Molecular Cloning, Bioinformatics Analysis And Preliminary Functional Characterization Of Vitamin K Epoxide Reductase Gene From Solanum Lycopersicum

Vitamin K epoxide reductase (VKOR) is an integral membrane protein widely existed inmammal endoplasmic reticulum. Homologues of Vitamin K epoxide reductase (VKOR)widely existed in plants. In the mutant, this gene plays a very important role in the growth ofplants. However, there is no report on other plant VKOR. In the present study, we focused onSolanum lycopersicum VKOR. The VKOR full length cDNA was cloned by rapidamplification of cDNA ends (RACE). We called it LeVKOR and registered in GenBank(JF951971). We conducted a series of bioinformatics analysis about LeVKOR and plant VKOR.In addition, we use prokaryotic expression and transgenic to preliminary research functions ofthis gene. The results as follows:(1) The VKOR full length cDNA was cloned by rapid amplification of cDNA ends(RACE). We found tomato cDNA of VKOR from NCBI, and designed primers to amplify thesequence. But the sequence we cloned does not exactly match to the opend database for lostthe termination codon. So we cloned VKOR full length cDNA by RACE. We found that thisgene is located on chromosome2of tomato, The full length of LeVKOR has7exonscontaining a1122bp ORF that encoded373amino acids. It has eight conserved cysteines,even four of the conserved cysteine is the very conservative CXXC motif.(2) We use online software to predict its signal peptide sequence and structure. Theresults showed that the first47amino acids from the N-terminus to act as a transit peptidetargeting the protein to the chloroplast. Sequence comparison and secondary structureindicated that the amino acid of VKOR possessed a transmembrane domain and a solubledomain. The predicted was very similar to the counterparts of AtVKOR. This research alsocompared the12plants VKOR sequences we can found from NCBI. We also did genephylogenetic analysis. We found that all plant have a transit peptide on the N-terminus, and most localized on chloroplast.(3) We did the suborganellar localization of LeVKOR. In the experiment, stroma andthylakoids were isolated from tomato plants, fractionated by SDS/PAGE, transferred to polymembrane, and visualized using anti-VKOR, or anti-D1(as a control for thylakoid proteins).The Western-blot revealed that LeVKOR was localized in the member of thylakoid, and thesoluble domain is faced on lumen.(4) Full-length of LeVKOR without transit peptide could catalyze the formation ofdisulfide bonds. We constructed the LeVKOR-pTrc99a vector, we performed functionalcomplementation assays in E.coli Dsb null strains. The motility complementation assay andthe X-gal assay showed that the full-length of LeVKOR without transit peptide could catalyzethe formation of disulfide bonds.(5) Constructed sense, antisense and RNAi plant expression vector and got transgenicseedling. We use pBI121and pFGC1008plasmid to construct sense, antisense and RNAiplant expression vector. We got transgenic plants by leaf disc transformation of cultivatedtomato using Agrobacterium tumefaciens. Now we have got T1transgenic seedling. Weidentified that the gene has antioxidant function by analysis of the LeVKOR over-expressionor less-expression in transgenic tomato.