Cloning And Analysis Of Zeaxanthin Epoxidase Gene And Its Promoter In Watermelon

Xanthophyll cycle exists in all higher plant and is metabolic process that has been preserved during the long-term evolution history. Heat dissipation is the most principal function of Xanthophyll cycle. The enzyme encoded by Zeaxanthin Epoxidase gene is one of the both key enzyme in xanthophyll cycle. In this study, Zeaxanthin Epoxidase gene in wild watermelon was cloned by RT-PCR and analyzed by bioinformatics. What is more, the promoter was isolated by using genome walking approach. The results are as follows:1. The cDNA fragments of ClZE gene were obtained by RT-PCR. Using RACE technique the whole ClZE gene was cloned. The nucleotide sequence of ClZE cDNA was 2,535 bp, which contained an open reading frame (ORF) of 1,998 bp encoding a putative polypeptide of 665 amino acids. The molecular mass of the predicted protein was 73.17 kDa and the isoelectric point was calculated to be 7.25. Its accession in GenBank is HM107775.2. The bioinformatic analysis showed that ClZE protein was stable and hasn′t signal peptide. There were one transmembrane domain and two conserved domain which were Pyr_redox superfamily and FHA.3. Homology comparison and cluster results showed that the ClZE gene and cucumber were grouped together.4. The prokaryotic expression vector pET-ClZE was successfully constructed. The recombinant ClZE was expressed successfully in E.coli with the induction of 1 mM IPTG after 3 hours at 28°C. The expression of protein increased significantly with the induction time, and got the most at 12 hours. Approximate 93 kDa recombined protein were found through SDS-PAGE.5. Using genome walking approach, the promoter region of ClZE with 1256 bp length was isolated. The conserved regulatory elements functional on transcriptional regulation, such as TATA box and CAAT box were identified. The low temperature and light response elements (LTRE1HVBLT49 and -10PEHVPSBD) were figured out in ClZE promoter, indicating that these elements possibly involved in low temperature and light of ClZE. 6. Using DNA recombinant technology, the binary cassette of ClZE-Gus, and series of cassettes in which the reporter gene Gus being driven by the deleted promoter fragments have been constructed. This work would provide the basis for the further exploration of the transcriptional regulation of ClZE in the future.