Cloning And Expression Analysis Of Walnut (Juglans Regia L.) JrVTE1Gene

Vitamin E is an essential lipid soluble compound for humans which is synthesized onlyby oxygenic photosynthetic organisms, and walnut (Juglans rehia L.) is a major Vitamin Esource. In this study, the tocopherol cyclase gene was isolated from walnut cultivar‘Xiangling’, cloned to pET28a and was transformed to E.coli BL21(DE3) for expression;Agrobacterium-mediated genetic tansformation system was established, and the JrVTE1genewas transformed into jujube (Zizyphus spinosus Hu) and pear (Pyrus communis L.)respectively. It has the important practical significance to improve crop nutrition value,excavate the excellent genetic resources of walnut. There are several works in the follow.1. Molecular cloning and characterization of JrVTE1gene from Walnut. The tocopherolcyclase gene was isolated from Walnut by RACE, and designated as JrVTE1. Sequence dataof JrVTE1had been deposited at GenBank under accession numbers of KC751543. Thefull-length cDNA sequence of JrVTE1contained1,749base pairs (bp). It was predicted toencode a protein of451amino acids. Multiple alignment of amino acid sequences indicatedthat JrVTE1shared high sequence identity with the VTE1s from Eucalyptus gunnii, Heveabrasiliensis, Glycine max, Arabidopsis Thaliana, Medicago truncatula and Sesamum indicum,which was about80％. JrVTE1was belonged to the tocopherol cyclase family, which wasused to catalyze2-methyl-6-based-benzoquinone (2M6PBQ) to δ-tocopherol or catalyze2,3-two methyl5-based-benzoquinone (2,3DM5PBQ) to γ-tocopherol in the synthesis of vitaminE.2. Expression study of real-time fluorescent quantitative of JrVTE1gene in J. Regiaembryonal tissue. Real-time fluorescent quantitative RT-PCR analysis showed that JrVTE1gene was expressed in three important periods, respectively. And it was highest in90d afterflorescence.3. Prokaryotic expression of JrVTE1gene. Prokaryotic expression vectorpET28a-JrVTE1was constructed and was transformed into E. coli BL21(DE3) for expression.The culture conditions of recombinant cells was optimized in37℃and was induced by 1mmol·L-1IPTG. The SDS-PAGE analysis showed that there was a specific protein in thecells which had recombinant plasmid pET28a-JrVTE1after2hours. And the quantity of theprotein was increased with the extension of time. But there was no specific expression proteinin the control ones. The molecular weight of protein expressed by pET28a-JrVTE1was49.5kD which was equal to the expected result, so it was the target protein.4. Plant expression vectors was constructed and transformed to wild jujube and pearsuccessfully. JrVTE1gene was cloned to pRI101-AN vector and was transformed into jujubeand pear tender leaves respectively by Agribacterium-mediated transformation. The JrVTE1transgenic jujube and pear were both identified by Kan-resistant and PCR, and there were9and25transgenic JrVTE1jujube and pear positive plants respectively.