Cloning Key Enzyme (PEPC,PPDK) Genes Of C₄ Photosynthesis From Barnyardgrass (Echinochloa) And PEPC Gene Transformation In Rice (Oryza Sativa) And Tobacco (Nicotiana Tabacum)

It is a very important research field that C4 cycle key genes were transformed into C3 plants by gene engineering to improve their photosynthesis, water and N use efficiency. Echinochloa is a typical C4 weed that has wide adaptation, stress tolerance and growth in rice field. However, its C4 cycle key genes have not been studied and transformed. In this study, the C4 key enzymes genes of Echinochloa were isolated, sequenced and tranformed into monocotyledon rice and dicotyledon tobacco, and the activities of the enzyme and photosynthetic characteristics were measured in transgenic plants. The results were as follow:A full-length cDNA of root type PEPC gene and a C4 PPDK gene were firstly isolated by RT-PCR method from Echinochloa crusgalli and Echinochlo frumentacea, respectively. The sequences have been submitted to the GenBank database with the accession numbers AY251482 and AY792619, which named Ecppc and Efpdk, respectively. E. crusgalli ppc gene contains a open reading frame of 2886 bp, encoding 961 amino acid residues and E. frumentacea pdk gene contains a open reading frame of 2838 bp, encoding 945 amino acid residues.Three promoters, pUbi, the promoter of ubiquitin which is a strong promoter expressed in all tissues, and pRbcS, the promoter of Rubisco small subunit which is a strong promoter regulated by light, While pZmp, the promoter of maize PEPC gene, were used to control the expression of the cloned Ppc gene from Echinochloa crusgalli. They were inserted in the poly-cloned site of plant expression vectors pCB. Three plant expression vectors containing exogenesis Ppc gene, pUbi-Eppc, pRbcS-Eppc, pZmp-Eppc, were constructed. All these vectors and another plant expression vector containing maize complete Ppc gene were transformed into rice and tobacco by Agrobactirium mediated transformation method.The marker gene Hygr and the transformed exogenesis Ppc gene were detected by PCR in regenerated plants. Correct products were amplified in most of them. RT-PCR of Ppc gene and Western blot were analyzed on some of the transgenic plants. The result indicated that the Ppc genes were successfully transformed into rice and transcripted and expressed correctly.PEPC activities of the leaves from most of the transgenic rice were measured. The result demonstrated that the exogenesis Ppc can express efficiently only with the complete genomic genes are used. The most of the PEPC activities of the transgenic rice expressed the complete maize Ppc gene are 10-fold higher than that of the non-transformants. While most of the PEPC activities of the transgenic rice expressed Echinochloa crusgalli cDNA of Ppc gene are higher than the non-transformed rice. A few were up to 7-fold higher than that of control. And in transgenic upland rice plants H65 the PEPC activities were higher than that in transgenic lowland rice plants Zhonghua8. It seems that the transformed C4 PEPC gene could expressed more efficiently in upland rice plants.Gas exchange was measured in some transgenic rice plants. Pn is closely related to stomatai conductance (Gs). This indicated that the expressed PEPC increase the opening of the stomatai. Anotherremarkable effect is the over-expression of Ppc gene in rice increasing its water use efficiency. This would improve the drought tolerance of the transgenic rice plants.On the contrary, the results of tobacco transformed with barnyardgrass cDNA are better than the complete maize Ppc gene. The tobacco plants, which transformed maize Ppc gene, have lower efficiency of regeneration and leaves turned yellowish and appeared wilted in their growthng. This is probability bcause of their abnormality chloroplasts. The results demonstrate that the complete maize Ppc gene cannot be expressed correctly in tabacco due to incorrect initiation and termination of transcription and incorrect splicing.So our study also indicated that to achieve high-level expression of the C4 enzyme in C3 plantds has some limitation. Only while trandgenes from phylogenetically closely related plants have to be used 10 achieve high-level expression...