Study On Cloning Of The CDNAs Of FBPase And GAPDH Genes And The Prokaryotic Expression From Camelia Oleifera

Camelia oleifera is an important tree in southern China edible oil species, whose seed oil is higher. Although isolated some genes from the camelia oleifera, but there are also a large number of genes identified have not been isolated, thus it is difficult to meet the requirements of improved varieties of camelia oleifera. FBPase and GAPDH is an important regulatory enzyme in the dark reaction of photosynthesis. It is helpful for revealing the process of photosynthesis in camelia oleifera and improving camelia oleifera production to clone FBPase and GAPDH gene, analyse the structure and function, and research FBPase and GAPDH gene regulation mechanism. To this end, we have carred out camelia oleifera FBPase and GAPDH gene cloning, structural features and activity of enzymes, prokaryotic expression studies, and the main findings are as follows:1. Camelia oleifera FBPase and GAPDH full-length gene cloning. Based on the laboratory's camelia oleifera cDNA library and EST library, designed primers on the software of Primer Premier 5.0, taked the "xianglin 1" near-mature seeds as materials and the extracted RNA after reverse transcription of cDNA as template, we won the FBPase gene and GAPDH gene fragment product with 5'RACE and 3'RACE, which was cloned into pMD-18T vector and transformed into E.coli DH5a. BLAST sequence alignment through online results confirmed the fragment products as the camelia oleifera full length cDNA of FBPase and GAPDH gene. The FBPase gene full cDNA sequence is 1356 bp with a ORF of 1023 bp, and the GAPDH gene full cDNA sequence is 1364 bp with a ORF of 1014 bp.2. Camelia oleifera FBPase and GAPDH biological function prediction. Application of some bioinformatics software online analysis, the results showed that: Camelia oleifera FBPase protein molecular weight is 37.7KDa, theoretical isoelectric point is 5.54, where the number of negatively charged amino acid residues 44, the number of positively charged amino acid residues 38, and it is a stable protein with three obvious transmembrane domain, two structural analysis of 12 of a-helices, and its encoded protein does not contain disulfide bonds, showing that it out of light regulation, suggesting that it is cytosolic FBPase, and its activity was inhibited by AMP, the best activity is pH 8.0; Camelia oleifera GAPDH protein isoelectric point is 5.08, the formula can be written as C3033H5054N1014O1282S216, and the protein is unstable, instability coefficient is 44.50. Its half-life of the protein in E.coli more than 10 hours, and the fat factor is 25.15, the average hydrophilicity is 0.692, without transmembrane regions, and secondary structure analysis with 7 a-helices.3. Camelia oleifera FBPase and GAPDH gene expression in the original fusion. We designed two pairs of specific expression with the choice of restriction enzymes Ndel and Hindâ…¢, and constructed the fusion expression vector of pET-28b-FBPase and pET-28b-GAPDH, which were successfully expressed in E.coli. The results showed:recombinant FBPase protein have a large number of expressionwith IPTG in one hour, and the iduction time increased with the expression level significantly increased; recombinant GAPDH protein have a significantly expression in two hours with IPTG, and followed by 3h,4h,5h, the incresing trend is ease.