Improve The Genetic Manipulation Of The Tobacco Of Co <sub> 2 </ Sub> Fixation Capacity

Based on the carbon numbers of first product produced in the photosynthesis darkreaction, photosynthesis is divided into two types-----C₃ photosynthesis and C₄photosynthesis. CO₂ is fixed in C₃ plant by C₃ photosynthesis and in C₄ plant by C₄ photosynthesis. The photosynthesis of C₄ plants has CO₂ condensation mechanism. In drought, high temperature and high light conditions, the efficiency of CO₂ fixation in C₄ plants is 1.5～2 times higher than in C₃ plants. However, most important of crops such as rice, wheat and soybean are C₃ plants. For a long time, one of the main purposes of plant scientists is how to transfer C₄ pathway mechanism into C₃ plant. Normal hybridization breeding is difficult to realize, but it is possible to transfer the key enzyme genes of CO₂ fixation existed in C₄ photosynthesis pathway into C₃ plant using plant gene engineering, making C₃ plants have C₄-like characteristics of CO₂ fixation. During the C₄ photosynthesis pathway, there are several key enzymes and translocators, such as phosphoenolpyruvate carboxylase(PEPC), phosphoenolpyruvate carboxykinase(PCK), Malate/Na translocator and phosphoenolpyruvate/phosphate translocator(PEP/Pi). The study used C₃ plant tobacco (wild type tobacco and transgenic PCK tobacco) as research material. PEPC and PEP/Pi were transformed into the tobacoo and overexpressed. Because of having appropriate transit peptide, expressed PEPC was located at cytoplasm (or chloroplast) of mesophyll cell, becoming the enzyme of CO₂ fixation in the C4 pathway; and PEP/Pi transporter was located at inner membrane of chloroplast, transporting OAA and PEP into chloroplast. The strategy would be used to install a C₄-like photosynthesis pathway and improve the CO₂ fixation in C₃ plant. The major results were as follows:The PEP/Pi transporter and PEPC were cloned from Arabidopsis and a thermophilic cystfiobacterium-Synechococcus Vulcanus, respectively. A constitutive plant expression vector pH35S-PEP/Pi and three light inducible plant expression vectors pH-rbcS-KsPEPC, pH-rbcS-SvPEPC and pH-rbcS-T-KsPEPC (SvPEPC is wild type, KsPEPC is mutant) were constructed using the Gateway technology. The plant expression vectors were transformed into wild type tobacco and transgenic PCK tobacco(P7) via Agrobacterium-mediated method. The transgenic plants were selected on MS mediumcontaining antibiotics and verified by PCR. We obtained eighteen transgenic PEP/Pi tobacco lines, sixteen transgenic PEP/Pi and PCK tobacco lines, twelve transgenic KsPEPC tobacco lines and fifteen transgenic KsPEPC and PCK tobacco lines.RT-PCR assays demonstrated these two PEP/Pi and PEPC genes were transcribed successfully in transgenic tobacco, and mensuration of enzyme activity showed that PEPC activity in the transgenic KsPEPC genes(located at cytoplasm) tobacco exhibited 3～5 folds higher than the control, indicating PEPC activity in transgenic tobacco harboring PEPC gene were enhanced. The leaves of pH-rbcS-T-KsPEPC transgenic plants became yellow and reticulated. The height of pH35S-PEP/Pi transgenic tobacco in the potted experiment under middle (5m Mol N /L) nitrogen nutrition condition were about 1～2 folds shorter than the control.All these results indicated that we had constructed a constitutive plant vector pH35S-PEP/Pi and three light inducible plant vectors pH-rbcS-KsPEPC, pH-rbcS-SvPEPC and pH-rbcS-T-KsPEPC. These vectors could be transformed into tobacco and expressed. PEPC activity in transgenic KsPEPC (located at cytoplasm) tobacco exhibited 3～5 folds higher than the control. At the same time, we obtained four kinds of transgenic single genes tobaccos(transgenic PEP/Pi,transgenic cytoplasm type KsPEPC,transgenic chloroplast type SvPEPC and KsPEPC), and four kinds of transgenic double genes tobacco(transgenic PEP/Pi+PCK, transgenic cytoplasm type KsPEPC+PCK, transgenic chloroplast type SvPEPC+PCK and transgenic KsPEPC+PCK). These transgenic tobaccos would provide good materials for future research. These results would be established the base of experiments for installing inducible C₄-like photosynthesis pathway in the lacking the "Kranz"-anatomy characteristic of C₃-plant.