利用生物信息学方法,通过电子克隆获得葡萄(Vitis vinifera Linn.) ( E)-β-丁香烯合酶基因的cDNA 序列;以从葡萄品种‘德引84-1’(‘Deyin 84-1’)果肉中提取的mRNA 为cDNA 模板, 利用特异PCR 技术克隆得到1 个全长1 880 bp 的基因,被命名为Vv-ECar(GenBank 登录号JF808010),该基因序列包括开放阅读框1 674 bp、3’非翻译编码区209 bp 和poly+(A) 28 bp,可编码557 个氨基酸。比对结果显示:葡萄Vv-ECar 基因的核苷酸序列与葡萄VvGwECar2 基因的同源性达93%,二者编码的氨基酸序列同源性达90. 8%,均含有植物萜类合酶家族共有的保守域DDXXD;葡萄Vv-ECar 与茶[Camellia sinensis (Linn.) O. Kuntze]和杨(Populus balsamifera subsp. trichocarpa×P.deltoids)的萜类合酶相关基因同源性均在73%以上;分子进化树的分析结果也显示葡萄Vv-ECar 基因编码的氨基酸序列与其他植物的同源序列具有高度保守性。半定量RT-PCR 和荧光定量PCR 分析结果显示:在葡萄果实发育的不同阶段均有Vv-ECar 基因的表达,但其相对表达量随果实的发育呈先低后高的趋势,其中在幼果期相对表达量最高。

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.