decrease in the contents of valine, tyrosine, cine, e, 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. ed ”Metabolic engineering with Dofl transcription factor in plants: 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 lation in transgenic Arabidopsis plants. Dofl expressing plants showed up-regulation of genes encoding enzymes for C skeleton tion, a marked increase of amino acid contents, and a reduction of the glucose level. The s t ative 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%), ting promotion of net N assimilation. r, effect of C N alteration on plant biomass or yield was not discussed.

Reference may be made to still another article by Takahashi, H., Takahara, K., Hashida, S., Hirabayashi, T., Fujimori, T., Kawai-Yamada, M., Yamaya, T., sawa, S. and Hirofumi Uchimiya, H., entitled ”Pleiotropic Modulation of carbon and nitrogen metabolism in Arabidopsis plants overexpressing the NAD kinase2 gene” by (2009, Plant Physiology. 151:100-113), wherein transgenic 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 proportional to NADK activity in NADK2 overexpressors and in the NADK2 mutant. Several metabolites associated with the calvin cycle were also higher in the overexpressors, anied by an increase in overall Rubisco activity. Furthermore, enhanced NADP (H) production due to NADK2 overexpression increased N assimilation. Gin and Glu nons, 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] m 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 The improvement in the C and N status of plants is a major n to improve productivity.

However, there is no report yet which show enhancement of C and N levels and uent improvement in the biomass and yield of plant.

Further, no attempt has been made to co-over express three genes, viz. AspAT, GS and PEPCase, leading to enhanced status of C and N, biomass, and yield.

IVES 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.

Another objective of the present invention is to provide an expression construct for co- pression 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 mediated 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 e.

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

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

SUMMARY OF THE INVENTION Accordingly, the present invention provides an expression uct represented by SEQ ID NO. 7 for co-expression of the genes AspAT, GS and e comprising nucleotide ces represented by SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, wherein SEQ ID NO: 1 represents AspAT genes, SEQ ID NO: 2 represents GS genes and SEQ ID NO: 3 represents PEPCase genes linked to t 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. ation] 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] m 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 represented by SEQ ID NO: 4.

In another embodiment of the present invention, the transcription terminator ce is represented by SEQ ID NO: 5.

In an ment, the present invention provides an expression construct prepared from the cytoso|ic AspATgene from soyabean, cytoso|ic GS gene from tobacoo and |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 embodiment of the present invention, the control sequence used is a constitutive promoter selected from the group consisting of CaMV 35$ promoter, rubisco promoter, ubiquitin promoter, actin promoter.

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

In still another embodiment of the present invention, a process for ing the expression construct wherein the process comprising the steps of: i) amplifying cDNA ces ng 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 ; iii) digesting independently the plasmid from the positive clones as obtained in step (ii) along with pCAMBIA 1302 and further ligating the digested gene products and pCAMBIA 1302 and transforming into E.coli DH5 0L cells; iv) sequencing the plasmid from the positive colonies obtained in step (iii) ming the inframe cloning of AspAT::pCAMBIA1302; GS::pCAMBIA1302 and PEPCase::pCAMBIA 1302. v) amplifying the products obtained 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] m MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam ation] amandam None set by amandam [Annotation] amandam MigrationNone set by m [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam ation] amandam ed set by amandam vi) cloning, digesting, ligating and sequencing was again performed independently for the amplified GS coding sequence to form GS+pCAMB|A1302 which was further digested and ligated with the ds of positive clones of amplified AspATcoding sequence to form A5pAT+GS+pCAMB|A1302 expression te; vii) ligating the ed plasmids of positive clones of amplified PEPCase 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 PEPCase were controlled by independent CaMV 35$ promoter and Nos transcriptional terminator to form single plant expression construct AspAT + GS + PEPCase represented 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 expression construct, wherein the said process comprising the steps of: a) transforming Agrobacterium tumefacians strain with the expression construct as claimed in claim 1; b) transforming the explants with the recombinant Agrobacterium tumefacians strain as obtained in step (a); c) selecting the ormed explants of step (b) to obtain the desired transformed plants having enhanced level of , nitrogen, biomass and yield of plants as compared to wild type plant.

In still another embodiment of the t invention, a process wherein the ormed plants display an increase of about 45-50% in PEPCase activity, atleast 55% in GS ty and 55-60% in AspAT activity 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 ed from a group consisting of GV3101 with ATCC number cterium tumefaciens (GV3101 (pMP90RK) (C58 derivative) ATCC® Number: 33970 Reference: Hayashi H, Czaja |, Lubenow H, Schell J ,Walden R. 1992.

In yet another embodiment of the t invention, the transformed plants are selected from the group consisting of grain crops, pulses, vegetable crops, oilseed crop and ornamens.

[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 [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by m In yet another embodiment, the transformed plants are ed 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 biomass, indicated by increased seed yield and/or pod yield.

In still another embodiment, the transformed plants display enhanced growth characteristics characterized by increased 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 , nitrogen, biomass and yield as compared to wild plants.

In still r embodiment of the present invention, the expression and functionality of over sed enzymes in transgenic plants is evaluated.

In yet another embodiment of the present invention, the selectable marker used is hpt gene mycin 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 ment of the present ion, the transgenic plants were igated for ent growth and yield parameters and compared to wild plants cultivated under the same conditions.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS Figure 1 represents a schematic view of T- DNA region of plant transformation vector A1302 for co-overexpression of AspAT, GS and PEPCase (a) and amplification of coding sequences for AspAT, GS and PEPCase from respective plant s (b) as discussed in es 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] amandam 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 days of sowing. Data is mean of five separate biological replicates with standard ion marked on each bar.

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

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

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

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

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

DETAILED DESCRIPTION OF THE INVENTION The present ion relates to genetic 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 e for concomitant alteration in the enzymes 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 foreign source can be ligated and isolated when needed. 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 another.

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

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

[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 ed set by amandam [Annotation] 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 ce of an ise toxic otic The term "transgenic plant" refers to genetically transformed plants with stable ation 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, wherein the enzyme RNA polymerase binds for the process of transcription. “Constitutive promoters” direct expression of the gene in all tissues and during all periods regardless of the nding environment and pment stage of the organism.

The term ’expression cassettes” refers to vector comprising of (a) a constitutive promoter; (b) all the three genes cloned 3' to the tutive promoter, (c) a polyadenylation signal located 3' to the coding sequence. and capable of passing genetic information on to successive tions.

’Wild-type" plants are untransformed plants.

The term "To" refers to the first set of genetically ormed 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 flowers of T 0 generation plants, previously selected as being transgenic. "T2” plants are generated from T1 plants, and so on.

The t 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 Sequence Sequence e sequence ID No.

AspAT cDNA atggcttctc acgacagcat ttct ccaacctccg cttctgattc cgtcttcaat 60 Represents 1 cacctcgttc gtgctcccga agatcctatc ctcggggtaa ctgtcgctta taacaaagat 120 sequence nucleotide ccaagtccag ttaagctcaa cttgggagtt ggtgcttacc gaactgagga aggaaaacct 180 cttgttttga atgtagtgag gcgagttgaa cagcaactca taaatgacgt gtcacgcaac 240 sequences of aaggaatata ttccgatcgt tgggcttgct gattttaata aattgagtgc taagcttatt 300 AspAT genes for tttggggctg acagccctgc tattcaagac aacagggtta ccactgttca atgcttgtct 360 ggaactggtt ctttaagagt tgggggtgaa tttttggcta atca ccaacggact 420 making an atatacttgc caac ttggggcaat aagg ttttcaactt agcaggcttg 480 expression tctgtcaaaa cataccgcta ctatgctcca gcaacacgag gacttgactt tcaaggactt 540 ctggaagacc ttggttctgc tccatctgga tctattgttt tgctacatgc atgcgcacat 600 aaccccactg gtgtggatcc aacccttgag gagc agattaggca gctaataaga 660 tcaaaagctt tgttaccttt ctttgacagt gcttatcagg gttttgctag tggaagtcta 720 gatgcagatg cccaacctgt tcgtttgttt gttgctgatg gaggcgaatt gctggtagca 780 caaagctatg caaagaatct gggtctttat ggggaacgtg ttggcgcctt aagcattgtc 840 tgcaagtcag ctgatgttgc aagcagggtt gagagccagc tgaagctagt gattaggccc 900 atgtactcaa gtcctcccat tcatggtgca tccattgtgg ctgccattct caaggaccgg 960 aatttgttca atgactggac tattgagttg aaggcaatgg ctgatcgcat catcagtatg 1020 cgccaagaac ttttcgatgc tttatgttcc agaggcacac ctggcgattg gagtcacatt 1080 caga ttggaatgtt tactttcact ggattgaatg cggaacaagt ttccttcatg 1140 actaaagagt tccatatata catgacatct gatgggagga ttagcatggc tggtctgagt 1200 tccaaaactg tcccacttct ggcggatgcg atacatgcag ctgtaacccg agttgtctaa 1260 GS cDNA atggctcatc tttcagatct cgttaatctc tctg actccactca gaaaattatt 60 Represents 2 gctgaataca tatggattgg tggatcagga atggacgtca ggagcaaagc actt 120 sequence nucleotide tctggacctg ttgatgatcc ttcaaagctt cccaaatgga attatgatgg ttctagcaca 180 ggacaagctc ctggagaaga cagtgaagag atcctatatc ctcaagcaat tttcaaggat 240 sequences of GS ccattcagaa ggggcaacaa tatcttggtc gatt gttacacccc agctggtgaa 300 genes for making cccattccaa caaacaaaag gcacagtgct gccaagattt tcagccaccc tgatgttgtt 360 gttgaggaac cctggtatgg tcttgagcaa acct tgttgcaaaa agatatcaat 420 an expression tggcctcttg gatggcctct tggtggtttt ccac agggaccata cgga 480 construct attggagctg gaaaggtctt tggacgcgat atcgttgact ctcattataa ggcatgtctc 540 ggga ttaacatcag tggtatcaat gtga tgcccggaca gtgggaattt 600 caagttggac cttcagttgg catttcagca gctgatgaat tgtgggcagc tcgttacatt 660 agga ttactgagat tgctggagtt tcat ttgaccccaa acctattccg 720 tgga atggtgctgg agctcacaca aactacagca caaagtctat gaggaatgaa 780 ggaggctatg aagtcattaa aatt gagaaccttg ggca caaggagcat 840 attgcagcat atggtgaagg caacgagcgt cgtctcactg gaagacacga aacagctgac 900 atcaacacat tcaaatgggg agttgcgaac cgtggtgcat ctattcgtgt gggaagagac 960 acggagagag aagggaaggg atacttcgag gataggaggc ctgcttcgaa tatggatcca 1020 ttcgtcgtga cttccatgat tgctgagacc actatcctat ccgagccttg a 1071 PEPCase atggcgtcga ccaaggctcc cggc gagaagcacc tcga cgcgcagctc 60 Represents 3 cgtcagctgg tcccaggcaa ggtctccgag gacgacaagc tcatcgagta cgatgcgctg 120 cDNA nucleotide ctcgtcgacc tcaa catcctccag gacctccacg ggcccagcct tcgcgaattt 180 sequence gtccaggagt gctacgaggt ctcagccgac tacgagggca aaggagacac gacgaagctg 240 sequences of ggcgagctcg gcgccaagct cacggggctg gcccccgccg tcct cgtggcgagc 300 PEPCase genes for tccatcctgc acatgctcaa caac ctggccgagg aggtgcagat cgcgcaccgc 360 cgccgcaaca gcaagctcaa gaaaggtggg gacg agggctccgc caccaccgag 420 making an tccgacatcg aggagacgct caagcgcctc gtgtccgagg tcggcaagtc ccccgaggag 480 expression gtgttcgagg cgctcaagaa cgtc gacctcgtct cgca tcctacgcag 540 tccgcccgcc gctcgctcct gcaaaaaaat gccaggatcc gtct gacccagctg 600 construct aatgccaagg acatcactga cgacgacaag caggagctcg atgaggctct gcagagagag 660 atccaagcag ccttcagaac cgatgaaatc aggagggcac aacccacccc gcaggccgaa 720 atgcgctatg gcta catccatgag actgtatgga agggtgtgcc taagttcttg 780 cgccgtgtgg atacagccct tatc ggcatcaatg agcgccttcc ctacaatgtt 840 tctctcattc ggttctcttc gggt ggtgaccgcg atcc aagagttacc 900 gtga caagagatgt atgcttgctg gccagaatga tggctgcaaa cttgtacatc 960 gatcagattg aagagctgat gtttgagctc tctatgtggc gctgcaacga tgagcttcgt 1020 gttcgtgccg aagagctcca cagttcgtct ggttccaaag ttaccaagta ttacatagaa 1080 ttctggaagc ctcc aaacgagccc taccgggtga tactaggcca tgtaagggac 1140 aagctgtaca acacacgcga gcgtgctcgc ctgg cttctggagt ttctgaaatt 1200 tcagcggaat cgtcatttac cagtatcgaa gagttccttg agccacttga gctgtgctac 1260 aaatcactgt gtgactgcgg cgacaaggcc atcgcggacg ggagcctcct ggacctcctg 1320 gtgt tcacgttcgg gctctccctg gtgaagctgg acatccggca ggagtcggag 1380 cggcacaccg acgtgatcga cgccatcacc ctcg gcatcgggtc gtaccgcgag 1440 tggcccgagg acaagaggca ggagtggctg ctgtcggagc tgcgaggcaa gcgcccgctg 1500 ctgcccccgg accttcccca gaccgacgag atcgccgacg tcatcggcgc cgtc 1560 gagc ccga cagcttcggc ccctacatca tctccatggc gacggccccc 1620 tcggacgtgc tcgccgtgga gctcctgcag cgcgagtgcg gcgtgcgcca gccgctgccc 1680 gtggtgccgc tgttcgagag gctggccgac ctgcagtcgg cgcccgcgtc cgtggagcgc 1740 ctcttctcgg ggta catggaccgg atcaagggca agcagcaggt cggc 1800 tactccgact ccggcaagga cgccggccgc ctgtccgcgg cgtggcagct gtacagggcg 1860 caggaggaga aggt ggccaagcgc gtca cctt 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 tcat ggacgagatg 2160 gcggtcgtgg ccacggagga gtaccgctcc gtca aggaggcgcg cttcgtcgag 2220 tacttcagat cggctacacc cgag tacgggagga tgaacatcgg cagccggcca 2280 gccaagagga ggcccggcgg cggcatcacg accctgcgcg ccatcccctg gatcttctcg 2340 tggacccaga ccaggttcca cctccccgtg ggag tcggcgccgc attcaagttc 2400 gccatcgaca aggacgtcag gaacttccag gtcctcaaag agatgtacaa cgagtggcca 2460 ttcttcaggg tcaccctgga cctgctggag atggttttcg ccaagggaga ccccggcatt 2520 gccggcttgt atgacgagct gcttgtggcg gaagaactca agccctttgg gaagcagctc 2580 agggacaaat acgtggagac acagcagctt caga tcgctgggca caaggatatt 2640 ggcg atccattcct gaagcagggg ctggtgctgc gcaaccccta catcaccacc 2700 ctgaacgtgt tccaggccta cacgctgaag cggataaggg accccaactt caaggtgacg 2760 ccccagccgc ccaa ggagttcgcc gacgagaaca agcccgccgg actggtcaag 2820 ctgaacccgg cgagcgagta cccgcccggc ctggaagaca cgctcatcct caccatgaag 2880 ggcatcgccg ccggcatgca gaacactggc tag 2913 CaMV 35S catggagtca aagattcaaa tagaggacct aacagaactc gccgtaaaga ctggcgaaca 60 Represents control 4 gttcatacag agtctcttac gactcaatga caagaagaaa atcttcgtca acatggtgga 120 er sequence gcacgacaca cttgtctact ccaaaaatat caaagataca gtctcagaag accaaagggc 180 ce aattgagact caaa gggtaatatc cggaaacctc ctcggattcc attgcccagc 240 tatctgtcac tttattgtga agatagtgga aaaggaaggt ggctcctaca aatgccatca 300 ttgcgataaa ggaaaggcca tcgttgaaga tgcc gacagtggtc ccaaagatgg 360 acccccaccc agca tcgtggaaaa agaagacgtt ccaaccacgt cttcaaagca 420 agtggattga tgtgatatct ccactgacgt aagggatgac tccc actatccttc 480 gcaagaccct tcctctatat aaggaagttc atttcatttg gagagaacac gggggact 538 nos cgttcaaaca tttggcaata aagtttctta agattgaatc ctgttgccgg tcttgcgatg 60 Represents 5 attatcatat aatttctgtt gaattacgtt aagcatgtaa taattaacat gtaatgcatg 120 (nopaline transcription acgttattta tgagatgggt ttttatgatt agagtcccgc aattatacat ttaatacgcg 180 synthase) atagaaaaca aaatatagcg cgcaaactag gataaattat cgcgcgcggt gtcatctatg 240 terminator 3'UTR TTACTAGATCGGG sequence (polyAsignal) sequence hygromycin ctatttcttt gccctcggac gagtgctggg gcgtcggttt tcgg cgagtacttc 60 Represents hpt 6 phosphotransfe tacacagcca tcggtccaga cggccgcgct tctgcgggcg atttgtgtac gcccgacagt 120 gene (hygromycin rase cccggctccg acga ttgcgtcgca tcgaccctgc gcccaagctg cgaa 180 attgccgtca ctct gttg gtcaagacca atgcggagca tatacgcccg 240 phosphotransferas gagtcgtggc gatcctgcaa gctccggatg cctccgctcg aagtagcgcg tctgctgctc 300 e)for hygromycin catacaagcc aaccacggcc tccagaagaa gatgttggcg acctcgtatt gggaatcccc 360 gaacatcgcc tcgctccagt caatgaccgc tgttatgcgg tccg tcaggacatt 420 resistance gttggagccg aaatccgcgt gcacgaggtg ccggacttcg gggcagtcct cggcccaaag 480 ctca tcgagagcct gcgcgacgga cgcactgacg gtgtcgtcca tcacagtttg 540 atac acatggggat cagcaatcgc gcatatgaaa tcacgccatg tagtgtattg 600 accgattcct tgcggtccga atgggccgaa cccgctcgtc tggctaagat cggccgcagc 660 gatcgcatcc atagcctccg cgaccggttg agcg ggcagttcgg tttcaggcag 720 gtcttgcaac gtgacaccct gtgcacggcg ggagatgcaa taggtcaggc tctcgctaaa 780 ation] amandam None set by amandam [Annotation] amandam ionNone 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 m [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam ctccccaatg lcaagcacxt ccggaatcgg gagcgcgcc gatgcaaagt gccgataaac 840 ataacgatct ttgtagaaac ca tcggcgca gctamacc acat 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 sequcrl ccs.

CIOHEd gcttctcacnacagcatdccncttctccaacctccncttcmattccgtcttcaatcacctcgttcntg ctcccga tatcctcngggt aactgtcgcttataacaaagatccaagtccagttaagctcaacttpgg u n dcr agttgglgcttaccga actgaggaaggaaaacctcttnttttgaatmagtgangcgngttnaacagcaact C0" "0| 0“ ca taaa tgacg tgtca cgcaacaaggaatatattccgatcgttgggctmctga tttlaataaattgamflct atttttggggctga tgctattca agacaacagggrraccactgttcaatgcttgtctgga ac Camv 3 SS tggttctttaagagttgggggtgaamttggctaaacactatcaccaacggactatatacflgccaa caccaa promoter ctlggngcaatcacccgaamttttcaacttagcapgcttgtctgtca aaacataccgctactatgctccagc ( ) a n d N as aacacgaggacttgactttcaaggacttctggaaga ccttggttctgctcca tctgga gttttgctaca ‘ tgcatgcgcacataaccccactrgmmgatccaacccnnagcaarmnagcagattaggcagcta ata an terminator atcaaa agcmgttacctttctttgaca mgcttatca tgctagtggaagtctagamcagatficcca a cctgttcgtttgtttgttgctgatggaggcgaattgctggtagcacaaagcta mcaaa gaatctngntct tt (a) in atggggaacgtgttggcgccttaagcattgtctgcaagtcagctgatgttgcaagcagggttgagagccanc PCAM BIA mgtgattaggccca tgtactcaagtcctcccattcatggtgcatcca ttgtggctgccattctca ag 1302 aamgttcaatnactggactattgagttgaaggcaatggctgatcgcatcatcagtatgcgccaag aacttttcgamcntatgtxccagamcacacctmcnatmgagtcacattatcaaacagattggaatgttt actltcactggattgaatgcfigaacaagtttcc‘ttcatgactaaagagttccatatatacatgacatctgatgg gaggattagcatggctggrctgagttccaaaactgtcccacttctggcngamcgatacatgcanctgtaa'or: gagttgtdaagg? Mtgaat:ggtgaccagctcgaamccccgatggguaaaca-mggcaataaagsnmaazanggarc gagggggtgggxafitggtfitcgtaxaazmmgmacgggaggajgtaataattaacaszag 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 ggcacagtgctgcca agattttcagccaccctga tgttgttgttgaggaaccctggtatg mmgagcaanaatacaccltmtgcaaaaagatatcaattggcctcttggatggcctcttggtggttttcct ggaccacaggga cca ta ciattgcgnaattgga gctggaaaggtcmggacgcgatatcgttgactctcatt ataaggcatgtctctatgctgggattaacatcagtggtatcaatggagaaglgatgcccggacagtgggaat ttcaagttgga ccttcagttggcatttcagcagctgatgaa ttgtggncagctcgttacattcttgagagga tt actgagattgctpgagttgtggtctcantgaccccaaacctattccgggtgactgnaatggtgctgga gctc acacaaactacagcacaaagtctarga gga atgaaggaggctatgaagtcattaaga aggcaangagaa ccttggactgaggca caagga gcatattgcagcatatggtgaaggcaacgagentcgtctcactgga agac /‘ [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 [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam ' ‘h'l gacacggagagagaagggaaggga tacttcgagaztaggaggcctgcttcgaatatggatccattcgtcgt gacttccatgattgctgagaccactatcctatccgagccttg- : - as Ianaxcatat aa ca aa‘aanaaca aa cat ac tamasg agataggggggggggaggccggcaattatacamaataggggatagaaaacaagataxa. aggggcaaacta catct acta an: aattaaactatcagt gmgacaggatatattggcfiigcgcgcaaatggcgaa tgctagagcagqtrgagcttggatcagattgtcg -..| ;-.-;v!.‘;-_ aLQ.‘1".','..'-, ".. El--'.:- . a z : - - - ‘ _ . . . -. mmcttgacca PEPCan: cod 3 sequznce ——> tgg-tatggcgtcgaccaaggctcccggccccggcgagaagcaccactccatqgaqzcgcagctc ctggtcccaggcaaggtctccgaggacgacaagctca(cgagtacgatgcgctgctcgtcgaccgc ttcctcaacatcctccaggacctccacgggcccagccttcgcgaatttgtccaggagtgctacgaggtctcag ‘ ccgacta cgagggcaa aggagacacgacgaagctgggcgagctcggcgccaagctcacggggctggcccc cgccgacgccatcctcgtggcgagctccazcctgcacalgctcaacczcgccaacctggccgaggaggtgca ga a ccgccgccgcaacagca agctcaagaaaggtgggttcgccgacgagggctccgcca ccacc gagtccg‘acatcgaggagacgctcaagcgcctcgtgtccgaggtcggcaagtcccccgaggaggtgttcga ggcgctcaagaaccagaccgtcgacctcgtcttcaccgcgcatcctacgcagtccgcccgccgctcgctcctg aatgccaggatccgaaattgtctgacccagctgaatgccaaggacatcactgacgacgacaagc aggagctcgatgaggctctgcagagagagatccaagcagccttcagaaccgatgaaatcaggagggcac ccccgca ggccgaaatgcgctatgggatgagctacatccatgagactgtatggaagggtg‘gcct an gttcttgcgccgtgtggatacagccctgaagaatatcggcatcaatgagcgccttccctacaatgtttctct gttctcttcttggatgggtggtgaccgcgaxggaaatccaagagtta ccccggaggtgacaagaga tgtatgcttgctggccagaatgatggctgca tacatcgatcagangaagagctgatgtttgagent ctatgtggcgctgcaacga(gagcltcgtgttcgtgccgaagagdccacagttcgtctggttccaaagttacc aagtattacatagaattckggaagcaaattcctccaaacgagccctaccgggtgatactaggocacgtaagg ctgtacaacacacgcgagcgtgctcgccatctgctggcttctggagtttctgaaamcagcggaat cgtcamaccagtatcga agagnccttgagccacttgagctgtgctacaaa tcactgtgtgactgcggcga ca aggccatcgcggacgggagcctcctggacctcctgcgccaggtgttcacgttcgggctctccctggtgaa catccggcaggagtcggagcggcacaccgacgtgatcgacgccatcaccacgcacctcggcatcg ggtcgtaccgcgagtggcccgaggacaagaggcaggagtggctgctgtcggagctgcgaggcaagcgccc gctgctgcccccggaccttccccagaccgacgagatcgccgacgtcatcggcgcgttccacgtéctcgcgga gctcccgcccgacagcttcggcccctacatcatctccatggcgacggccccctcggacgtgctcgccgtggag ctcctg cagcgcgagtgcggcgtgcgccagccgctgcccgtggtgccgctgttcgagasgctggccgacctg cagtcggcgcccgcgtccgtggagcgcctmctcggtggactggtacatggaccggatcaagggca agcag caggtcatggtcggctactccgactccggcaaggacgccggccgcctgtcc gcggcgtggcagctgtacagggcgca ggaggagatggcgcaggtggccaagcgctacggcgtcaagctca ccttgttccacggccgcggaggcaccgtgggcaggggtggcgggcccacgcaccttgccatcctgtcccagc cgccgga cacca tcaacgggtccatccgtgtgacggtgcagggcgaggtcatcgagttctgcncggggagg agcacctgtgcttccagactctgcagcgcncacggccgccacgctggagcacggcatgcacccgccggtct ctcccaagcccgagtggcgcaagctcatggacgagalggcggtcgtggccacggaggagtaccgctccmc gtcgtcaaggaggcgcgcttcgtcgagtacttcagatcggctacaccggagaccgagtacgggaggatgaa catcggcagccggccagccaagaggaggcccggcggcggcatcacgaccctgcgcgccatcccctggatct ‘ctcgtggacccagaccaggttccacctccccgtgtggctgggagtcggcgccgcancaagncgccatcga ca agga cgtcaggaacttccaggtcctcaa agagatgtacaacgagtggccattcttcagggtcaccctgga cctgctggagatggttttcgccaagggagaccccggcattgccggcttgtatgacgagctgcttgtggcggaa gaaacaagcocmgggaagcagctcagggacaaa‘acgtggagacacagcagcttctcctccagatcgct gggcacaaggatattcttgaaggcgatccattcctgaagcaggggctggtgctgcgcaacccctacatcacc accctgaacgtgttccaggcctacacgctgaagcggata cccaacttca 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 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 m [Annotation] amandam ed set by amandam momma: d 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' o 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 5‘-GCCACGTGTTAGACAACTCGGGTTACAGCTG-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, including restriction site for enzyme Bglll. _W—_—_ Reverse primer for amplification of PEPCase maize PEPCase ’-AGACTAGTGCCAGTGTTCTGCATGCCGGCGG3’ 13 59:! R . coding sequence, Including restriction site for enzyme Spei.

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

Reverse rio'r'fl‘ amplification of Nos S’—GGAC’I'AGTTTAATTCCCGA'I'Cl‘AGTAACA’l'AGA'l'GJ‘ terminator seq uence. 16 Including 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.

Reverse 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 hygromycin ' - GATGTTGGCGACCl'CGTATTGG -3' 20 phosphotransferase for screening transgenic plants.

Forward primer for PEPCase Exp 5' — ACG'i'CAGGAACITCCAGGTC -3‘ maize C, used F for RT-PCR based evaluation of [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 e transgene expression.

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

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

Reverse primer for tobacco GS, used for RT~PCR based ' - GGCAGCACTGTGCCTT -3' 24 tion 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 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 [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 [Annotation] amandam Unmarked set by amandam RT-PCR baséd evaluation of transgene expression MAS!lDSISASPTSASDSVFNHLVRAPEDPILGVTVAYNKDPSPVK|.NI.GVGAYRTEEG Represents Proteins WRRVE of AspAT genes QQLINDVSRNKEYIPNGLADFNKLSAKLIFGADSPAIQDNRVTTVQCLSGTGSLRVGG EFlAKHYHQRT IYLPTPTWGNHEKVFNLAGLSVKTYRYYAPATRGLDFQGLLEDLGSAPSGSIVLLIMCA AspATPr HNPTGVDPTLE QWEOJRQLIRSKALLPFFDSAYQGFASGSLDADAQPVRLFVADGGELLVAQSYAKNLG LYGERVGALSIV CKSADVASRVESQLKLVIRPMYSSPPIHGASIVA/\ILKDRNLFNDWTIELKAMADRIISM RQELFDALG RGTPGDWSHIIKQIGMFTFTGLNAEQVSFMTKEHIIYMTSDGRISMAGLSSKTVPLLA DAIHAAVTRW _‘ _ __ MAIILSDLVNINLSDSTQKIIAEYIWIGGSGMDVRSKARTLSGPVDDPSKLPKWNYDG Represents Proteins SSTGQAPGEDSEE of 65 genes ILYPQAIFKDPFRRGNNILVICDCYTPAGEPIPTNKRIiSMKlFSHPDWVEEPWYGLEQ EYTLLQKDIN WPLGWPIGGFPGPQGPYYCGIGAGKVFGRDIVDSHYKACLYAGINISGINGEVMPGQ GSPr PSVGISA 30 ADELWAARYILERITEIAGVVVSF GDWNGAGAI ITNYSTKSMRNEGGYEVI KK AIENLGLRHKEH IAAVGEGNERRLTGRHETADINTFKWGVANRGASI RVGRDTEREGKGYFEDRRPASN MDPFWI'SMIAET TILSEP PGPGEKHHSIDAQLRQLVPGKVSEDDKLIEYDALUEIFLNILQDLI (GPSIRE Represents Proteins FVQEOIEVSAD of PEPCase genes YEGKGDTTKLGELGAKLTGLAPADAILVASSILI lMlN EVQIAHRRRNSKLKKG GFADEGSATI'E SDIEETLKRLVSEVG KSPEEVFEALKNQTVDLVFTN(PTQSARRSLLQKNARIRNCLTQL DDDK QELDEALQREIQAAFRTDEIRRAQPTPQAEMRYG MSYIHETVWKGVPKFLRRVDTAL KNIGINERLPVNV SLIRFSSWMGGDROGNPRVTPEVTRDVCLLARMMAANLYIDQIEELMFELSMWRCN DELRVRAEELHSSS GSKVTKYYIEFWKQIPPNEPYRVILGIWROKLYNTRERARHLLASGVSEISAESSFTSIEE FLEPLELCY KSLCDCGDKAIADGSLLDLLRQVFTFGLSLVKLDIRQESERHTDVIDAIT‘I’HLGIGSYRE PEPCdsePr WPEDKRQEWL LSELRG KRPLLPPDLPQTDE IADVIGAFHVLAELPPDSFG ATAPSDVLAVELLQR ECGVRQPLP VVPLFERLADLOSAPASVERLFSVDWYM DRIKGKQQVMVGYSDSGKDAGRLSAAW QLYRAQEEMAQVAKR YGVKLTLFHGRGGTVGRGGGPTHLAILSQPPDTINGSIRVTVQGEVI EFCFGEEHLCFQ AATLE HGMHPPVSPKPEWRKLMDEMAWATEEYRSVWKEARFVEYFRSATPETEYGRMNI GSRPAKRRPGGGIT WIFSWTQTRFI ILPVWLGVGAAFKFAIDKDVRNFQVLKEMVNEWPFFRVTLD LLEMVFAKGDPGI AGLYDELLVAEELKPFGKQLR DKYVETQQLLLOJAGHKDILEGDPFLKQGLVLRNPYITI' LNVFQAYTLK RIRDPNFKVTPQPPLSKEFADEN KPAGLVKLNPASEYPPGLEDTLI LTMKGIAAGMQN [Annotation] m 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] m None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam Amplification and cloning ofAspA T gene Nucleotide sequence encoding soyabean cytosolic AspAT gene (SEQ ID NO: 1) was obtained from the NCBI database of tide sequences (GenBank Accession No. AFO34210.1; (hgpzllwwwncbinlm.nih.gov/nuccore/AF034210.l) RNA from soyabean plant was isolated using iRIS Plant RNA Kit (Ghawana et al., US Patent no 0344NF2004/LN). cDNA was sized 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, lnvitrogen, USA) following the manufacturer’s instructions. The full coding region of AspAT was then amplified 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 I) and Pmll G) is incorporated in the coding sequence for AspA T. 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 305econds, extension at 72 °C for 1 minute 20 seconds, with a final ion of 72 °C for 7 s. The amplification product was cloned in to pGEM-T easy vector (Promega, USA). Plasmid from the positive clones and pCAMBIA 1302 plasmid were ed with Bglll and Pmll and digested products isolated from an agarose gel electrophoresis were ligated and transformed in to E. coli DHSa cells which were obtained from Takara Bio Company, Japan (Cat. No. 9057). d 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 AI302 and resulting 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 database of nucleotide sequences (GenBank Accession No. X95932.1; ihttp://www.ncbi.nlm.nih.gov/nuccore/X95932.l) .RNA from o 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 oligo(dT)lz..3 and 400 U of reverse transcriptase cript II (Invitrogen) afier digesting with 2 U DNase I (amplification grade, Invitrogen, USA) following the manufacturer‘s instructions.

[Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by m ation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] m Unmarked set by amandam [Annotation] m None set by m [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by m 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 ement of ‘A’ nucleotide by ‘G ‘ at posiu'on 15.

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 305econds, extension at 72 °C for lminute 10 s, 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 colonies and binary vector pCAMBIA 1302 were digested 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 on 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.

Amplification and cloning of maize PEPCase gene Nucleotide sequence encoding maize PEPCase gene (SEQ ID NO: 3) was ed from the NCBI database of nucleotide ces (NCBI Reference Sequence: NM_OOIlll948.I; (http://www.ncbi.nIm.nih.gov/nuccore/NM 948.” 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 digesting with 2 U DNase I (amplification grade, Invitrogen, USA) following the cturer’s instructions.

The full coding region of PEPCase was amplified fiom maize cDNA using primers PEPCase 331" F with ction 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 denaturating 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 amplification product was cloned in to pGEM-T easy vector (Promega, USA). Plasmid from the positive clones and pCAMBIA 1302 plasmids were digested with BgIII and SpeI and digested product isolated [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by m [Annotation] amandam Unmarked set by amandam [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 from an e gel electrophoresis were ligated and then transformed in to E. coli DHSo cells. Transformants were sequenced to verify the in frame cloning of the PEPCase coding sequence and resulting vector was designated as PEPCasezzpCAMBlA 1302.

EXAMPLE 4 Assembly of expression tes for Asp/1T, GS and e in single pCAMBIA 1302 vector (generous g1]? from “Centre for Application of lar Biology to International Agriculture ", A ustralia) A stepwise method for amplification and integration of expression cassettes each for AspA T, GS and PEPCase in to single plant ormation vector A 1302 is described as follows: GS expression cassette comprising CaMV3SS promoter, downstream cloned GS and nopaline synthase (hereinafler, referred as “Nos”) terminator was amplified from 08:: pCAMBlA 1302 vector ( Example 2 ), using primers 358 5,,“ F( SEQ ID NO: l4) and NosT Am, R (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 e primer to facilitate the subcloning 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 t was cloned in to pGEM-T easy vector (Promega, USA). Plasmids from the positive clones was digested with 81221 and Pmll, and the digested t was then isolated from an agarose gel electrophoresis and ligated in to Spel and PmlI sites of pCAMBIA 1302 vector. The ligation product 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 minutes °C for 30 seconds, , 30 cycles of 94 [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by m [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] m Unmarked set by amandam annealing at 59 °C for 30 s, 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 digestion 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 PEPCase gene from e:: pCAMBIA 1302 vector (example 3) was amplified with the primers 358 A“! F (SEQ ID NO: 17) having restriction site for AscI (GQCGCGCC) and PEPCase awe. R (SEQ ID NO: 18) having restriction site for Bb VCI (CCTCAGC). ‘ Qiagen High ty Taq polymerase enzyme was used for the PCR using the following conditions: initial denaturation at 94 °C for 3 minutes , 30 cycles of 94 °C for 30 seconds, annealing at 60 °C for 30 seconds, ion at 72 °C for 4 minutes, 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 was ed with AscI (GGCGCGCC) and BbVCI (CCTCAGC) and digested product isolated from an agarose gel electrophoresis d upstream of Nos terminator sequence of destination pCAMBIA. 1302 previously cloned with GS and AspAT sion cassettes. The ligation product was transformed in to E. coli DHSa cells and transformants sequenced 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 e. A hygromycin resistance gene (SEQ ID No.6) was included as a selectable marker for screening enic plants.

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

Raising of transgenic 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 s: cDNA sequences encoding soybean AspAT gene (SEQ ID NO: 1), o 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 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 [Annotation] amandam ionNone set by amandam [Annotation] amandam Unmarked set by amandam vector. The elements for sion cassette for AspAT, GS and PEPCase were then amplified and assembled in to ation pCAMBlAl302 such that genes AspAT, GS and PEPCase were controlled by independent CaMV 35$ promoter and Nos transcriptional terminator.

Agrobacterium mediated 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 n R. 1992 using standard triparental mating method.

Briefly, E. coli DHSa cells harboring the recombinant construct AspAT + G8 + PEPCase and those harboring helper plasmid 3 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 inant 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 cteria harboring AspA'I‘ + GS + PEPCase using vaCuum infiltration method. Briefly, liquid S-ml cultures were established from single transformed Agrobaclerium colony and grown in YEM medium supplemented with SOug/ml kanamycin, SOug/ml rifampicin at 28°C up to the late thmic phase. Next, 1 ml of bacterial suspension Was diluted with 100 ml of YEB culture medium supplemented with the same antibiotics. The culture was grown overnight until their optical 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 th 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 erred to growth chamber and grown under controlled long day conditions (16-h light at C and 8-h darkness at 20°C) for seed set.

Selection of y transformant To transgenic Arabidopsis plant: Seeds from transformed plants were surface ized by immersion 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 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 followed by immersion in 10% (v/v) sodium hypochlorite solution. Seeds were then washed four times with sterile led water and sown onto 1% agar containing MS medium supplemented with hygromycin B at a cancentration of 20 pg ml'I (Sigma # H3274). Seeds were then stratified 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 germination. After 14.days, hygromycin resistant seedlings were selected as putative primary ormants (To) sand transferred to pots containing vermiculite, e and at mix (1:111) and grown to maturity under cantrolled conditiou of light, temperature and humidity for growth and seed set.

Raising T1 and '1'; generation AspAT + GS + PEPCase transgenic plants: Seeds harvested from To transgenic plants were germinated on MS + hygromycin B (at a cancentration of 20ug ml") plates and transgenic lines exhibiting a segregation ratio of 3:1 (scored by their ivity to hygromycin B) were selected to raise T1 generation of transgenic plants . gous enic 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 transformed with AspAT + GS + PEPCase Arabidopsis plants from two independent transgenic lines ormed with AspAT + GS + PEPCase were selected to verify the insertion of transgenes in to plant . The genomic DNA was isolated using DNeasy Plant mini kit (QIAGEN Co.). PCR was carried out by using the isolated DNA as te with primers hpt F (SEQ ID NO: 19) and hp! R (SEQ ID NO: 20) annealing to the hygromycin transferaes (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 s, annealing at 58 “C for 30 secOnds, extension at 72 °C for 1 minute, * 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 ation] 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 EXAMPLE? Evaluation of AspAT + GS + PEPCase transgenics by reverse transcriptase - rase chain reaction (RT-PCR) RNA analysis of transformants was done to confirm the expression of Asp/1T, GS and PEPCase. 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 dT).2.lg and 400 U of reverse riptase Superscript II (lnvitrogen) afier digesting with 2 U DNase I (amplification grade, lnvitrogen, USA) following the manufacturer’s instructions). Expression of enes 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 c0ntrol 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 ents wild and L1 and L2 ent two transgenic lines. The amplification of RT—PCR products were ed only in trangenics confirming the expression of introduced genes.

EXAMPLE 8 Enzymatic assays from wild type and AspAT + GS + PEPCasetransgenic opsis plants Enzymatic assays were performed with AspAT + GS + PEPCase enic and wild plants as follows: PEPCase Activity Measurement: Frozen leaf samples (200 mg) ground with a mortar and pestle in lml of extraction buffer containing 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 initiated by the addition of 5 mM PEP.

[Annotation] m 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] m Unmarked set by amandam [Annotation] amandam None set by m [Annotation] amandam MigrationNone set by m [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 assayed in an MDH-coupled reaction essentially as described by Ireland and Joy (1990). Briefly the reaction e 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- tarate. Assay control was run by excluding the 2-oxoglutarate from the reaction mix.

GS Activity Measurement: GS (glutamine synthetase) was extracted in the grinding 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 insoluble PVPP (2% w/v). Enzyme assay was performed as described earlier by Lea et a1. (1990) and the activity was calculated from the standard curve prepared with y—glutamylhydroxamate.

The results of the analyses are shown in the Figure 5A to SC, an se of about 45 to 50% in e activity, 55% in GS ty 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 enic 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 seedlings were transferred to pots ning 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 harvested from 65 —days old plants and dried at 80 °C for 48 hrs. The quantitative 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 analysis 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 .

EXAMPLE 10 Investigation of growth and yield in wild and AspAT+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 60-days old plants. Across ent parameters evaluated, AspAT + GS + PEPCase plants showed enhanced growth ation] m 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] m Unmarked set by m [Annotation] amandam None set by amandam [Annotation] 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. enic 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 transgenic 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 invention provides an innovative approach wherein overexpression of PEPCase provides a carbon skeleton to capture 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 tivity both in terms of plant seed and plant biomass production.

The

Claims (11)

Claims:

1. 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 5 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 ed to wild type or untransformed plant.

2. An expression uct as claimed in claim 1, wherein the control sequence ses 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 d in claim 1, wherein the said control sequence is a constitutive promoter selected from the group ting of CaMV 35S promoter, o promoter, ubiquitin promoter, actin er.

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, n 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 ndently 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 pCAMBIA 1302 and further ligating the 30 ed gene products 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, ng 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 digested plasmids of positive clones of amplified PEPCase 10 coding sequence with the destination pCAMBIA1302 which was previously cloned with the AspAT +GS+ expression cassette as obtained in step (vi) such that the genes AspA, GS and PEPCase were controlled by independent CaMV 35S promoter and Nos riptional terminator to form single plant expression construct AspAT + GS + PEPCase 15 represented by SEQ ID NO: 7.

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

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

9. A process as claimed in claim 7, wherein the transformed plants display an increase of about 45-50% in PEPCase activity, atleast 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 s as claimed in claim 7, wherein the transformed plants as ed to wild type display 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 transformed plants display enhanced growth characteristics terized by increased shoot fresh weight, shoot dry weight, root fresh and dry weight as compared to wild type or untransformed plant.