Cloning and expression analysis of (E)-β-caryophyllene synthase gene from Vitis vinifera fruit.

Using bioinformatics method,cDNA sequence of(E)-β-caryophyllene synthase gene of Vitis vinifera Linn.was obtained by the silico cloning method.Taking mRNA isolated from flesh of V.vinifera 'Deyin 84-1' as the cDNA template,a gene with the full-length of 1 880 bp was cloned by means of specific PCR,which is named Vv-ECar(GenBank accession No.JF808010).And this gene includes an opening reading frame of 1 674 bp,3′ untranslated coding region of 209 bp and poly+(A) of 28 bp,and it can encode 557 amino acids.The alignment result shows that the homology of nucleotide sequence of Vv-ECar gene of V.vinifera with that of VvGwECar2 gene of V.vinifera reaches 93%,and the homology of amino acid sequence encoded by two genes reaches 90.8% which all have the common conserved box DDXXD of plant terpenoid synthase family.The homology between Vv-ECar of V.vinifera and terpenoid synthase related genes of Camellia sinensis(Linn.) O.Kuntze and Populus balsamifera subsp.trichocarpa×P.deltoids is more than 73%.And also,the result of molecular phylogenetic tree indicates that there is highly conservation in the amino acid sequences encoded by Vv-ECar of V.vinifera with homologous sequence of other plants.The analysis results of semi-quantitative RT-PCR and fluorescence quantitative PCR show that Vv-ECar gene expression exists in different development stages of V.vinifera fruit,but its relative expression amount appears the trend of decreasing firstly and then increasing with fruit developing with the highest relative expression amount in young fruit stage.