Cloning And Analysis Of Cotton Polyphenol Oxidase And Its Promoter

Large areas of planting Bt cotton make the status of cotton pests changed, Apolygus lucorum, cotton aphid, leafhoppers et.such as the increase of the secondary pests of our counlry’s cotton production safety constitute a new threat. Based on the cotton (CCRI49) as test materials, the application of molecular biology, bioinformatics methods, cloned the cotton induction delense genes polyphenol oxidase genes and its promoter. It lays the foundation for study the specific role of PPO in collon defense system. The main results are as follows:1.Through race technology and electronic cloning, we cloned a PPO gene full-length cDNA from CCR149.It was named GhPPO (GenBank accession: JQ345705).The longest ORF1797bp gene and encoded a putative protein of598aa. The gene5’UTR including102bp,3’ UTR including l23bp.Construclion of the prokaryolic expression vector pGEX-4T-1-PPO, in E.coli induces the expression of the PPO protein was about67kD, and prediction of consistent molecular weight.2. In the genome of colton (CCR149) as a template, design primer get polyphenol oxidase gene sequence and cDNA sequence is the same, namely the cotton PPO genes the same as the other reports of dicotyledons PPO genes no introns. Using ANTMEPRO5.0application software analyzes the amino acid sequence GhPPO coding areas with function site. Application ChloroP1.1) Server presumably the collon contains N-PPO sequence the guide peptide (chloroplast transit peptides), at the same lime, Wolf pSORT application for sub-cellular localization, GhPPO prolein expression mainly exist in the chloroplasts, presumably PPO and cell photosynlhelic metabolism cotton on, but specific and cells to positioning fluorescence microscopic lest, and the specific mechanism remains to be studied further.3. A variety of plant PPO gene promoter have be cloned, but collon polyphenol oxidase promoter of the cloning has not been reported. In this paper the method of chroniosome movement of step cloned cotton PPO promoter sequence gene, named for GhPPO-P, application PlantCARE and PLACE of software GhPPO-P molecular slmclure is analyzed, and!he role of the prediction of components have special express root sequence, light effect promoter (GT-1site.1-DOX, etc.), heating promoter (CAAT BOX, AT-rich active components series), cold effect promoter (CCGAC modif, G-BOX structure, etc), water effect promoter (ARE, GC rich. GT rich. etc), plant defend mechanism relaled components (W-BOX, WUN-motif, etc.).4. Successfully constructed plant expression vector of cotton Polyphenol Oxidase genes, then the next step we will transfor it in Arabidopsis to test and verify its function。...