Abstract:
The assimilated C and N largely influence plant growth and crop yields. Previous attempts to alter the carbon and nitrogen status of the plants attempted with one or two genes The present invention involves simultaneous co-overexpression of three genes wherein one gene (PEPCase) efficiently capture CO2 whereas the other two encode for enzymes (Asp AT and GS) involved in nitrogen assimilation. The combined effect is the enhancement of carbon and nitrogen status of the plant and the productivity.

Description:
[0001]    The following specification particularly describes the invention and the manner in which it is to be performed: 
       FIELD OF THE INVENTION 
       [0002]    The present invention relates to an expression construct for enhancing the carbon (C), nitrogen (N), biomass and yield of plants. 
         [0003]    Further, the present invention provides the process for enhancement of C and N levels and subsequent improvement in the biomass and yield of plant by using the aforesaid expression construct which utilizes co-overexpression of genes from enzymes phosphoenolpyruvate carboxylase (hereinafter, referred as “PEPCase”), glutamine synthetase (hereinafter, referred as “GS”) and aspartate aminotransferase (hereinafter, referred as “AspAT”). In particular, the present invention is directed to transgenic plants where nucleic acid sequences encoding the said proteins are expressed in plant cells. More particularly, the present invention relates to the transformation of a plant with genetic construct involving co-overexpression of three genes wherein one gene PEPCase encodes enzyme responsible to capture CO 2  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 control of constitutive promoter comprising plant  Arabidopsis thaliana  transformed with AspAT+GS+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 
       [0004]    The present invention relates to a transformed plant with co-overexpression of three genes, viz. 
         [0005]    AspAT, GS 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 β-carboxylation of phosphoenolpyruvate (hereinafter, referred as “PEP”) in the presence of HCO 3   −  and Mg 2   +  to yield oxaloacetate (hereinafter, referred as “OAA”) and inorganic phosphate (hereinafter, 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 opening, fruit ripening and seed maturation. The leaves of C4 and CAM plants contain high levels of PEPCase, which catalyze the initial CO 2  fixation of photosynthesis. The much lower levels of PEPCase seen in the leaves of C3 plants contribute to an anaplerotic function and play a role in regulation of the cellular pH. 
         [0006]    GS (EC 6.3.1.2) catalyses the ATP-dependent condensation of ammonia (hereinafter, referred as “NH 3 ”) with glutamate (hereinafter, referred as “Glu”) to produce glutamine (hereinafter, referred as “Gln”). Subsequently, glutamate synthase (GOGAT) transfers the amide group of Gln to α-ketoglutarate producing two molecules of Glu. Both Gln and Glu are the primary source of organic N for proteins, nucleic acid and chlorophyll. 
         [0007]    AspAT (EC 2.6.1.1) catalyzes the reversible transfer of the amino group of asparate (hereinafter, referred as “Asp”) to α-ketoglutarate to form OAA and Glu. In plants, AspAT has been proposed to play several metabolic roles including: recycling of C skeletons during NH 3   +  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. 
         [0008]    Plant performance in terms of biomass production, yield or harvest index depends upon number of internal and environmental factors. Among all these factors, plant C and N level is one of the important factors governing plant productivity. The emerging details 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 status of C, N, biomass and yield in plants. 
         [0009]    Table 1 illustrates the status of information available on the various strategies to improve C and/or N and biomass in different plants. 
         [0000]    
       
         
               
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                   
                 Transformation 
                   
                   
               
               
                 Functions 
                 System adopted 
                 Results 
                 Reference 
               
               
                   
               
             
             
               
                 NAD kinase2 
                   Arabidopsis  NADK2 
                 NADK2 overexpressors 
                 Takahashi, H., 
               
               
                 (NADK2) 
                 overexpressor and 
                 were characterized by 
                 Takahara, K., 
               
               
                 Catalyzes the 
                 nadk2 mutant were 
                 increase in calvin cycle 
                 Hashida, S., 
               
               
                 synthesis of 
                 studied to investigate 
                 intermediates and 
                 Hirabayashi, T., 
               
               
                 NADP from NAD 
                 the impact of altering 
                 amino acid like Glu 
                 Fujimori, T., 
               
               
                 in chloroplasts 
                 NADP level on plant 
                 and Gln. However, 
                 Yamada, M. K., 
               
               
                   
                 metabolism. 
                 there is no clear 
                 Yamaya, T., 
               
               
                   
                   
                 evidence on role of 
                 Yanagisawa, S. 
               
               
                   
                   
                 NADK2 influencing C 
                 and Uchimiy, H. 
               
               
                   
                   
                 and N metabolism. 
                 2009. Plant 
               
               
                   
                   
                   
                 Physiol. 151: 100- 
               
               
                   
                   
                   
                 113. 
               
               
                 Dof 1 
                 Maize Dof1 cDNA was 
                 Dof1 overexpression 
                 Yanagisawa, S., 
               
               
                 Dof1 is a 
                 overexpressed in 
                 in  Arabidopsis  has led 
                 Akiyama, A., 
               
               
                 transcription 
                   Arabidopsis  plants 
                 to co-operative 
                 Kisaka, H., 
               
               
                 activator for 
                 under derivative of 
                 modification of plant C 
                 Uchimiya, H. and 
               
               
                 multiple gene 
                 the 35S promoter 
                 and N content, with 
                 Miwa, T. 2004. 
               
               
                 expressions 
                 designated as 
                 improved growth 
                 Proc. Natl. Acad. 
               
               
                 associated with 
                 35SC4PPDK. 
                 under low N 
                 Sci. USA. 101: 
               
               
                 the organic acid 
                   
                 conditions. However, 
                 7833-7838 
               
               
                 metabolism, 
                   
                 effect of CN 
               
               
                 including 
                   
                 alteration on plant 
               
               
                 PEPCase. 
                   
                 biomass or yield was 
               
               
                   
                   
                 not discussed. 
               
               
                 GS 
                 i.) A soybean cytosolic 
                 Over expression of 
                 Vincent, R., 
               
               
                 GS catalyses the 
                 GS gene (GS15) fused 
                 cytosolic GS 
                 Fraisier, V., 
               
               
                 ATP- dependent 
                 with the constitutive 
                 accelerated plant 
                 Chaillou, S., 
               
               
                 condensation of 
                 CaMV 35S promoter in 
                 development, leading 
                 Limami, M. A., 
               
               
                 NH 3  with (Glu) to 
                 order to direct its over- 
                 to early senescence 
                 Deleens, E., 
               
               
                 produce (Gln). 
                 expression in  Lotus   
                 and premature 
                 Phillipson, B., 
               
               
                   
                   corniculatus  L. plants. 
                 flowering when grown 
                 Douat, C., 
               
               
                   
                   
                 NH 4   +  rich medium. 
                 Boutin, J.-P. and 
               
               
                   
                   
                 Limitation of C 
                 Hirel, B. 
               
               
                   
                   
                 skeleton and energy 
                 1997. Planta. 
               
               
                   
                   
                 for enhanced NH 4   +   
                 201: 424-433. 
               
               
                   
                   
                 assimilation were 
               
               
                   
                   
                 anticipated. 
               
               
                   
                 ii.) A pea cytosolic GS 
                 Overexpression of 
                 Oliveira, I.., 
               
               
                   
                 gene was 
                 cytosolic GS in relation 
                 Brears, T., 
               
               
                   
                 overexpressed in 
                 to N, light and 
                 Knight, T., Clark, 
               
               
                   
                 tobacco plants 
                 photorespiration 
                 A. and Coruzzi, 
               
               
                   
                   
                 suggested an 
                 G. 2002, Plant 
               
               
                   
                   
                 alternative route to 
                 Physiol. 
               
               
                   
                   
                 chloroplastic GS for 
                 129: 1170-1180 
               
               
                   
                   
                 assimilation of 
               
               
                   
                   
                 photorespiratory 
               
               
                   
                   
                 ammonium. 
               
               
                   
                 iii.) Full-length cDNAs 
                 An increased metabolic 
                 Cai, H., Zhou, Y., 
               
               
                   
                 encoding rice cytosolic 
                 level in GS- 
                 Xiao, J., Li, X., 
               
               
                   
                 GS genes (OsGS1;1 
                 overexpressed plants 
                 Zhang, Q. and 
               
               
                   
                 and OsGS1;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 35S 
                 total amino acids and 
               
               
                   
                 promoter. 
                 total N content in the 
               
               
                   
                   
                 whole plant. However, 
               
               
                   
                   
                 decrease in both grain 
               
               
                   
                   
                 yield production and 
               
               
                   
                   
                 total amino acids were 
               
               
                   
                   
                 observed in seeds of 
               
               
                   
                   
                 GS-overexpressed 
               
               
                   
                   
                 plants compared with 
               
               
                   
                   
                 wild-type plants. 
               
               
                   
                 iv) cDNA encoding alfa 
                 Transgenic plants 
                 Fuentes, S., Allen, 
               
               
                   
                 alfa cytosolic GS over 
                 grew better under N 
                 D., Ortiz-Lopez, A. 
               
               
                   
                 expressed in tobacco 
                 starvation by 
                 and Hernandez, 
               
               
                   
                 plants 
                 maintaining 
                 G. 2001. J. Exp. 
               
               
                   
                   
                 photosynthesis at rate 
                 Bot. 52: 1071- 
               
               
                   
                   
                 comparable to those 
                 1081. 
               
               
                   
                   
                 of plants under high N, 
               
               
                   
                   
                 while photosynthesis 
               
               
                   
                   
                 in control plants was 
               
               
                   
                   
                 inhibited by 40-50%. 
               
               
                   
                   
                 These results further 
               
               
                   
                   
                 reflect the need for 
               
               
                   
                   
                 cooperative 
               
               
                   
                   
                 modification of CN 
               
               
                   
                   
                 metabolism for 
               
               
                   
                   
                 developing plants with 
               
               
                   
                   
                 better agronomic 
               
               
                   
                   
                 traits. 
               
               
                 PEPCase 
                 i) The intact maize 
                 Transgenic plants 
                 Agarie, S., Miura, 
               
               
                 PEPCase catalyses 
                 gene encoding C4- 
                 exhibited higher 
                 A., Sumikura, R., 
               
               
                 the β- 
                 specific PEPCase used 
                 PEPCase activity with 
                 Tsukamoto, S., 
               
               
                 carboxylation of 
                 for transformation of 
                 reduced O 2  inhibition 
                 Nose, A., Arima, S., 
               
               
                 PEP in the 
                 rice plants 
                 of photosynthesis. It 
                 Matsuoka, M. and 
               
               
                 presence of HCO 3   −   
                   
                 was found that the 
                 Tokutomi, M. M. 
               
               
                 and Mg 2   +  to yield 
                   
                 reduced O 2  inhibition 
                 2002. Plant Sci. 
               
               
                 OAA and Pi. 
                   
                 photosynthesis was 
                 162: 257-265. 
               
               
                   
                   
                 primarily due to 
               
               
                   
                   
                 reduction of Pi rather 
               
               
                   
                   
                 than increase in the 
               
               
                   
                   
                 partial direct fixation 
               
               
                   
                   
                 of atmospheric CO 2   
               
               
                   
                   
                 via the enhanced 
               
               
                   
                   
                 maize PEPCase. 
               
               
                   
                   
                 However, no report on 
               
               
                   
                   
                 biomass accumulation 
               
               
                   
                   
                 or yield as a 
               
               
                   
                   
                 consequence of 
               
               
                   
                   
                 PEPCase 
               
               
                   
                   
                 overexpression was 
               
               
                   
                   
                 reported. 
               
               
                   
                 ii) Maize PEPCase 
                 Higher levels of maize 
                 Hudspeth, 
               
               
                   
                 introduced in to 
                 PEPCase transcript of 
                 R. L., Grula, 
               
               
                   
                 tobacco plants under 
                 the correct size were 
                 J. W., Dai, Z., 
               
               
                   
                 the control 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 
                 458-464 
               
               
                   
                 promoter. 
                 fold incerase in 
               
               
                   
                   
                 PEPCase activities in 
               
               
                   
                   
                 leaf, transgenic plants 
               
               
                   
                   
                 had significantly 
               
               
                   
                   
                 elevated levels of 
               
               
                   
                   
                 titratable acidity and 
               
               
                   
                   
                 malic acid. However, 
               
               
                   
                   
                 these biochemical 
               
               
                   
                   
                 differences did not 
               
               
                   
                   
                 produce any significant 
               
               
                   
                   
                 physiological changes 
               
               
                   
                   
                 with respect to 
               
               
                   
                   
                 photosynthetic rate or 
               
               
                   
                   
                 CO 2  compensation 
               
               
                   
                   
                 point. 
               
               
                 AspAT 
                 i)  Panicum miliaceum   
                 mAspAT- or cAspAT- 
                 Sentoku, N., 
               
               
                 AspAT 
                 L. mitochondrial and 
                 transformed plants 
                 Taniguchi, M., 
               
               
                 catalyzes 
                 cytosolic AspAT 
                 had about threefold or 
                 Sugiyama, T., 
               
               
                 the reversible 
                 (mAspAT and cAspAT, 
                 3.5-fold higher AspAT 
                 Ishimaru, K., 
               
               
                 transfer of the 
                 respectively) genes 
                 activity in 
                 Ohsugi, R., 
               
               
                 amino group of 
                 were expressed in 
                 the leaf than non- 
                 Takaiwa, F. and 
               
               
                 (Asp) to a- 
                 tobacco plants under 
                 transformed plants, 
                 Toki, S. 2000. 
               
               
                 ketoglutarate to 
                 CaMV 35S promoter. 
                 respectively. 
                 Plant Cell Rep. 
               
               
                 form OAA and 
                   
                 Leaves of both 
                 19: 598-603. 
               
               
                 Glu 
                   
                 transformed plants 
               
               
                   
                   
                 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) 
                 control 
                 Xiao, J. 
               
               
                   
                 and one AspAT gene 
                 plants, the 
                 Li, X., Zhang, Q. 
               
               
                   
                 from  E. coli  (EcAAT) 
                 transformants showed 
                 and Lian, X. 2009. 
               
               
                   
                 were over expressed 
                 significantly increased 
                 Theor Appl 
               
               
                   
                 under CaMV 35S 
                 leaf AspAT activity and 
                 Genet. 118: 1381- 
               
               
                   
                 promoter in rice 
                 greater seed amino 
                 1390 
               
               
                   
                 plants. 
                 acid and protein 
               
               
                   
                   
                 contents. However, 
               
               
                   
                   
                 influence of CN level 
               
               
                   
                   
                 on biomass or yield 
               
               
                   
                   
                 was not discussed. 
               
               
                   
               
             
          
         
       
     
         [0010]    Higher activity of PEPCase shall facilate CO 2  capturing and makes the carbon backbone available for routing of nitrogen in to organic form through joint activity of AspAT and GS. As a result, the inventors have found that object of the present invention can be attained by concomitant increase in expression of genes encoding AspAT, GS and PEPCase to establish the present invention. 
         [0011]    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, Z., Edwards, G. E. and Ku, M. S. B., entitled “Expression of miaze phosphoenolpyruvate carboxylase in transgenic tobacoo” (1992, Plant Physiology, 98: 458-464), wherein PEPCase from maize was expressed under a tobacco ( Nicotiana plumbaginifolia ) chlorophyll a/b binding protein gene promoter in tobacco plants. Up to two fold higher activity of PEPCase was observed in the transgenic leaves as compared to non-transformants with elevated levels of titratable acidity and malic acid. However, these biochemical differences did not produce any significant physiological changes with respect to photosynthetic rate or CO 2  compensation point. 
         [0012]    Reference may be made to article by Lebouteiller, B., Dupont, A. G., Pierre, J. N., Bleton, J., Tchapla, A., Maucourt, M. and Moing, A., Rolin, D., and Vidal, J. entitled “Physiological impacts of modulating phosphoenolpyruvate carboxylase levels in leaves and seeds of  Arabidopsis thaliana ” (2007, Plant Science, 172:256-272,), wherein the PEPCase of sorghum was expressed under CaMV 35S promoter in  Arabidopsis  plant. The leaves of the primary transformants showed up to ten-fold increase in PEPCase activity and up to 30% increase in the dry weight and total protein content of seeds. However, the transformants (primary and progeny) did not show any improved growth phenotype or modification in seed production per plant 
         [0013]    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  changes amino acid metabolism” (2004, Planta, 219: 440-419.), wherein the cyanobacterial  Synechococcus vulcanus  phosphoenolpyruvate carboxylase (SvPEPCase) with diminished sensitivity to feed back inhibition, was over expressed under the control of CaMV 35S promoter in  Arabidopsis  plant. One third of the T 1  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 C 4  photosynthesis, which is normally sensitive to a feedback inhibitor, L-malate. The growth inhibition of SvPEPC transformed T 2  plants was presumed to be primarily due to a decreased availability of phosphoenolpyruvate (PEP), one of the precursors for the shikimate pathway for the synthesis of aromatic amino acids and phenylpropanoids. 
         [0014]    Reference may be made to yet another article by Fukayama, H., Hatch, M. D., Tamai, T., Tsuchida, H., Sudoh, S., Furbank, R. T. and Miyao, M., entitled “Activity regulation and 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 PEPCase in transgenic rice leaves underwent activity regulation through protein phosphorylation in manner similar to endogenous rice PEPCase but contrary to that occurring in maize leaves, being downregulated in the light and upregulated in the dark. Compared with untransformed rice, the level of PEP was slightly lower and the product (OAA) was slightly higher in transgenic rice, suggesting that maize PEPCase was functioning even though it remained dephosphorylated and less active in the light.  14 CO 2  labeling experiments indicated that maize PEPCase did not contribute significantly to the photosynthetic CO 2  fixation of transgenic rice plants. Rather, it slightly lowered the CO 2  assimilation rate. This effect was ascribable to the stimulation of respiration in the light, which was more marked at lower O2 concentrations. It was concluded that overproduction of PEPCase does not directly affect photosynthesis significantly but it suppresses photosynthesis indirectly by stimulating respiration in the light. 
         [0015]    Reference may be made to yet another article by Vincent, R., Fraisier, V., Chaillou, S., Limami, M. A., Deleens, E., Phillipson, B., Douat, C., Boutin, J. P. and Hirel, B., entitled “Overexpression of a soybean gene encoding cytosolic glutamine synthetase in shoots of transgenic  Lotus corniculatus  L. plants triggers changes in ammonium assimilation and plant development” (1997, Planta. 201:424-433), wherein a soyabean cytosolic GS gene GS15 was fused with CaMV 35S promoter to achieve constitutive expression in the  lotus corniculatus  L. plants. On growing the transgenic plants under different N regimes an increase in free amino acids and ammonium was observed accompanied by a decrease in soluble carbohydrates in the transgenic plants cultivated with 12 mM NH 4+  in comparison to the wild type grown under the same conditions. Labelling experiments revealed that both ammonium uptake in the roots and the subsequent translocation of amino acids to the shoots was lower in plants over expressing GS. However the early floral development in the transformed 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 energy for enhanced NH 4   +  assimilation were anticipated. 
         [0016]    Reference may be made to yet another article by Fuentes, S. I., Allen, D. J., Ortiz-Lopez, A. and Hernandez, G., entitled “Overexpression of cytosolic glutamine synthetase increases photosynthesis and growth at low nitrogen conditions” (2001, Journal of Experimental Botany, 52:1071-1081), wherein the alfa alfa GS driven by constitutive CaMV 35S promoter introduced 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 observed. 
         [0017]    Reference may be made to yet another article by Oliveira, I., Brears, T., Knight, T., Clark, A. and Coruzzi, G., entitled “Overexpression of cytosolic glutamine synthetase. Relation to nitrogen, light, and photorespiration” (2002, Plant Physiology, 129: 1170-1180), wherein the overexpression of pea cytosolic GS was studied in relation to nitrogen, light and photorespiration. Tobacco plants, which ectopically overexpress cytosolic GS1 in leaves, display a light-dependent improved growth phenotype under N-limiting and N-non-limiting conditions as evident by increase in fresh weight, dry weight, and leaf soluble protein. The cytosolic GS1 transgenic plants also exhibit an increase in the CO 2  photorespiratory burst and an increase in levels of photorespiratory intermediates, suggesting changes in photorespiration. However, the effect of stimulation of photorespiration by GS overexression on plant productivity was not discussed. 
         [0018]    Reference may be made to yet another article by Cai, H., Zhou, Y., Xiao, J., Li, X., Zhang, Q. and Lian, X., entitled “Overexpressed glutamine synthetase gene modifies nitrogen metabolism and abiotic stress response in rice” (2009, Plant Cell Reports. 28: 527-537), wherein the full-length cDNAs encoding rice ( Oryza sativa ) cytosolic GS genes (OsGS1;1 and OsGS1;2) along with  E. coli  GS gene (glnA) were overexpressed in the rice plant under constitutive CaMV 35S promoter. An increased metabolic level in GS-overexpressed plants was obtained, which showed higher total GS activities and soluble protein concentrations in leaves and higher total amino acids and total N content in the whole plant. However, decrease in both grain yield production and total amino acids were observed in seeds of GS-overexpressed plants compared with wild-type plants. 
         [0019]    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: 598-603), wherein the effects of the overexpression of  Panicum  mitochondrial and cytoplasmic AspAT (mAspAT and cAspAT respectively) under the control of CaMV 35S promoter were evaluated 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. Interestingly, 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. 
         [0020]    Reference may be made to yet another article by Zhou, Y., Cai, H., Xiao, J., Li, X., Zhang, Q. and Lian, X., entitled “Over-expression of aspartate aminotransferase genes in rice resulted in altered nitrogen metabolism and increased amino acid content in seeds” (2009, Theoretical and Applied Genetics, 118:1381-1390), wherein three AspAT genes from rice (OsAAT1-3) encoding chloroplastic, cytoplasmic, and mitochondrial AspAT isoenzymes, respectively and one AspAT gene from  E. coli  (EcAAT) were overexpressed in rice plant under the control of CaMV 35S promoter. The OsAAT1, OsAAT2, and EcAAT transformants showed significantly increased 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 content in OsAAT3 over-expressed plants. 
         [0021]    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), wherein AspAT5 encoding the chloroplast AspAT from Soyabean was linked to CaMV 35S promoter 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 T 3  seeds. However, a decrease in the contents of valine, tyrosine, isoleucine, leucine, and phenylalanine by several folds was also observed. Further, there is no report on effect of overexpression of AspAt on plant growth and productivity. 
         [0022]    Reference may be made to yet another article by Yanagisawa, S., Akiyama, A., Kawaka, H., Uchimiya, H. and Miwa, T. entitled “Metabolic engineering with Dof1 transcription factor in plants: Improved nitrogen assimilation and growth under low-nitrogen conditions” (2004, Proceedings of the National Academy of Sciences (USA), 101:7833-7838), wherein over-expression of Dof1 transcription factor from maize improves N assimilation in transgenic  Arabidopsis  plants. Dof1 expressing plants showed up-regulation of genes encoding enzymes for C skeleton production, a marked increase of amino acid contents, and a reduction of the glucose level. The results suggest cooperative modification of C and N metabolisms on the basis of their intimate link. Elementary analysis revealed that the N content increased in the Dof1 transgenic plants (≈30%), indicating promotion of net N assimilation. However, effect of C N alteration on plant biomass or yield was not discussed. 
         [0023]    Reference may be made to still another article by Takahashi, H., Takahara, K., Hashida, S., Hirabayashi, T., Fujimori, T., Kawai-Yamada, M., Yamaya, T., Yanagisawa, 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, accompanied by an increase in overall Rubisco activity. Furthermore, enhanced NADP(H) production due to NADK2 overexpression increased N assimilation. Gln and Glu concentrations, 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. 
         [0024]    The improvement in the C and N status of plants is a major concern to improve productivity. However, there is no report yet which show enhancement of C and N levels and subsequent improvement in the biomass and yield of plant. 
         [0025]    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. 
       OBJECTIVES OF THE INVENTION 
       [0026]    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 of the hitherto known prior art as detailed above. 
         [0027]    Another objective of the present invention is to provide an expression construct for co-overexpression of AspAT (SEQ ID NO: 1), GS (SEQ ID NO: 2). and PEPcase (SEQ ID NO: 3) wherein PEPCase efficiently captures CO 2  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 enhancement of C and N status with improved biomass and yield of plants. 
         [0028]    Yet another objective of the present invention is to raise transgenic plant exhibiting co-overexpression of genes AspAT, GS and PEPCase. 
         [0029]    Still another objective of the present invention is to evaluate the expression of AspAT, GS and PEPCase genes in transgenic plants. 
         [0030]    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 
       [0031]    Accordingly, the present invention provides an expression construct represented by SEQ ID NO. 7 for co-expression of the genes AspAT, GS and PEPCase comprising nucleotide sequences represented by SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, wherein SEQ ID NO: 1 represents AspAT genes, SEQ ID NO: 2 represents GS genes and SEQ ID NO: 3 represents PEPCase genes 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. 
         [0032]    In an embodiment of the present invention, the control sequence is preferably represented by SEQ ID NO: 4. 
         [0033]    In another embodiment of the present invention, the transcription terminator sequence is represented by SEQ ID NO: 5. 
         [0034]    In an embodiment, the present invention provides an expression construct prepared from the cytosolic AspATgene from soyabean, cytosolic GS gene from tobacoo and cytosolic PEPCase gene from maize. 
         [0035]    In another embodiment of the present invention, the polynucleotide having SEQ ID No: 7 is overexpressed in plants. 
         [0036]    In still another embodiment of the present invention, the control sequence used is a constitutive promoter selected from the group consisting of CaMV 35S promoter, rubisco promoter, ubiquitin promoter, actin promoter. 
         [0037]    In still another embodiment of the present invention, the terminator used is preferably selected from the group consisting of Nos terminator and CaMV 3′ UTR. 
         [0038]    In still another embodiment of the present invention, a process for preparing the expression construct wherein the process comprising the steps of:
       i) amplifying cDNA sequences encoding genes represented by SEQ ID NO: 1 using primers represented by SEQ ID NO: 10 and SEQ ID NO: 11, SEQ ID NO: 2 using primers represented by SEQ ID NO: 8 and SEQ ID NO: 9 and SEQ ID NO: 3 using primers represented by SEQ ID NO: 12 and SEQ ID NO: 13;   ii) cloning independently the amplified product of SEQ ID NO: 1, 2 and 3 as obtained in
           step (i) into pGEM-T easy vector;   
           iii) digesting independently the plasmid from the positive clones as obtained in step (ii) along with pCAMBIA 1302 and further ligating the digested 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.   v) amplifying the products obtained in step (iv) by using primers represented by SEQ ID NO: 10 and SEQ ID NO: 16; SEQ ID NO: 14 and SEQ ID NO: 15 and SEQ ID NO: 17 and SEQ ID NO: 18.   vi) cloning, digesting, ligating and sequencing was again performed 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 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 transcriptional terminator to form single plant expression construct AspAT+GS+PEPCase represented by SEQ ID NO: 7.       
 
         [0047]    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 transformed explants of step (b) to obtain the desired transformed plants having enhanced level of carbon, nitrogen, biomass and yield of plants as compared to wild type plant.   In still another embodiment of the present invention, a process wherein the transformed plants display an increase of about 45-50% in PEPCase activity, at least 55% in GS activity and 55-60% in AspAT activity as compared to wild type, resulting in increase in carbon and nitrogen levels in the plant.       
 
         [0052]    In another embodiment of the present invention, the  Agrobacterium  strain provided is selected from a group consisting of GV3101 with ATCC number  Agrobacterium tumefaciens  (GV3101 (pMP90RK) (C58 derivative) ATCC® Number: 33970 Reference: Hayashi H, Czaja I, Lubenow H, Schell J, Walden R. 1992. 
         [0053]    In yet another embodiment of the present invention, the transformed plants are selected from the group consisting of grain crops, pulses, vegetable crops, oilseed crop and ornamentals. 
         [0054]    In yet another embodiment, the transformed plants are selected from the group consisting of  arabidopsis , tomato, potato, tobacco, maize, wheat, rice, cotton, mustard, pigeon pea, cowpea, pea, sugarcane, soya bean and sorghum. 
         [0055]    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. 
         [0056]    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. 
         [0057]    In yet another embodiment of the present invention, the transformed plant shows enhanced levels of carbon, nitrogen, biomass and yield as compared to wild plants. 
         [0058]    In still another embodiment of the present invention, the expression and functionality of over expressed enzymes in transgenic plants is evaluated. 
         [0059]    In yet another embodiment of the present invention, the selectable marker used is hpt gene (hygromycin phosphotransferase) represented by SEQ ID NO: 6 for hygromycin resistance controlled by duplicated CaMV 35S promoter and terminated by CaMV 3′UTR (polyA signal). 
         [0060]    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. 
         [0061]    In a further embodiment of the present invention, the transgenic plants were investigated for different growth and yield parameters and compared to wild plants cultivated under the same conditions. 
     
    
     
       BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS 
         [0062]      FIG. 1  represents a schematic view of T-DNA region of plant transformation vector pCAMBIA1302 for co-overexpression of AspAT, GS and PEPCase (a) and amplification of coding sequences for AspAT, GS and PEPCase from respective plant sources (b) as discussed in Examples 1 to 4. 
           [0063]      FIG. 2  represents DNA analysis (a) and RNA analysis (b) of WT, L1 and L2, where WT=wild; L1 and L2=two different transgenic lines co-overexpressing AspAT, GS and PEPCase. 
           [0064]      FIG. 3  represents 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 days of sowing. Data is mean of five separate biological replicates with standard deviation marked on each bar. 
           [0065]      FIG. 4  represents AspAT activity (a) GS activity (b) and PEPCase activity (d) of WT, L1 and L2 at 42 days of sowing. Data is mean of three separate biological replicates with standard deviation marked on each bar. 
           [0066]      FIG. 5  represents Analyses of N (a) and C (b) content from different plant parts of WT, L1 and L2 lines at 65 days of sowing. Data is mean of three separate biological replicates with standard deviation marked on each bar. 
           [0067]      FIG. 6  represents a representative WT and AspAT+GS+PEPCase transgenic plants at 75 days of sowing. 
           [0068]      FIG. 7  represents pod number (a) and seed yield (b) in WT, L1 and L2 at 75 days of sowing. Data is mean of five separate biological replicated with standard deviation marked on each bar. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0069]    The present invention 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 PEPCase 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. 
         [0070]    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  E. coli . The vector in the present invention was used to transfer the gene from one source to another. 
         [0071]    The term “gene” refers to the sequence of nucleic acids that can produce a polypeptide chain. 
         [0072]    The term “gene expression” refers to the level/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. sequence of deoxyribonucleic acid). When the gene was transcribed in higher amounts as compared to the control, it was referred to as “over-expression” of gene. 
         [0073]    The term “selectable marker” refers to a gene, which allows a cell to survive in the presence of an otherwise toxic antibiotic 
         [0074]    The term “transgenic plant” refers to genetically transformed plants with stable integration of introduced gene in to its genome The term “promoter” refers to the specific DNA sequence, usually located upstream (5′) to the DNA sequence involved in transcription, 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 surrounding environment and development stage of the organism. 
         [0075]    The term ‘expression cassettes” refers to vector comprising of (a) a constitutive promoter; (b) all the three genes cloned 3′ to the constitutive promoter, (c) a polyadenylation signal located 3′ to the coding sequence. 
         [0076]    and capable of passing genetic information on to successive generations. 
         [0077]    ‘Wild-type” plants are untransformed plants. 
         [0078]    The term “T 0 ” refers to the first set of genetically transformed plants that can be identified and selected upon growth in presence of a selection agent antibiotic, for which the transgenic plant contains the corresponding resistance gene. The term “T 1 ” refers to the generation of plants obtained after self-fertilization of the flowers of T 0  generation plants, previously selected as being transgenic. “T 2 ” plants are generated from T 1  plants, and so on. The present invention will be illustrated in greater details by the following examples. 
       EXAMPLES 
       [0079]    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. 
         [0080]    Sequences of the primers used in the present invention are listed as follows: 
         [0000]    
       
         
               
               
               
               
             
           
               
                   
               
               
                   
                   
                   
                 Se- 
               
               
                 Name 
                   
                   
                 quence  
               
               
                 of the 
                   
                   
                 ID 
               
               
                 sequence 
                 Sequence 
                 Purpose 
                 No. 
               
               
                   
               
             
             
               
                 AspAT  
                 atggcttctc acgacagcat ctccgcttct ccaacctccg cttctgattc cgtcttcaat 60 
                 Represents 
                 1 
               
               
                 cDNA 
                 cacctcgttc gtgctcccga agatcctatc ctcggggtaa ctgtcgctta taacaaagat 120 
                 nucleotide 
                   
               
               
                 sequence 
                 ccaagtccag ttaagctcaa cttgggagtt ggtgcttacc gaactgagga aggaaaacct 180 
                 sequences 
                   
               
               
                   
                 cttgttttga atgtagtgag gcgagttgaa cagcaactca taaatgacgt gtcacgcaac 240 
                 of AspAT  
                   
               
               
                   
                 aaggaatata ttccgatcgt tgggcttgct gattttaata aattgagtgc taagcttatt 300 
                 genes 
                   
               
               
                   
                 tttggggctg acagccctgc tattcaagac aacagggtta ccactgttca atgcttgtct 360 
                 for 
                   
               
               
                   
                 ggaactggtt ctttaagagt tgggggtgaa tttttggcta aacactatca ccaacggact 420 
                 making 
                   
               
               
                   
                 atatacttgc caacaccaac ttggggcaat cacccgaagg ttttcaactt agcaggcttg 480 
                 an 
                   
               
               
                   
                 tctgtcaaaa cataccgcta ctatgctcca gcaacacgag gacttgactt tcaaggactt 540 
                 expression 
                   
               
               
                   
                 ctggaagacc ttggttctgc tccatctgga tctattgttt tgctacatgc atgcgcacat 600 
                   
                   
               
               
                   
                 aaccccactg gtgtggatcc aacccttgag caatgggagc 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 
                   
                   
               
               
                   
                 atcaaacaga ttggaatgtt tactttcact ggattgaatg cggaacaagt ttccttcatg 1140 
                   
                   
               
               
                   
                 actaaagagt tccatatata catgacatct gatgggagga ttagcatggc tggtctgagt 1200 
                   
                   
               
               
                   
                 tccaaaactg tcccacttct ggcggatgcg atacatgcag ctgtaacccg agttgtctaa 1260 
                   
                   
               
               
                   
               
               
                 GS  
                 atggctcatc tttcagatct cgttaatctc aatctctctg actccactca gaaaattatt 60 
                 Represents 
                 2 
               
               
                 cDNA 
                 gctgaataca tatggattgg tggatcagga atggacgtca ggagcaaagc cagaacactt 120 
                 nucleotide 
                   
               
               
                 sequence 
                 tctggacctg ttgatgatcc ttcaaagctt cccaaatgga attatgatgg ttctagcaca 180 
                 sequences 
                   
               
               
                   
                 ggacaagctc ctggagaaga cagtgaagag atcctatatc ctcaagcaat tttcaaggat 240 
                 of GS  
                   
               
               
                   
                 ccattcagaa ggggcaacaa tatcttggtc atttgtgatt gttacacccc agctggtgaa 300 
                 genes 
                   
               
               
                   
                 cccattccaa caaacaaaag gcacagtgct gccaagattt tcagccaccc tgatgttgtt 360 
                 for   
                   
               
               
                   
                 gttgaggaac cctggtatgg tcttgagcaa gaatacacct tgttgcaaaa agatatcaat 420 
                 making 
                   
               
               
                   
                 tggcctcttg gatggcctct tggtggtttt cctggaccac agggaccata ctattgcgga 480 
                 an 
                   
               
               
                   
                 attggagctg gaaaggtctt tggacgcgat atcgttgact ctcattataa ggcatgtctc 540 
                 expression 
                   
               
               
                   
                 tatgctggga ttaacatcag tggtatcaat ggagaagtga tgcccggaca gtgggaattt 600 
                 construct 
                   
               
               
                   
                 caagttggac cttcagttgg catttcagca gctgatgaat tgtgggcagc tcgttacatt 660 
                   
                   
               
               
                   
                 cttgagagga ttactgagat tgctggagtt gtggtctcat ttgaccccaa acctattccg 720 
                   
                   
               
               
                   
                 ggtgactgga atggtgctgg agctcacaca aactacagca caaagtctat gaggaatgaa 780 
                   
                   
               
               
                   
                 ggaggctatg aagtcattaa gaaggcaatt gagaaccttg gactgaggca 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 
                 ctcgtcgacc gcttcctcaa catcctccag gacctccacg ggcccagcct tcgcgaattt 180 
                 Represents 
                 3 
               
               
                 cDNA 
                 gtccaggagt gctacgaggt ctcagccgac tacgagggca aaggagacac gacgaagctg 240 
                 nucleotide 
                   
               
               
                 sequence 
                 ggcgagctcg gcgccaagct cacggggctg gcccccgccg acgccatcct cgtggcgagc 300 
                 sequences 
                   
               
               
                   
                 tccatcctgc acatgctcaa cctcgccaac ctggccgagg aggtgcagat cgcgcaccgc 360 
                 of   
                   
               
               
                   
                 cgccgcaaca gcaagctcaa gaaaggtggg ttcgccgacg agggctccgc caccaccgag 420 
                 PEPCase 
                   
               
               
                   
                 tccgacatcg aggagacgct caagcgcctc gtgtccgagg tcggcaagtc ccccgaggag 480 
                 genes 
                   
               
               
                   
                 gtgttcgagg cgctcaagaa ccagaccgtc gacctcgtct tcaccgcgca tcctacgcag 540 
                 for  
                   
               
               
                   
                 tccgcccgcc gctcgctcct gcaaaaaaat gccaggatcc gaaattgtct gacccagctg 600 
                 making 
                   
               
               
                   
                 aatgccaagg acatcactga cgacgacaag caggagctcg atgaggctct gcagagagag 660 
                 an  
                   
               
               
                   
                 atccaagcag ccttcagaac cgatgaaatc aggagggcac aacccacccc gcaggccgaa 720 
                 expression 
                   
               
               
                   
                 atgcgctatg ggatgagcta catccatgag actgtatgga agggtgtgcc taagttcttg 780 
                 construct 
                   
               
               
                   
                 cgccgtgtgg atacagccct gaagaatatc ggcatcaatg agcgccttcc ctacaatgtt 840 
                   
                   
               
               
                   
                 tctctcattc ggttctcttc ttggatgggt ggtgaccgcg atggaaatcc aagagttacc 900 
                   
                   
               
               
                   
                 ccggaggtga caagagatgt atgcttgctg gccagaatga tggctgcaaa cttgtacatc 960 
                   
                   
               
               
                   
                 gatcagattg aagagctgat gtttgagctc tctatgtggc gctgcaacga tgagcttcgt 1020 
                   
                   
               
               
                   
                 gttcgtgccg aagagctcca cagttcgtct ggttccaaag ttaccaagta ttacatagaa 1080 
                   
                   
               
               
                   
                 ttctggaagc aaattcctcc aaacgagccc taccgggtga tactaggcca tgtaagggac 1140 
                   
                   
               
               
                   
                 aagctgtaca acacacgcga gcgtgctcgc catctgctgg cttctggagt ttctgaaatt 1200 
                   
                   
               
               
                   
                 tcagcggaat cgtcatttac cagtatcgaa gagttccttg agccacttga gctgtgctac 1260 
                   
                   
               
               
                   
                 aaatcactgt gtgactgcgg cgacaaggcc atcgcggacg ggagcctctt ggacctcctg 1320 
                   
                   
               
               
                   
                 cgccaggtgt tcacgttcgg gctctccctg gtgaagctgg acatccggca ggagtcggag 1380 
                   
                   
               
               
                   
                 cggcacaccg acgtgatcga cgccatcacc acgcacctcg gcatcgggtc gtaccgcgag 1440 
                   
                   
               
               
                   
                 tggcccgagg acaagaggca ggagtggctg ctgtcggagc tgcgaggcaa gcgcccgctg 1500 
                   
                   
               
               
                   
                 ctgcccccgg accttcccca gaccgacgag atcgccgacg tcatcggcgc gttccacgtc 1560 
                   
                   
               
               
                   
                 ctcgcggagc tcccgcccga cagcttcggc ccctacatca tctccatggc gacggccccc 1620 
                   
                   
               
               
                   
                 tcggacgtgc tcgccgtgga gctcctgcag cgcgagtgcg gcgtgcgcca gccgctgccc 1680 
                   
                   
               
               
                   
                 gtggtgccgc tgttcgagag gctggccgac ctgcagtcgg cgcccgcgtc cgtggagcgc 1740 
                   
                   
               
               
                   
                 ctcttctcgg tggactggta catggaccgg atcaagggca agcagcaggt catggtcggc 1800 
                   
                   
               
               
                   
                 tactccgact ccggcaagga cgccggccgc ctgtccgcgg cgtggcagct gtacagggcg 1860 
                   
                   
               
               
                   
                 caggaggaga tggcgcaggt ggccaagcgc tacggcgtca agctcacctt gttccacggc 1920 
                   
                   
               
               
                   
                 cgcggaggca ccgtgggcag gggtggcggg cccacgcacc ttgccatcct gtcccagccg 1980 
                   
                   
               
               
                   
                 ccggacacca tcaacgggtc catccgtgtg acggtgcagg gcgaggtcat cgagttctgc 2040 
                   
                   
               
               
                   
                 ttcggggagg agcacctgtg cttccagact ctgcagcgct tcacggccgc cacgctggag 2100 
                   
                   
               
               
                   
                 cacggcatgc acccgccggt ctctcccaag cccgagtggc gcaagctcat ggacgagatg 2160 
                   
                   
               
               
                   
                 gcggtcgtgg ccacggagga gtaccgctcc gtcgtcgtca aggaggcgcg cttcgtcgag 2220 
                   
                   
               
               
                   
                 tacttcagat cggctacacc ggagaccgag tacgggagga tgaacatcgg cagccggcca 2280 
                   
                   
               
               
                   
                 gccaagagga ggcccggcgg cggcatcacg accctgcgcg ccatcccctg gatcttctcg 2340 
                   
                   
               
               
                   
                 tggacccaga ccaggttcca cctccccgtg tggctgggag tcggcgccgc attcaagttc 2400 
                   
                   
               
               
                   
                 gccatcgaca aggacgtcag gaacttccag gtcctcaaag agatgtacaa cgagtggcca 2460 
                   
                   
               
               
                   
                 ttcttcaggg tcaccctgga cctgctggag atggttttcg ccaagggaga ccccggcatt 2520 
                   
                   
               
               
                   
                 gccggcttgt atgacgagct gcttgtggcg gaagaactca agccctttgg gaagcagctc 2580 
                   
                   
               
               
                   
                 agggacaaat acgtggagac acagcagctt ctcctccaga tcgctgggca caaggatatt 2640 
                   
                   
               
               
                   
                 cttgaaggcg atccattcct gaagcagggg ctggtgctgc gcaaccccta catcaccacc 2700 
                   
                   
               
               
                   
                 ctgaacgtgt tccaggccta cacgctgaag cggataaggg accccaactt caaggtgacg 2760 
                   
                   
               
               
                   
                 ccccagccgc cgctgtccaa 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 
                 4 
               
               
                 promoter 
                 gttcatacag agtctcttac gactcaatga caagaagaaa atcttcgtca acatggtgga 120 
                 control 
                   
               
               
                 sequence 
                 gcacgacaca cttgtctact ccaaaaatat caaagataca gtctcagaag accaaagggc 180 
                 sequence 
                   
               
               
                   
                 aattgagact tttcaacaaa gggtaatatc cggaaacctc ctcggattcc attgcccagc 240 
                   
                   
               
               
                   
                 tatctgtcac tttattgtga agatagtgga aaaggaaggt ggctcctaca aatgccatca 300 
                   
                   
               
               
                   
                 ttgcgataaa ggaaaggcca tcgttgaaga tgcctctgcc gacagtggtc ccaaagatgg 360 
                   
                   
               
               
                   
                 acccccaccc acgaggagca tcgtggaaaa agaagacgtt ccaaccacgt cttcaaagca 420 
                   
                   
               
               
                   
                 agtggattga tgtgatatct ccactgacgt aagggatgac gcacaatccc actatccttc 480 
                   
                   
               
               
                   
                 gcaagaccct tcctctatat aaggaagttc atttcatttg gagagaacac gggggact   538 
                   
                   
               
               
                   
               
               
                 nos 
                 cgttcaaaca tttggcaata aagtttctta agattgaatc ctgttgccgg tcttgcgatg 60 
                 Represents 
                 5 
               
               
                 (nopaline 
                 attatcatat aatttctgtt gaattacgtt aagcatgtaa taattaacat gtaatgcatg 120 
                 tran- 
                   
               
               
                 synthase) 
                 acgttattta tgagatgggt ttttatgatt agagtcccgc aattatacat ttaatacgcg 180 
                 scription 
                   
               
               
                 3′UTR 
                 atagaaaaca aaatatagcg cgcaaactag gataaattat cgcgcgcggt gtcatctatg 240 
                 terminator 
                   
               
               
                 (poly- 
                   
                 sequence 
                   
               
               
                 Asignal) 
                   
                   
                   
               
               
                 sequence 
                   
                   
                   
               
               
                   
               
               
                 hygro- 
                 ctatttcttt gccctcggac gagtgctggg gcgtcggttt ccactatcgg cgagtacttc 60 
                 Represents 
                 6 
               
               
                 mycin- 
                 tacacagcca tcggtccaga cggccgcgct tctgcgggcg atttgtgtac gcccgacagt 120 
                 hpt gene 
                   
               
               
                 phospho- 
                 cccggctccg gatcggacga ttgcgtcgca tcgaccctgc gcccaagctg catcatcgaa 180 
                 (hygro- 
                   
               
               
                 trans- 
                 attgccgtca accaagctct gatagagttg gtcaagacca atgcggagca tatacgcccg 240 
                 mycin 
                   
               
               
                 ferase 
                 gagtcgtggc gatcctgcaa gctccggatg cctccgctcg aagtagcgcg tctgctgctc 300 
                 phospho- 
                   
               
               
                   
                 catacaagcc aaccacggcc tccagaagaa gatgttggcg acctcgtatt gggaatcccc 360 
                 trans- 
                   
               
               
                   
                 gaacatcgcc tcgctccagt caatgaccgc tgttatgcgg ccattgtccg tcaggacatt 420 
                 ferase) 
                   
               
               
                   
                 gttggagccg aaatccgcgt gcacgaggtg ccggacttcg gggcagtcct cggcccaaag 480 
                 for  
                   
               
               
                   
                 catcagctca tcgagagcct gcgcgacgga cgcactgacg gtgtcgtcca tcacagtttg 540 
                 hygromycin 
                   
               
               
                   
                 ccagtgatac acatggggat cagcaatcgc gcatatgaaa tcacgccatg tagtgtattg 600 
                 resistance 
                   
               
               
                   
                 accgattcct tgcggtccga atgggccgaa cccgctcgtc tggctaagat cggccgcagc 660 
                   
                   
               
               
                   
                 gatcgcatcc atagcctccg cgaccggttg tagaacagcg ggcagttcgg tttcaggcag 720 
                   
                   
               
               
                   
                 gtcttgcaac gtgacaccct gtgcacggcg ggagatgcca taggtcaggc tctcgctaaa 780 
                   
                   
               
               
                   
                 ctccccaatg tcaagcactt ccggaatcgg gagcgcggcc gatgcaaagt gccgataaac 840 
                   
                   
               
               
                   
                 ataacgatct ttgtagaaac catcggcgca gctatttacc cgcaggacat atccacgccc 900 
                   
                   
               
               
                   
                 tcctacatcg aagctgaaag cacgagattc ttcgccctcc gagagctgca tcaggtcgga 960 
                   
                   
               
               
                   
                 gacgctgtcg aacttttcga tcagaaactt ctcgacagac gtcgcggtga gttcaggctt 1020 
                   
                   
               
               
                   
                 tttcat 1026 
                   
                   
               
               
                   
               
               
                 express- 
                 
                   
                     catggagtcaaagattcaaatagaggacctaacagaactcgccgtaaagactggcgaacagttcataca 
                   
                 
                 Represents 
                 7 
               
               
                 ion  
                 
                   
                     gagtctcttacgactcaatgacaagaagaaaatcttcgtcaacatggtggagcacgacacacttgtctact 
                   
                 
                 an  
                   
               
               
                 cassettes 
                 
                   
                     ccaaaaatatcaaagatacagtctcagaagaccaaagggcaattgagacttttcaacaaagggtaatatc 
                   
                 
                 expression 
                   
               
               
                 for  
                 
                   
                     cggaaacctcctcggattccattgcccagctatctgtcactttattgtgaagatagtggaaaaggaaggtgg 
                   
                 
                 construct  
                   
               
               
                 AspAT,  
                 
                   
                     ctcctacaaatgccatcattgcgataaaggaaaggccatcgttgaagatgcctctgccgacagtggtccca 
                   
                 
                 for 
                   
               
               
                 GS and 
                 
                   
                     aagatggacccccacccacgaggagcatcgtggaaaaagaagacgttccaaccacgtcttcaaagcaag 
                   
                 
                 co-  
                   
               
               
                 PEPCase 
                 
                   
                     tggattgatgtgatatctccactgacgtaagggatgacgcacaatcccactatccttcgcaagaccct 
                   
                 
                 expression 
                   
               
               
                 coding 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 of 
                   
               
               
                 se- 
                 gcttctcacgacagcatctccgcttctccaacctccgcttctgattccgtcttcaatcacctcgttcgtg 
                 the genes 
                   
               
               
                 quences, 
                 ctcccgaagatcctatcctcggggtaactgtcgcttataacaaagatccaagtccagttaagctcaacttggg 
                 AspAT, 
                   
               
               
                 cloned  
                 agttggtgcttaccgaactgaggaaggaaaacctcttgttttgaatgtagtgaggcgagttgaacagcaact 
                 GS and  
                   
               
               
                 under 
                 cataaatgacgtgtcacgcaacaaggaatatattccgatcgttgggcttgctgattttaataaattgagtgct 
                 PEPCase 
                   
               
               
                 control  
                 aagcttatttttggggctgacagccctgctattcaagacaacagggttaccactgttcaatgcttgtctggaac 
                 SpeI 
                   
               
               
                 of 
                 tggttctttaagagttgggggtgaatttttggctaaacactatcaccaacggactatatacttgccaacaccaa 
                   
                   
               
               
                 CamV 35S 
                 cttggggcaatcacccgaaggttttcaacttagcaggcttgtctgtcaaaacataccgctactatgctccagc 
                   
                   
               
               
                 promoter 
                 aacacgaggacttgactttcaaggacttctggaagaccttggttctgctccatctggatctattgttttgctaca 
                   
                   
               
               
                 (   )  
                 tgcatgcgcacataaccccactggtgtggatccaacccttgagcaatgggagcagattaggcagctaataag 
                   
                   
               
               
                 and 
                 atcaaaagctttgttacctttctttgacagtgcttatcagggttttgctagtggaagtctagatgcagatgccca 
                   
                   
               
               
                 Nos 
                 acctgttcgtttgtttgttgctgatggaggcgaattgctggtagcacaaagctatgcaaagaatctgggtcttt 
                   
                   
               
               
                 termin- 
                 atggggaacgtgttggcgccttaagcattgtctgcaagtcagctgatgttgcaagcagggttgagagccagc 
                   
                   
               
               
                 ator 
                 tgaagctagtgattaggcccatgtactcaagtcctcccattcatggtgcatccattgtggctgccattctcaag 
                   
                   
               
               
                 (   ) in 
                 gaccggaatttgttcaatgactggactattgagttgaaggcaatggctgatcgcatcatcagtatgcgccaag 
                   
                   
               
               
                 pCAMBIA 
                 aacttttcgatgctttatgttccagaggcacacctggcgattggagtcacattatcaaacagattggaatgttt 
                   
                   
               
               
                 1302 
                 actttcactggattgaatgcggaacaagtttccttcatgactaaagagttccatatatacatgacatctgatgg 
                 PrnII 
                   
               
               
                   
                 gaggattagcatggctggtctgagttccaaaactgtcccacttctggcggatgcgatacatgcagctgtaacc 
                   
                   
               
               
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
                   
                   
               
               
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
                   
                   
               
               
                   
                 
                   
                     ctgttgccggtcttgcgatgattatcatataatttctgttgaattacgttaagcatgtaataattaacatgtaa 
                   
                 
                   
                   
               
               
                   
                 
                   
                     tgcatgacgttatttatgagatgggtttttatgattagagtcccgcaattatacatttaatacgcgatagaaa 
                   
                 
                   
                   
               
               
                   
                 
                   
                     acaaaatatagcgcgcaaactaggataaattatcgcgcgcggtgtcatctatgt 
                   
                 
                   
                   
               
               
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
                   
                   
               
               
                   
                 gccttcagtttagctt   catggagtcaaagattcaaatagaggacctaacagaactcgccgtaaagactggc     
                   
                   
               
               
                   
                 
                   
                     gaacagttcatacagagtctcttacgactcaatgacaagaagaaaatcttcgtcaacatggtggagcacg 
                   
                 
                   
                   
               
               
                   
                 
                   
                     acacacttg 
                   
                 
                   
                   
               
               
                   
                 
                   
                     tctactccaaaaatatcaaagatacagtctcagaagaccaaagggcaattgagacttttcaacaaagggt 
                   
                 
                   
                   
               
               
                   
                 
                   
                     aatatccggaaacctcctcggattccattgcccagctatctgtcactttattgtgaagatagtggaaaagga 
                   
                 
                   
                   
               
               
                   
                 
                   
                     aggtggctcctacaaatgccatcattgcgataaaggaaaggccatcgttgaagatgcctctgccgacagtg 
                   
                 
                   
                   
               
               
                   
                 
                   
                     gtcccaaagatggacccccacccacgaggagcatcgtggaaaaagaagacgttccaaccacgtcttcaaa 
                   
                 
                 NeaI 
                   
               
               
                   
                 
                   
                     gcaagtggattgatgtgatatctccactgacgtaagggatgacgcacaatcccactatccttcgcaagacc 
                   
                 
                   
                   
               
               
                   
                 
                   
                     c 
                   
                 
                   
                   
               
               
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
                   
                   
               
               
                   
                 gatctcgttaatctcaatctctctgactccactcagaaaattattgctgaatacatatggattggtggatcagg 
                   
                   
               
               
                   
                 aatggacgtcaggagcaaagccagaacactttctggacctgttgatgatccttcaaagcttcccaaatggaa 
                   
                   
               
               
                   
                 ttatgatggttctagcacaggacaagctcctggagaagacagtgaagagatcctatatcctcaagcaattttc 
                   
                   
               
               
                   
                 aaggatccattcagaaggggcaacaatatcttggtcatttgtgattgttacaccccagctggtgaacccattc 
                   
                   
               
               
                   
                 caacaaacaaaaggcacagtgctgccaagattttcagccaccctgatgttgttgttgaggaaccctggtatg 
                   
                   
               
               
                   
                 gtcttgagcaagaatacaccttgttgcaaaaagatatcaattggcctcttggatggcctcttggtggttttcct 
                   
                   
               
               
                   
                 ggaccacagggaccatactattgcggaattggagctggaaaggtctttggacgcgatatcgttgactctcatt 
                   
                   
               
               
                   
                 ataaggcatgtctctatgctgggattaacatcagtggtatcaatggagaagtgatgcccggacagtgggaat 
                   
                   
               
               
                   
                 ttcaagttggaccttcagttggcatttcagcagctgatgaattgtgggcagctcgttacattcttgagaggatt 
                   
                   
               
               
                   
                 actgagattgctggagttgtggtctcatttgaccccaaacctattccgggtgactggaatggtgctggagctc 
                   
                   
               
               
                   
                 acacaaactacagcacaaagtctatgaggaatgaaggaggctatgaagtcattaagaaggcaattgagaa 
                   
                   
               
               
                   
                 ccttggactgaggcacaaggagcatattgcagcatatggtgaaggcaacgagcgtcgtctcactggaagac 
                 BstEII 
                   
               
               
                   
                 acgaaacagctgacatcaacacattcaaatggggagttgcgaaccgtggtgcatctattcgtgtgggaaga 
                   
                   
               
               
                   
                 gacacggagagagaagggaagggatacttcgaggataggaggcctgcttcgaatatggatccattcgtcgt 
                   
                   
               
               
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
                   
                   
               
               
                   
                 ctcgaatttccccgat   cgttcaaacatttggcaataaagtttcttaagattgaatcctgttgccggtcttgcga     
                   
                   
               
               
                   
                 
                   
                     tgattatcatataatttctgttgaattacgttaagcatgtaataattaacatgtaatgcatgacgttatttatg 
                   
                 
                   
                   
               
               
                   
                 
                   
                     agatgggtttttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatata 
                   
                 
                   
                   
               
               
                   
                 
                   
                     gcgcgcaaactaggataattatcgcgcgcggtgtcatctatgttactagatcggg 
                   
                   aattaaactatcagt 
                 
                   
                   
               
               
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
                   
                   
               
               
                   
                 tttcccgccttcagtttagctt   catggagtcaaagattcaaatagaggacctaacagaactcgccgtaaaga     
                   
                   
               
               
                   
                 
                   
                     ctggcgaacagttcatacagagtctcttacgactcaatgacaagaagaaaatcttcgtcaacatggtggag 
                   
                 
                   
                   
               
               
                   
                 
                   
                     cacgacacacttgtctactccaaaaatatcaaagatacagtctcagaagaccaaagggcaattgagactt 
                   
                 
                   
                   
               
               
                   
                 
                   
                     ttcaacaaagggtaatatccggaaacctcctcggattccattgcccagctatctgtcactttattgtgaagat 
                   
                 
                   
                   
               
               
                   
                 
                   
                     agtggaaaaggaaggtggctcctacaaatgccatcattgcgataaaggaaaggccatcgttgaagatgcc 
                   
                 
                   
                   
               
               
                   
                 
                   
                     tctgccgacagtggtcccaaagatggacccccacccacgaggagcatcgtggaaaaagaagacgttccaa 
                   
                 
                   
                   
               
               
                   
                 
                   
                     ccacgtcttcaaagcaagtggattgatgtgatatctccactgacgtaagggatgacgcacaatcccactat 
                   
                 
                   
                   
               
               
                   
                 
                   
                     ccttcgcaagacccttcctctatataaggaagttcatttcatttggagagaacacgggggact 
                   
                   cttgacca 
                 
                   
                   
               
               
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
                   
                   
               
               
                   
                 cgtcagctggtcccaggcaaggtctccgaggacgacaagctcatcgagtacgatgcgctgctcgtcgaccgc 
                   
                   
               
               
                   
                 ttcctcaacatcctccaggacctccacgggcccagccttcgcgaatttgtccaggagtgctacgaggtctcag 
                   
                   
               
               
                   
                 ccgactacgagggcaaaggagacacgacgaagctgggcgagctcggcgccaagctcacggggctggcccc 
                   
                   
               
               
                   
                 cgccgacgccatcctcgtggcgagctccatcctgcacatgctcaacctcgccaacctggccgaggaggtgca 
                   
                   
               
               
                   
                 gatcgcgcaccgccgccgcaacagcaagctcaagaaaggtgggttcgccgacgagggctccgccaccacc 
                   
                   
               
               
                   
                 gagtccgacatcgaggagacgctcaagcgcctcgtgtccgaggtcggcaagtcccccgaggaggtgttcga 
                   
                   
               
               
                   
                 ggcgctcaagaaccagaccgtcgacctcgtcttcaccgcgcatcctacgcagtccgcccgccgctcgctcctg 
                   
                   
               
               
                   
                 caaaaaaatgccaggatccgaaattgtctgacccagctgaatgccaaggacatcactgacgacgacaagc 
                   
                   
               
               
                   
                 aggagctcgatgaggctctgeagagagagatccaagcagccttcagaaccgatgaaatcaggagggcac 
                   
                   
               
               
                   
                 aacccaccccgcaggccgaaatgcgctatgggatgagctacatccatgagactgtatggaagggtgtgcct 
                   
                   
               
               
                   
                 aagttcttgcgccgtgtggatacagccctgaagaatatcggcatcaatgagcgccttccctacaatgtttctct 
                   
                   
               
               
                   
                 cattcggttctcttcttggatgggtggtgaccgcgatggaaatccaagagttaccccggaggtgacaagaga 
                   
                   
               
               
                   
                 tgtatgcttgctggccagaatgatggctgcaaacttgtacatcgatcagattgaagagctgatgtttgagctct 
                   
                   
               
               
                   
                 ctatgtggcgctgcaacgatgagcttcgtgttcgtgccgaagagctccacagttcgtctggttccaaagttacc 
                   
                   
               
               
                   
                 aagtattacatagaattctggaagcaaattcctccaaacgagccctaccgggtgatactaggccatgtaagg 
                   
                   
               
               
                   
                 gacaagctgtacaacacacgcgagcgtgctcgccatctgctggcttctggagtttctgaaatttcagcggaat 
                   
                   
               
               
                   
                 cgtcatttaccagtatcgaagagttccttgagccacttgagctgtgctacaaatcactgtgtgactgcggcga 
                   
                   
               
               
                   
                 caaggccatcgcggacgggagcctcctggacctcctgcgccaggtgttcacgttcgggctctccctggtgaa 
                   
                   
               
               
                   
                 gctggacatccggcaggagtcggagcggcacaccgacgtgatcgacgccatcaccacgcacctcggcatcg 
                   
                   
               
               
                   
                 ggtcgtaccgcgagtggcccgaggacaagaggcaggagtggctgctgtcggagctgcgaggcaagcgccc 
                   
                   
               
               
                   
                 gctgctgcccccggaccttccccagaccgacgagatcgccgacgtcatcggcgcgttccacgtcctcgcgga 
                   
                   
               
               
                   
                 gctcccgcccgacagcttcggcccctacatcatctccatggcgacggccccctcggacgtgctcgccgtggag 
                   
                   
               
               
                   
                 ctcctgcagcgcgagtgcggcgtgcgccagccgetgcccgtggtgccgctgttcgagaggctggccgacctg 
                   
                   
               
               
                   
                 cagtcggcgcccgcgtccgtggagcgcctcttctcggtggactggtacatggaccggatcaagggcaagcag 
                   
                   
               
               
                   
                 caggtcatggtcggctactccgactccggcaaggacgccggccgcctgtcc 
                   
                   
               
               
                   
                 gcggcgtggcagctgtacagggcgcaggaggagatggcgcaggtggccaagcgctacggcgtcaagctca 
                   
                   
               
               
                   
                 ccttgttccacggccgcggaggcaccgtgggcaggggtggcgggcccacgcaccttgccatcctgtcccagc 
                   
                   
               
               
                   
                 cgccggacaccatcaacgggtccatccgtgtgacggtgcagggcgaggtcatcgagttctgcttcggggagg 
                   
                   
               
               
                   
                 agcacctgtgcttccagactctgcagcgcttcacggccgccacgctggagcacggcatgcacccgccggtct 
                   
                   
               
               
                   
                 ctcccaagcccgagtggcgcaagctcatggacgagatggcggtcgtggccacggaggagtaccgctccgtc 
                   
                   
               
               
                   
                 gtcgtcaaggaggcgcgcttcgtcgagtacttcagatcggctacaccggagaccgagtacgggaggatgaa 
                   
                   
               
               
                   
                 catcggcagccggccagccaagaggaggcccggcggcggcatcacgaccctgcgcgccatcccctggatct 
                   
                   
               
               
                   
                 tctcgtggacccagaccaggttccacctccccgtgtggctgggagtcggcgccgcattcaagttcgccatcga 
                   
                   
               
               
                   
                 caaggacgtcaggaacttccaggtcctcaaagagatgtacaacgagtggccattcttcagggtcaccctgga 
                   
                   
               
               
                   
                 cctgctggagtggttttcgccaagggagaccccggcattgccggcttgtatgacgagctgcttgtggcggaa 
                   
                   
               
               
                   
                 gaactcaagccctttgggaagcagctcagggacaaatacgtggagacacagcagcttctccttccagatcgct 
                   
                   
               
               
                   
                 gggcacaaggatattcttgaaggcgatccattcctgaagcaggggctggtgctgcgcaacccctacatcacc 
                   
                   
               
               
                   
                 accctgaacgtgttccaggcctacacgctgaagcggataagggaccccaacttcaaggtgacgccccagcc 
                   
                   
               
               
                   
                 gccgctgtccaaggagttcgccgacgagaacaagcccgccggactggtcaagctgaacccgg 
                   
                   
               
               
                   
                 cgagcgagtacccgcccggcctggaagacacgctcatcctcaccatgaagggcatcgccgccggcatgcag 
                   
                   
               
               
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
                   
                   
               
               
                   
                 
                   
                     caataaagtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttgaattacgt 
                   
                 
                   
                   
               
               
                   
                 
                   
                     taagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggtttttatgattagagtcccgca 
                   
                 
                   
                   
               
               
                   
                 
                   
                     attatacatttaatacgcgatagaaaacaaaatatagcgcgcaaactaggataaattatcgcgcgcggtg 
                   
                 
                   
                   
               
               
                   
                 
                   
                     tcatctatgttactagatcggg 
                   
                 
                   
                   
               
               
                   
               
               
                 GS NcoI  F 
                 5′-TG CCATGG CTCATCTTTCGGATCTCGTT-3′ 
                 Forward  
                 8 
               
               
                   
                   
                 primer 
                   
               
               
                   
                   
                 for  
                   
               
               
                   
                   
                 amplifi-  
                   
               
               
                   
                   
                 cation  
                   
               
               
                   
                   
                 of   
                   
               
               
                   
                   
                 tobacco  
                   
               
               
                   
                   
                 GS 
                   
               
               
                   
                   
                 coding 
                   
               
               
                   
                   
                 sequence, 
                   
               
               
                   
                   
                 including 
                   
               
               
                   
                   
                 restriction 
                   
               
               
                   
                   
                 site 
                   
               
               
                   
                   
                 for 
                   
               
               
                   
                   
                 enzyme  
                   
               
               
                   
                   
                 NcoI. 
                   
               
               
                   
               
               
                 GS BstEII  R 
                 5′-G GGTGACC TCAAGGCTCGGATAGGATAGTG -3′ 
                 Reverse  
                 9 
               
               
                   
                   
                 primer 
                   
               
               
                   
                   
                 for   
                   
               
               
                   
                   
                 amplifi-  
                   
               
               
                   
                   
                 cation  
                   
               
               
                   
                   
                 of 
                   
               
               
                   
                   
                 tobacco  
                   
               
               
                   
                   
                 GS 
                   
               
               
                   
                   
                 coding 
                   
               
               
                   
                   
                 sequence, 
                   
               
               
                   
                   
                 including 
                   
               
               
                   
                   
                 restriction 
                   
               
               
                   
                   
                 site  
                   
               
               
                   
                   
                 for 
                   
               
               
                   
                   
                 enzyme 
                   
               
               
                   
                   
                 BstEII. 
                   
               
               
                   
               
               
                 AspAT BgIII    
                 5′-CAT AGATCT TATGGCTTCTCACGACAGCATCT -3′ 
                 Forward  
                 10  
               
               
                 F 
                   
                 primer  
                   
               
               
                   
                   
                 for  
                   
               
               
                   
                   
                 amplifi-  
                   
               
               
                   
                   
                 cation  
                   
               
               
                   
                   
                 of  
                   
               
               
                   
                   
                 Soyabean   
                   
               
               
                   
                   
                 AspAT 
                   
               
               
                   
                   
                 coding 
                   
               
               
                   
                   
                 sequence, 
                   
               
               
                   
                   
                 including 
                   
               
               
                   
                   
                 restriction 
                   
               
               
                   
                   
                 site 
                   
               
               
                   
                   
                 for 
                   
               
               
                   
                   
                 enzyme 
                   
               
               
                   
                   
                 BgIII. 
                   
               
               
                   
               
               
                 AspAT PmII    
                 5′-GC CACGTG TTAGACAACTCGGGTTACAGCTG-3′ 
                 Reverse  
                 11  
               
               
                 R 
                   
                 primer  
                   
               
               
                   
                   
                 for  
                   
               
               
                   
                   
                 amplifi-  
                   
               
               
                   
                   
                 cation 
                   
               
               
                   
                   
                 of  
                   
               
               
                   
                   
                 Soyabean   
                   
               
               
                   
                   
                 AspAT 
                   
               
               
                   
                   
                 coding 
                   
               
               
                   
                   
                 sequence,  
                   
               
               
                   
                   
                 including 
                   
               
               
                   
                   
                 restriction 
                   
               
               
                   
                   
                 site 
                   
               
               
                   
                   
                 for 
                   
               
               
                   
                   
                 enzyme 
                   
               
               
                   
                   
                 PmII. 
                   
               
               
                   
               
               
                 PEP-  
                 5′-AT AGATCT TATGGCGTCGACCAAGGCTCCG -3′ 
                 Forward  
                 12  
               
               
                 Case BgIII   
                   
                 primer 
                   
               
               
                 F 
                   
                 for   
                   
               
               
                   
                   
                 amplifi- 
                   
               
               
                   
                   
                 cation  
                   
               
               
                   
                   
                 of maize    
                   
               
               
                   
                   
                 PEPCase 
                   
               
               
                   
                   
                 coding  
                   
               
               
                   
                   
                 sequence, 
                   
               
               
                   
                   
                 including 
                   
               
               
                   
                   
                 restriction 
                   
               
               
                   
                   
                 site 
                   
               
               
                   
                   
                 for 
                   
               
               
                   
                   
                 enzyme 
                   
               
               
                   
                   
                 BgIII. 
                   
               
               
                   
               
               
                 PEP-  
                 5′-AG ACTAGT GCCAGTGTTCTGCATGCCGGCGG3′ 
                 Reverse  
                 13  
               
               
                 Case SpeI   
                   
                 primer  
                   
               
               
                 R 
                   
                 for  
                   
               
               
                   
                   
                 amplifi-  
                   
               
               
                   
                   
                 cation  
                   
               
               
                   
                   
                 of maize  
                   
               
               
                   
                   
                 PEPCase 
                   
               
               
                   
                   
                 coding 
                   
               
               
                   
                   
                 sequence, 
                   
               
               
                   
                   
                 including 
                   
               
               
                   
                   
                 restriction 
                   
               
               
                   
                   
                 site 
                   
               
               
                   
                   
                 for  
                   
               
               
                   
                   
                 enzyme  
                   
               
               
                   
                   
                 SpeI. 
                   
               
               
                   
               
               
                 35S SpeI  F 
                 5′-GG ACTAGT AATGGCGAATGCTAGAGCAGCTTGAG -3′ 
                 Forward  
                 14  
               
               
                   
                   
                 primer 
                   
               
               
                   
                   
                 for   
                   
               
               
                   
                   
                 amplifi- 
                   
               
               
                   
                   
                 cation 
                   
               
               
                   
                   
                 of  
                   
               
               
                   
                   
                 CaMV 35S    
                   
               
               
                   
                   
                 promoter  
                   
               
               
                   
                   
                 sequence, 
                   
               
               
                   
                   
                 including 
                   
               
               
                   
                   
                 restriction 
                   
               
               
                   
                   
                 site 
                   
               
               
                   
                   
                 for 
                   
               
               
                   
                   
                 enzyme 
                   
               
               
                   
                   
                 SpeI. 
                   
               
               
                   
               
               
                 NosT AscI,   
                 5′-GC CACGTG T CCTCAGC T GGCGCGCC CGCCAATATATCCTGTCAAACACTGATAGT-3′ 
                 Reverse  
                 15  
               
               
                   BbvCI,PmII  R 
                   
                 primer  
                   
               
               
                   
                   
                 for  
                   
               
               
                   
                   
                 amplifi- 
                   
               
               
                   
                   
                 cation 
                   
               
               
                   
                   
                 of Nos  
                   
               
               
                   
                   
                 terminator  
                   
               
               
                   
                   
                 sequence,    
                   
               
               
                   
                   
                 including 
                   
               
               
                   
                   
                 restriction 
                   
               
               
                   
                   
                 site 
                   
               
               
                   
                   
                 for 
                   
               
               
                   
                   
                 enzyme  
                   
               
               
                   
                   
                 AscI, 
                   
               
               
                   
                   
                 BbvCI 
                   
               
               
                   
                   
                 PmII 
                   
               
               
                   
               
               
                 NosT SpeI  R 
                 5′-GG ACTAGT TTAATTCCCGATC TAGTAACA TAGATG-3′ 
                 Reverse  
                 16  
               
               
                   
                   
                 primer  
                   
               
               
                   
                   
                 for  
                   
               
               
                   
                   
                 amplifi-  
                   
               
               
                   
                   
                 cation   
                   
               
               
                   
                   
                 of Nos  
                   
               
               
                   
                   
                 terminator 
                   
               
               
                   
                   
                 sequence, 
                   
               
               
                   
                   
                 including 
                   
               
               
                   
                   
                 restriction 
                   
               
               
                   
                   
                 site 
                   
               
               
                   
                   
                 for 
                   
               
               
                   
                   
                 enzyme 
                   
               
               
                   
                   
                 SpeI. 
                   
               
               
                   
               
               
                 35G AscI  F 
                 5′-ATCF GGCGCGCC AATGGCGAATGCTAGAGCAGCTTGAG -3′ 
                 Forward  
                 17  
               
               
                   
                   
                 primer  
                   
               
               
                   
                   
                 for 
                   
               
               
                   
                   
                 amplifi- 
                   
               
               
                   
                   
                 cation of  
                   
               
               
                   
                   
                 CaMV 35S   
                   
               
               
                   
                   
                 promoter 
                   
               
               
                   
                   
                 sequence, 
                   
               
               
                   
                   
                 including 
                   
               
               
                   
                   
                 restriction 
                   
               
               
                   
                   
                 site  
                   
               
               
                   
                   
                 for 
                   
               
               
                   
                   
                 enzyme  
                   
               
               
                   
                   
                 AscI. 
                   
               
               
                   
               
               
                 PEP- 
                 5′-GTG CCTCAGCC TAGCCAGTGTTCTGCATGCCGG -3′ 
                 Reverse  
                 18  
               
               
                 Case BbvCI   
                   
                 primer 
                   
               
               
                 R 
                   
                 for  
                   
               
               
                   
                   
                 amplifi-  
                   
               
               
                   
                   
                 cation  
                   
               
               
                   
                   
                 of maize  
                   
               
               
                   
                   
                 PEPCase   
                   
               
               
                   
                   
                 coding 
                   
               
               
                   
                   
                 sequence, 
                   
               
               
                   
                   
                 including 
                   
               
               
                   
                   
                 restriction 
                   
               
               
                   
                   
                 site 
                   
               
               
                   
                   
                 for 
                   
               
               
                   
                   
                 enzyme 
                   
               
               
                   
                   
                 BbvCI. 
                   
               
               
                   
               
               
                 hpt F 
                 5′-GAGGGCGAAGAATCTCGTGC -3′ 
                 Forward  
                 19  
               
               
                   
                   
                 primer 
                   
               
               
                   
                   
                 for   
                   
               
               
                   
                   
                 amplifi- 
                   
               
               
                   
                   
                 cation of  
                   
               
               
                   
                   
                 hygromycin  
                   
               
               
                   
                   
                 phospho- 
                   
               
               
                   
                   
                 trans- 
                   
               
               
                   
                   
                 ferase 
                   
               
               
                   
                   
                 for 
                   
               
               
                   
                   
                 screening 
                   
               
               
                   
                   
                 transgenic 
                   
               
               
                   
                   
                 plants. 
                   
               
               
                   
               
               
                 hpt R 
                 5′-GATECTGGCGACCTCGTATTGG -3′ 
                 Reverse  
                 20  
               
               
                   
                   
                 primer  
                   
               
               
                   
                   
                 for  
                   
               
               
                   
                   
                 amplifi-  
                   
               
               
                   
                   
                 cation of  
                   
               
               
                   
                   
                 hygromycin  
                   
               
               
                   
                   
                 phospho- 
                   
               
               
                   
                   
                 trans- 
                   
               
               
                   
                   
                 ferase 
                   
               
               
                   
                   
                 for 
                   
               
               
                   
                   
                 screening 
                   
               
               
                   
                   
                 transgenic 
                   
               
               
                   
                   
                 plants. 
                   
               
               
                   
               
               
                 PEPCase  
                 5′-ACGTCAGGAACTTCCAGGTIC-3′ 
                 Forward  
                 21  
               
               
                 Exp F 
                   
                 primer   
                   
               
               
                   
                   
                 for  
                   
               
               
                   
                   
                 maize  
                   
               
               
                   
                   
                 PEPCase,  
                   
               
               
                   
                   
                 used for 
                   
               
               
                   
                   
                 RT-PCR  
                   
               
               
                   
                   
                 based 
                   
               
               
                   
                   
                 evaluation 
                   
               
               
                   
                   
                 of 
                   
               
               
                   
                   
                 PEPCase 
                   
               
               
                   
                   
                 transgene 
                   
               
               
                   
                   
                 expression. 
                   
               
               
                   
               
               
                 PEPCase  
                 5′-CTTGTTCTCGTCGGCGAAC-3′ 
                 Reverse  
                 22  
               
               
                 Exp R 
                   
                 primer   
                   
               
               
                   
                   
                 for   
                   
               
               
                   
                   
                 maize  
                   
               
               
                   
                   
                 PEPCase,  
                   
               
               
                   
                   
                 used for 
                   
               
               
                   
                   
                 RT-PCR 
                   
               
               
                   
                   
                 based 
                   
               
               
                   
                   
                 evaluation 
                   
               
               
                   
                   
                 of 
                   
               
               
                   
                   
                 PEPCase 
                   
               
               
                   
                   
                 transgene 
                   
               
               
                   
                   
                 expression. 
                   
               
               
                   
               
               
                 GS Exp  
                 5′-ACTTTCTGGACCTGTTGAT-3′ 
                 Forward  
                 23  
               
               
                 F 
                   
                 primer  
                   
               
               
                   
                   
                 for 
                   
               
               
                   
                   
                 tobacco  
                   
               
               
                   
                   
                 GS, 
                   
               
               
                   
                   
                 used for 
                   
               
               
                   
                   
                 RT-PCR  
                   
               
               
                   
                   
                 based 
                   
               
               
                   
                   
                 evaluation 
                   
               
               
                   
                   
                 of GS 
                   
               
               
                   
                   
                 transgene 
                   
               
               
                   
                   
                 expression. 
                   
               
               
                   
               
               
                 GS Exp  
                 5′-GGCAGCACTGTGCCTT-3′ 
                 Reverse  
                 24  
               
               
                 R 
                   
                 primer   
                   
               
               
                   
                   
                 for 
                   
               
               
                   
                   
                 tobacco 
                   
               
               
                   
                   
                 GS, 
                   
               
               
                   
                   
                 used for 
                   
               
               
                   
                   
                 RT-PCR 
                   
               
               
                   
                   
                 based 
                   
               
               
                   
                   
                 evaluation  
                   
               
               
                   
                   
                 of GS 
                   
               
               
                   
                   
                 transgene  
                   
               
               
                   
                   
                 expression. 
                   
               
               
                   
               
               
                 AspAT  
                 5′-ATGGCTTCTCACGACAGCATC-3′ 
                 Forward  
                 25  
               
               
                 Exp F 
                   
                 primer   
                   
               
               
                   
                   
                 for  
                   
               
               
                   
                   
                 soyabean 
                   
               
               
                   
                   
                 AspAT, 
                   
               
               
                   
                   
                 used for 
                   
               
               
                   
                   
                 RT-PCR 
                   
               
               
                   
                   
                 based 
                   
               
               
                   
                   
                 evaluation  
                   
               
               
                   
                   
                 of GS 
                   
               
               
                   
                   
                 transgene 
                   
               
               
                   
                   
                 expression. 
                   
               
               
                   
               
               
                 AspAT  
                 5′-TTGCGTGACACGTCATTTATGAGT-3′ 
                 Reverse  
                 26  
               
               
                 Exp R 
                   
                 primer  
                   
               
               
                   
                   
                 for  
                   
               
               
                   
                   
                 soyabean  
                   
               
               
                   
                   
                 AspAT, 
                   
               
               
                   
                   
                 used for 
                   
               
               
                   
                   
                 RT-PCR 
                   
               
               
                   
                   
                 based  
                   
               
               
                   
                   
                 evaluation 
                   
               
               
                   
                   
                 of 
                   
               
               
                   
                   
                 GS  
                   
               
               
                   
                   
                 transgene 
                   
               
               
                   
                   
                 expression. 
                   
               
               
                   
               
               
                 26S F 
                 5′-CACAATGATAGGAAGAGCCGAC-3′ 
                 Forward  
                 27  
               
               
                   
                   
                 primer   
                   
               
               
                   
                   
                 for 
                   
               
               
                   
                   
                 26SrRNA,  
                   
               
               
                   
                   
                 used as 
                   
               
               
                   
                   
                 internal 
                   
               
               
                   
                   
                 control 
                   
               
               
                   
                   
                 for 
                   
               
               
                   
                   
                 RT-PCR  
                   
               
               
                   
                   
                 based 
                   
               
               
                   
                   
                 evaluation  
                   
               
               
                   
                   
                 of 
                   
               
               
                   
                   
                 transgene 
                   
               
               
                   
                   
                 expression. 
                   
               
               
                   
               
               
                 26S R 
                 5′-CAAGGGAACGGGCTTGGCAGAATC-3′ 
                 Reverse  
                 28  
               
               
                   
                   
                 primer  
                   
               
               
                   
                   
                 for   
                   
               
               
                   
                   
                 26SrRNA,   
                   
               
               
                   
                   
                 used as 
                   
               
               
                   
                   
                 internal 
                   
               
               
                   
                   
                 control 
                   
               
               
                   
                   
                 for 
                   
               
               
                   
                   
                 RT-PCR 
                   
               
               
                   
                   
                 based 
                   
               
               
                   
                   
                 evaluation  
                   
               
               
                   
                   
                 of 
                   
               
               
                   
                   
                 transgene 
                   
               
               
                   
                   
                 expression 
                   
               
               
                   
               
               
                 AspAT  
                 MASHDSISASPTSASDSVFNHLVRAPEDPILGVTVAYNKDPSPVKLNLGVGAYRTEEG 
                 Represents 
                 29  
               
               
                 Pr 
                 KPLVLNVVRRVE 
                 Proteins 
                   
               
               
                   
                 QQLINDVSRNKEYIPIVGLADFNKLSAKLIFGADSPAIQDNRVTTVQCLSGTGSLRVGG 
                 of AspAT  
                   
               
               
                   
                 EFLAKHYHQRT 
                 genes 
                   
               
               
                   
                 IYLPTPTWGNHPKVFNLAGLSVKTYRYYAPATRGLDFQGLLEDLGSAPSGSIVLLHACA 
                   
                   
               
               
                   
                 HNPTGVDPTLE 
                   
                   
               
               
                   
                 QWEQIRQLIRSKALLPFFDSAYQGFASGSLDADAQPVRLFVADGGELLVAQSYAKNLG 
                   
                   
               
               
                   
                 LYGERVGALSIV 
                   
                   
               
               
                   
                 CKSADVASRVESQLKLVIRPMYSSPPIHGASIVAAILKDRNLFNDWTIELKAMADRIISM 
                   
                   
               
               
                   
                 RQELFDALCS 
                   
                   
               
               
                   
                 RGTPGDWSHIIKQIGMFTFTGLNAEQVSFMTKEFHIYMTSDGRISMAGLSSKTVPLLA 
                   
                   
               
               
                   
                 DAIHAAVTRVV 
                   
                   
               
               
                   
               
               
                 GSPr 
                 MAHLSDLVNLNLSDSTQKIIAEYIWIGGSGMDVRSKARTLSGPVDDPSKLPKWNYDG 
                 Represents 
                 30  
               
               
                   
                 SSTGQAPGEDSEE 
                 Proteins 
                   
               
               
                   
                 ILYPQAIFKDPFRRGNNILVICDCYTPAGEPIPTNKRHSAAKIFSHPDVVVEEPWYGLEQ 
                 of GS  
                   
               
               
                   
                 EYTLLQKDIN 
                 genes 
                   
               
               
                   
                 WPLGWPLGGFPGPQGPYYCGIGAGKVFGRDIVDSHYKACLYAGINISGINGEVMPGQ 
                   
                   
               
               
                   
                 WEFQVGPSVGISA 
                   
                   
               
               
                   
                 ADELWAARYILERITEIAGVVVSFDPKPIPGDWNGAGAHTNYSTKSMRNEGGYEVIKK 
                   
                   
               
               
                   
                 AIENLGLRHKEH 
                   
                   
               
               
                   
                 IAAYGEGNERRLTGRHETADINTFKWGVANRGASIRVGRDTEREGKGYFEDRRPASN 
                   
                   
               
               
                   
                 MDPFVVTSMIAET 
                   
                   
               
               
                   
                 TILSEP 
                   
                   
               
               
                   
               
               
                 PEPCase 
                 MASTKAPGPGEKHHSIDAQLRQLVPGKVSEDDKLIEYDALLVDRFLNILQDLHGPSLRE 
                 Represents 
                 31  
               
               
                 Pr 
                 FVQECYEVSAD 
                 Proteins 
                   
               
               
                   
                 YEGKGDTTKLGELGAKLTGLAPADAILVASSILHMLNLANLAEEVQIAHRRRNSKLKKG 
                 of PEPCase  
                   
               
               
                   
                 GFADEGSATTE 
                 genes 
                   
               
               
                   
                 SDIEETLKRLVSEVGKSPEEVFEALKNQTVDLVFTAHPTQSARRSLLQKNARIRNCLTQL 
                   
                   
               
               
                   
                 NAKDITDDDK 
                   
                   
               
               
                   
                 QELDEALQREIQAAFRTDEIRRAQPTPQAEMRYGMSYIHETVWKGVPKFLRRVDTAL 
                   
                   
               
               
                   
                 KNIGINERLPYNV 
                   
                   
               
               
                   
                 SLIRFSSWMGGDRDGNPRVTPEVTRDVCLLARMMAANLYIDQIEELMFELSMWRCN 
                   
                   
               
               
                   
                 DELRVRAEELHSSS 
                   
                   
               
               
                   
                 GSKVTKYYIEFWKQIPPNEPYRVILGHVRDKLYNTRERARHLLASGVSEISAESSFTSIEE 
                   
                   
               
               
                   
                 FLEPLELCY 
                   
                   
               
               
                   
                 KSLCDCGDKAIADGSLLDLLRQVFTFGLSLVKLDIRQESERHTDVIDAITTHLGIGSYRE 
                   
                   
               
               
                   
                 WPEDKRQEWL 
                   
                   
               
               
                   
                 LSELRGKRPLLPPDLPQTDEIADVIGAFHVLAELPPDSFGPYIISMATAPSDVLAVELLQR 
                   
                   
               
               
                   
                 ECGVRQPLP 
                   
                   
               
               
                   
                 VVPLFERLADLQSAPASVERLFSVDWYMDRIKGKQQVMVGYSDSGKDAGRLSAAW 
                   
                   
               
               
                   
                 QLYRAQEEMAQVAKR 
                   
                   
               
               
                   
                 YGVKLTLFHGRGGTVGRGGGPTHLAILSQPPDTINGSIRVTVQGEVIEFCFGEEHLCFQ 
                   
                   
               
               
                   
                 TLQRFTAATLE 
                   
                   
               
               
                   
                 HGMHPPVSPKPEWRKLMDEMAVVATEEYRSVVVKEARFVEYFRSATPETEYGRMNI 
                   
                   
               
               
                   
                 GSRPAKRRPGGGIT 
                   
                   
               
               
                   
                 TLRAIPWIFSWTQTRFHLPVWLGVGAAFKFAIDKDVRNFQVLKEMYNEWPFFRVTLD 
                   
                   
               
               
                   
                 LLEMVFAKGDPGI 
                   
                   
               
               
                   
                 AGLYDELLVAEELKPFGKQLRDKYVETQQLLLQIAGHKDILEGDPFLKQGLVLRNPYITT 
                   
                   
               
               
                   
                 LNVFQAYTLK 
                   
                   
               
               
                   
                 RIRDPNFKVTPQPPLSKEFADENKPAGLVKLNPASEYPPGLEDTLILTMKGIAAGMQN 
                   
                   
               
               
                   
                 TG 
               
               
                   
               
             
          
         
       
     
       Example 1 
     Amplification and Cloning of AspAT Gene 
       [0081]    Nucleotide sequence encoding soyabean cytosolic AspAT gene (SEQ ID NO: 1) was obtained from the NCBI database of nucleotide sequences (GenBank Accession No. AF034210.1; (http://www.ncbi.nlm.nih.gov/nuccore/AF034210.1) RNA from soyabean plant was isolated using IRIS Plant RNA Kit (Ghawana et al., US Patent no 0344NF2004/IN). cDNA was synthesized using total RNA preparations (2 μg) in the presence of 1 μg oligo(dT) 12-18  and 400 U of reverse transcriptase Superscript II (Invitrogen) after digesting with 2 U DNase I (amplification grade, Invitrogen, USA) following the manufacturer&#39;s instructions. The full coding region of AspAT was then amplified from soyabean cDNA using primers AspAT BgfII  F (SEQ ID NO: 10) and AspAT Pmfl  R (SEQ ID NO: 11) such that restriction sites BglII ( AGATCT ) and PmlI ( CACGTG ) is incorporated in the coding sequence for AspAT. 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 1 minute 20 seconds, with a final extension of 72° C. for 7 minutes. The amplification product was cloned in to pGEM-T easy vector (Promega, USA). Plasmid from the positive clones and pCAMBIA 1302 plasmid were digested with BglII and PmlI and digested products isolated from an agarose gel electrophoresis were ligated and transformed in to  E. coli  DH5α cells which were obtained from Takara Bio Company, Japan (Cat. No. 9057). Plasmid from the positive colonies were sequenced to verify the in frame cloning of the AspAT coding sequence placed between CaMV 35S promoter (SEQ ID NO: 4) and Nos terminator (SEQ ID NO: 5) of pCAMBIA1302 and resulting vector was designated as AspAT::pCAMBIA1302. 
       Example 2 
     Amplification and Cloning of GS Gene 
       [0082]    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; (http://www.ncbi.nlm.nih.gov/nuccore/X95932.1). RNA from tobacco plant was isolated using IRIS Plant RNA Kit (Ghawana et al., US Patent no 0344NF2004/IN). cDNA was synthesized using total RNA preparations (2 μg) in the presence of 1 μg oligo(dT) 12-18  and 400 U of reverse transcriptase Superscript II (Invitrogen) after digesting with 2 U DNase I (amplification grade, Invitrogen, USA) following the manufacturer&#39;s instructions. 
         [0083]    The full coding region of GS was amplified from tobacco cDNA using primers GS NcoI  F with restriction sites NcoI ( CCATGG ) (SEQ ID NO: 8) and GS BstEII  R with restriction sites for BstEII ( GGTGACC ) (SEQ ID NO: 9). GS NcoI  F primers was modified so as to eliminate the BglII site by replacement of ‘A’ nucleotide by ‘G’ at position 15. 
         [0084]    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 1 minute 10 seconds, 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 NcoI and BstEII and digested product isolated from an agarose gel electrophoresis were ligated such that GS is placed downstream of CaMV 35S promoter of pCAMBIA vector. The ligation product was transformed in to  E. coli  DH5α cells and transformants were sequenced to verify the in frame cloning of the GS coding sequence and the resulting vector was designated as GS::pCAMBIA1302. 
       Example 3 
     Amplification and Cloning of Maize PEPCase Gene 
       [0085]    Nucleotide sequence encoding maize PEPCase gene (SEQ ID NO: 3) was obtained from the NCBI database of nucleotide sequences (NCBI Reference Sequence: NM — 001111948.1; (http://www.ncbi.nlm.nih.gov/nuccore/NM — 001111948.1) RNA from 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 μg) in the presence of 1 μg oligo(dT) 12-18  and 400 U of reverse transcriptase Superscript II (Invitrogen) after digesting with 2 U DNase I (amplification grade, Invitrogen, USA) following the manufacturer&#39;s instructions. 
         [0086]    The full coding region of PEPCase was amplified from maize cDNA using primers PEPCase BglII  F with restriction sites for BglII ( AGATCT ) (SEQ ID NO: 12) and PEPCase SpeI  R with restricition sites for SpeI ( ACTAGT ) (SEQ ID NO: 13). Qiagen High Fidelity Taq polymerase enzyme supplemented with Q-solution (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 BglII and SpeI and digested product isolated from an agarose gel electrophoresis were ligated and then transformed in to  E. coli  DH5α cells. Transformants were sequenced to verify the in frame cloning of the PEPCase coding sequence and resulting vector was designated as PEPCase::pCAMBIA 1302. 
       Example 4 
     Assembly of Expression Cassettes for AspAT, GS and PEPCase in Single pCAMBIA 1302 Vector (Generous Gift from “Centre for Application of Molecular Biology to International Agriculture”, Australia) 
       [0087]    A stepwise method for amplification and integration of expression cassettes each for AspAT, GS and PEPCase in to single plant transformation vector pCAMBIA 1302 is described as follows: 
         [0088]    GS expression cassette comprising CaMV35S promoter, downstream cloned GS and nopaline synthase (hereinafter, referred as “Nos”) terminator was amplified from GS:: pCAMBIA 1302 vector 
         [0089]    (Example 2), using primers 35 SpeI  F (SEQ ID NO: 14) and NosT AscI, BbvCI, PmlI  R (SEQ ID NO: 15). The primers were designed to incorporate the SpeI ( ACTAGT ) in the forward primer and AscI ( GGCGCGCC ), BbvCI ( CCTCAGC ) and PmlI ( CACGTG ) in reverse primer to facilitate the subcloning of GS expression cassette in to SpeI and PmlI sites of pCAMBIA 1302 vector as well as to create the additional restriction sites (AscI, BbvCI) at 3′ end in the vector backbone. Qiagen High Fidelity Taq polymerase enzyme was used for the PCR using the following conditions: initial desaturating at 94° C. for 3 minutes, 30 cycles of 94° C. for 30 seconds, annealing at 59° C. for 30 seconds, extension at 72° C. for 2 minutes, with a final extension of 72° C. for 7 minutes. The amplification product was cloned in to pGEM-T easy vector (Promega, USA). Plasmids from the positive clones was digested with SpeI and PmlI, and the digested product was then isolated from an agarose gel electrophoresis and ligated in to SpeI and PmlI sites of pCAMBIA 1302 vector. The ligation product was transformed in to  E. coli  DH5α cells and transformants were verified by sequencing of plasmid. 
         [0090]    AspAT coding sequence along with 3′Nos terminator sequence was amplified from AspAT:: pCAMBIA 1302 vector (Example 1) using primers AspAT BglII  F (SEQ ID NO: 10) and NosT SpeI  (SEQ ID NO: 16) with restriction sites for BglII ( AGATCT ) and SpeI ( ACTAGT ) respectively. 
         [0091]    Qiagen High Fidelity 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 59° C. for 30 seconds, extension at 72° C. for 2 minutes, with a final extension of 72° C. for 7 minutes. The amplification product was cloned in to pGEM-T easy vector (Promega, USA). Plasmids from the positive clones upon digestion with BglII and SpeI, cloned downstream of CaMV 35S promoter of destination pCAMBIA 1302 (previously cloned with GS expression cassette). The ligation product was then transformed in to  E. coli  DH5α cells and transformants were sequenced to verify the in frame cloning of the AspAT coding sequence. 
         [0092]    CaMV 35S promoter along with the downstream cloned PEPCase gene from PEPCase:: pCAMBIA 1302 vector (example 3) was amplified with the primers 35S AscI  F (SEQ ID NO: 17) having restriction site for AscI ( GGCGCGCC ) and PEPCase BBvCI  R (SEQ ID NO: 18) having restriction site for BbVCI ( CCTCAGC ). 
         [0093]    Qiagen High Fidelity 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, extension 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 digested with AscI ( GGCGCGCC ) and BbVCI ( CCTCAGC ) and digested product isolated from an agarose gel electrophoresis ligated upstream of Nos terminator sequence of destination pCAMBIA 1302 previously cloned with GS and AspAT expression cassettes. The ligation product was transformed in to  E. coli  DH5α 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 PEPcase. A hygromycin resistance gene (SEQ ID NO. 6) was included as a selectable marker for screening transgenic plants. Schematic diagram of expression construct is shown in  FIG. 1 , 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. 
       Example 5 
     Raising of Transgenic  Arabidopsis  Plants Co-Over Expressing Genes AspAT, GS and PEPCase 
       [0094]    Generation of Plant Expression Vector (AspAT+GS+PEPCase) 
         [0095]    Briefly, the plant expression vector was constructed as follows: cDNA sequences encoding soybean AspAT gene (SEQ ID NO: 1), tobacco cytosolic GS gene (SEQ ID NO: 2) and maize PEPCase gene (SEQ ID NO: 3), were first independently cloned in to pCAMBIA 1302 vector. The elements for expression cassette for AspAT, GS and PEPCase were then amplified and assembled in to destination pCAMBIA1302 such that genes AspAT, GS and PEPCase were controlled by independent CaMV 35S promoter and Nos transcriptional terminator. 
         [0096]      Agrobacterium  Mediated Plant Transformation: 
         [0097]    AspAT+GS+PEPCase were transferred to  Agrobacterium tumefaciens  strain GV3101 with ATCC number  Agrobacterium tumefaciens  (GV3101 (pMP90RK) (C58 derivative) ATCC® Number: 33970 Reference: Hayashi H, Czaja I, Lubenow H, Schell J, Walden R. 1992 using standard triparental mating method. 
         [0098]    Briefly,  E. coli  DH5α cells harboring the recombinant construct AspAT+GS+PEPCase and those harboring helper plasmid pRK2013 were cultured overnight at 37° C.  Agrobacterium  strain GV3101 grown at 28° C. for 48 hrs. All the three cultures were then pelleted, washed, and mixed, followed by plating on YEM (Yeast Extract Mannitol) plates supplemented with the antibiotics kanamycin (50 ug/ml) and rifampcin (50 ug/ml). Antibiotic resistant colonies were verified by colony PCR to assure the transformation of  Agrobacterium  with the recombinant construct AspAT+GS+PEPCase. 
         [0099]      Arabidopsis  Seeds of the Columbia Ecotype were Generous Gift by Dr. Christine H Foyer Of, IACR-Rothamsted, Harpenden, UK 
         [0100]      Arabidopsis  plants were transformed with  Agrobacteria  harboring AspAT+GS+PEPCase using vacuum infiltration method. Briefly, liquid 5-ml cultures were established from single transformed  Agrobacterium  colony and grown in YEM medium supplemented with 50 ug/ml kanamycin, 50 ug/ml rifampicin at 28° C. up to the late logarithmic 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 strength MS (Murashige and Skoog) medium with 2% sucrose, 0.05% MES (Sigma,) and 0.01% of Silwet L-77 (Lehle Seeds, United States).  Arabidopsis  inflorescences were dipped in bacterial suspension and infiltrated under vacuum for 10 minutes. Plants were then transferred to growth chamber and grown under controlled long day conditions (16-h light at 22-23° C. and 8-h darkness at 20° C.) for seed set. 
         [0101]    Selection of Primary Transformant T o  Transgenic  Arabidopsis  Plant: 
         [0102]    Seeds from transformed plants were surface sterilized by immersion in 70% (v/v) ethanol for 2 min, followed by immersion in 10% (v/v) sodium hypochlorite solution. Seeds were then washed four times with sterile distilled water and sown onto 1% agar containing MS medium supplemented with hygromycin B at a concentration of 20 μml −1  (Sigma # H3274). Seeds were then stratified for 2 days in the dark at 4° C. After stratification plates were transferred to a growth chamber with 16 h light and 8 h dark cycle for germination. After 14-days, hygromycin resistant seedlings were selected as putative primary transformants (T 0 ) and transferred to pots containing vermiculite, perlite and cocopeat mix (1:1:1) and grown to maturity under controlled condition of light, temperature and humidity for growth and seed set. 
         [0103]    Raising T1 and T 2  Generation AspAT+GS+PEPCase Transgenic Plants: 
         [0104]    Seeds harvested from T 0  transgenic plants were germinated on MS+hygromycin B (at a concentration of 20 μml −1 ) plates and transgenic lines exhibiting a segregation ratio of 3:1 (scored by their sensitivity to hygromycin B) were selected to raise T1 generation of transgenic plants. Homozygous transgenic plants were obtained in the T 2  generation and evaluated for different physiological and biochemical parameters in comparison to wild control plants. 
       Example 6 
     Analysis of the Genomic DNA from  Arabidopsis thaliana  Plants Transformed with AspAT+CS+PEPCase 
       [0105]      Arabidopsis  plants from two independent transgenic lines transformed with AspAT+GS+PEPCase were selected to verify the insertion of transgenes in to plant genome. The genomic DNA was isolated using DNeasy Plant mini kit (QIAGEN Co.). PCR was carried out by using the isolated DNA as template with primers hpt F (SEQ ID NO: 19) and hpt R (SEQ ID NO: 20) annealing to the hygromycin phosphtransferaes (hpt) gene (SEQ ID NO: 6) (plant selection marker from pCAMBIA 1302 vector). 
         [0106]    PCR cycling conditions defined by initial denaturation at 94° C. for 3 minutes, 28 cycles of 94° C. for 30 seconds, annealing at 58° C. for 30 seconds, extension at 72° C. for 1 minute, with a final extension of 72° C. for 7 minutes. 
         [0107]    The result is shown in  FIG. 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. 
       Example 7 
     Evaluation of AspAT+GS+PEPCase Transgenics by Reverse Transcriptase—Polymerase Chain Reaction (RT-PCR) 
       [0108]    RNA analysis of transformants was done to confirm the expression of AspAT, GS and PEPCase. Total RNA was isolated from leaf and root of transgenic plants using iRIS Plant RNA Kit (Ghawana et al., US Patent no 0344NF2004/IN). cDNA was synthesized using total RNA preparations (2 μg) in the presence of 1 μg oligo(dT) 12-18  and 400 U of reverse transcriptase Superscript II (Invitrogen) after digesting with 2 U DNase I (amplification grade, Invitrogen, USA) following the manufacturer&#39;s instructions). Expression of transgenes was evaluated using gene specific primer for AspAT, GS and PEPCase, designated as PEPCase Exp F (SEQ ID NO: 21), PEPCase Exp R (SEQ ID NO: 22), GS Exp F (SEQ ID NO: 23), GS Exp R (SEQ ID NO: 24), AspAT Exp F (SEQ ID NO: 25) and AspAT ExpR (SEQ ID NO: 26). As a positive control for RT-PCR, 26S rRNA was amplified using primers 26S F (SEQ ID NO: 27) and 26S R (SEQ ID NO: 28). 
         [0109]    The results of analyses are shown in  FIG. 2B , in which WT represents wild and L1 and L2 represent two transgenic lines. The amplification of RT-PCR products were observed only in trangenics confirming the expression of introduced genes. 
       Example 8 
     Enzymatic Assays from Wild Type and AspAT+GS+PEPCase Transgenic  Arabidopsis  Plants 
       [0110]    Enzymatic assays were performed with AspAT+GS+PEPCase transgenic and wild plants as follows: 
         [0111]    PEPCase Activity Measurement: Frozen leaf samples (200 mg) ground with a mortar and pestle in 1 ml of extraction buffer containing 50 mM Tris-Cl buffer (pH 7.5), 1.0 mM MgCl2, 5.0 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 MgCl2, 5 mM DTT, 1 mM NaHCO 3 , 5 mM glucose-6-phosphate, 0.2 mM NADH, 2 units MDH, 0.1 units lactate dehydrogenase and crude extract. The reaction was initiated by the addition of 5 mM PEP. 
         [0112]    AspAT Activity Measurement: Extraction buffer for AspAT consisted of 200 mM Tris-Cl buffer (pH 7.5), 2.0 mM EDTA and 20% glycerol. 
         [0113]    The enzyme was assayed in an MDH-coupled reaction essentially as described by Ireland and Joy (1990). Briefly the reaction mixture contained 10 mM 2-oxoglutarate, 2 mM aspartate, 0.2 mM NADH, and 50 mM HEPES buffer (pH 8.0). Reaction was started by addition of 2-oxoglutarate. Assay control was run by excluding the 2-oxoglutarate from the reaction mix. 
         [0114]    GS Activity Measurement: 
         [0115]    GS (glutamine synthetase) was extracted in the grinding medium containing 50 mM Tris-Cl buffer (pH 7.8), 1 mM EDTA, 10 mM MgSO 4 , 5 mM sodium glutamate, 10% (v/v) glycerol and insoluble PVPP (2% w/v). Enzyme assay was performed as described earlier by Lea et al. (1990) and the activity was calculated from the standard curve prepared with γ-glutamylhydroxamate. 
         [0116]    The results of the analyses are shown in the  FIG. 5A to 5C , an increase of about 45 to 50% in PEPCase activity, 55% in GS activity and 55 to 60% in AspAT activity was observed with two independent AspAT+GS+PEPCase transgenic plants compared to wild plants. 
       Example 9 
       [0117]    C and N Analyses in Wild and AspAT+GS+PEPCase Transgenic  Arabidopsis  Plants 
         [0118]    Seeds of AspAT+GS+PEPCase transformed  Arabiopdsis thaliana  plants and wild control plants were germinated on half strength MS plates supplemented with 20 g/l sucrose. 14 days-old seedlings were transferred to pots containing mix of vermiculite; perlite and coco peat in the ratio of 1:1:1 and grown under long-day conditions comprising 16 hours of light period at 22° C. and 8 hours of dark period at 20° C. maintained in the  Arabidopsis  growth chamber. Different plant parts including rosette leaf; stem, cauline leaf and green pods were 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  FIG. 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 plants. 
       Example 10 
     Investigation of Growth and Yield in Wild and AspAT+GS+PEPCase Transgenic Plants 
       [0119]    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 different parameters evaluated, AspAT+GS+PEPCase plants showed enhanced growth characteristics. In particular, the transgenic plants have more number of leaves per rosette having larger area. Transgenic plants exhibited about 70% increase in the shoot fresh weight with 60% increase in the shoot dry weight whereas the increase of about 40% and 30% was observed in the root fresh and dry weight respectively (shown in  FIG. 3 ). 
         [0120]    Total number of pods from 72-days old AspAT+GS+PEPCase transgenic plants was calculated and compared to untransformed wild plants (shown in  FIG. 7   a ). Furthermore total seed yield (total seed weight per plant) was also measured for transgenic and control plants. Across both the parameters, AspAT+GS+PEPCase transgenic  Arabidopsis  plant showed increase in yield compared to wild plants as shown in  FIG. 7   b.    
       ADVANTAGES OF THE INVENTION 
       [0000]    
       
         
           
             1. There 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 of AspAT and GS. 
             3. The improved capacity of plant for carbon and nitrogen capture was also reflected in improved plant productivity both in terms of plant seed and plant biomass production.