Studies Of Construction Of Overexpression Vectors Containing RubisCO Activase Gene (RCA) And Genetic Transformation

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) is a key enzyme that catalyses the photosynthetic assimilation of atmospheric CO2 in chloroplasts of higher plants. The stability of RubisCO activity may be the main way to adapt to stress. RubisCO is activated only when the state with activation .RubisCO with non-activation must go through the activation of RCA, and then shows its carboxylation/oxygenase activity. So RCA plays an important role in the control of photosynthesis .We cloned RCA gene from cucumber leaf and construction of the sense expression vector introduce into cucumber by ovary-injection. Effect of low light intensity, suboptimal temperature and low light intensity, low temperature and low light intensity on photosynthesis and gene expression. The results showed as follows.1. Specific primers were designed based on the sequence of the rbcL and rbcS in Genbank, With RT-PCR methods, A 549bp rbcL cDNA fragment and a 490bp rbcS cDNA fragment were amplified from cucumber (Cucumis sativus L.). They were registified in GenBank with accession numbers EF208123 and EF208124 respectively.2. Specific primers were designed based on the sequence of the RCA in Genbank, With RT-PCR methods, the full-length cDNA of RCA was amplified from cucumber (Cucumis sativus L.). It's accession number in GenBank is EF421974.3. The cloned cDNA of RCA was introduced into binary vector pCAMBIA1301 under its promoter (CaMV 35S), giving an expressing plasmid containing the sense RCA gene. The sense vector were constructed and introduced into tobacco and cucumber using Agrobacterium-mediated plant transformation and ovary-injection, respectively.4. Quantitative of low light intensity, suboptimal temperature and low light intensity, low temperature and low light intensity material gene expression is determinated by real time PCR. Low light intensity, suboptimal temperature and low light intensity stress all lead to decreased obviously.5. Pn of low light intensity, suboptimal temperature and low light intensity, low temperature and low light intensity stress showed similar trend with relative expression of rbcL,rbcS and RCA. It shows that the decreace of were the important reason for Pn decreace of cucumber seedlings.