Chloroplast Expression Vector Construction Of CbbM Gene From Rhodospirillum Rubrum In Maize And CbbM Gene Expression Analysis

Maize is the world’s largest food crop and it is of vital importance to human food security. Due to the limitation of arable land area, maize production is faced with the pressure of increasing the level of yield per unit area. Exploring the mechanisms to improve maize photosynthetic efficiency from the photosynthetic carbon assimilation process is an important way to improve their yield levels. Rubisco is the rate-limiting enzyme of the Calvin cycle, which directly affects the activity of photosynthetic efficiency. Form Ⅱ Rubisco large subunit gene was cloned from Rhodospirillum rubrum and maize chloroplast expression vector was constructed in this article, laying the foundation for the bundle sheath cells of maize Rubisco heterologous gene expression, the analysis of CO2 fixation mechanism and also providing ideas for genetically modified maize photosynthesis.The main results are as follows:1. Rhodospirillum rubrum Form Ⅱ Rubisco subunit gene (CbbM) encoding sequence (1401bp) was successfully cloned using the PCR method,the homology with maize Rubisco large subunit gene was 39.18%.2. Maize chloroplast Rubisco subunit (RbcL) promoter and terminator as the promoter and terminator of CbbM maize expression were successfully cloned using the PCR method, and finally the three sequences were assembled into recombinant gene in the pBluescript Ⅱ KS (+) vector.3. The trnA^ trnl chloroplast DNA fragments were selected as the homologous recombination to insert CbbM recombinant gene into the maize chloroplast DNA sequences by analyzing maize chloroplast genome sequence. The trnA and trnl fragments were successfully cloned from the whole genome of maize, and were connected to the pMD19-T vector respectively.4. According to the literature, aadA was selected as the marker gene,16SrRNA gene promoter from maizes as a promoter sequence and RbcL terminator as a termination sequence. The 16SrRNA gene promoter and RbcL terminator were successfully cloned from the whole genome of maize, and were connected to the pMD19-T vector respectively.5. Finally, by constructing the intermediate vector, CbbM recombinant gene and aadA marker gene were successfully connected to the vector pBluescript Ⅱ KS (+),maize chloroplast CbbM gene expression vector was constructed, and successfully expressed in E. coli and its resistance has been initially verified.6. The CbbM gene, of Rhodospirillum rubrum was analyzed by using the real-time PCR method, the results showed that:At 20℃,25℃,30℃,35℃ and 40℃ condition, the highest relative expression of CbbM was at 25 ℃; Under the light intensity of 0 lux,1000 lux,2000 lux,3000 lux,4000 lux and 50001ux, the highest relative expression of CbbM was in 4000 lux; Under the condition of 0.5 g/L,0.75 g/L,1 g/L, 1.25 g/L and 1.5 g/L medium chloride content, the highest relative expression of CbbM was in 1.25 g/L; Under the condition of 1 g/L,2.5 g/L,4 g/L,5.5 g/L and 7 g/L medium malic acid content, the highest relative expression of CbbM was in 4 g/L.