茉莉花MVD基因及其启动子的克隆与表达

Cloning and expression of MVD gene and its promoter in Jasminum sambac

根据茉莉花转录组数据,采用RT-PCR技术,克隆了茉莉花甲羟戊酸焦磷酸脱羧酶(MVD)基因,命名为JsMVD(GenBank登录号为MH311041.1).采用染色体步移技术分离JsMVD基因5′端上游调控序列,通过实时荧光定量PCR技术检测JsMVD基因在茉莉花植株不同组织及不同激素处理下的表达水平.测序结果表明,JsMVD基因全长cDNA序列的长度为1 500 bp,包含长度为1 269 bp的开放阅读框(ORF),共编码422个氨基酸,亚细胞定位预测该蛋白可能位于细胞质上.JsMVD蛋白含有GHMP激酶N-端和C-端保守结构域,具有多个保守氨基酸残基及ATP结合位点,与野生油橄榄的相似性最高,相似系数达到88%,且进化树显示两者的亲缘关系最近.JsMVD基因5′端启动子序列长度为893 bp,该调控序列包含多种植物激素响应元件和光响应元件.实时荧光定量PCR技术检测结果表明,JsMVD基因在成熟花中的表达量最高,从高到低依次为成熟花、花蕾、茎、叶、根,且受GA、IAA和ABA不同程度的诱导.

Based on jasmine transcriptome,diphosphomevalonate decarboxylase(MVD)gene was cloned using RT PCR.The up stream regulation sequences of Jasminum sambac 5′end was isolated by Genome Walking Technique,and its expression treated with different hormones in different tissues were quantified using RT qPCR.The results showed that JsMVD gene(GenBank accession number MH311041.1)was 1 500 bp in length,which contained 1 269 bp open read frame and encoded 422 amino acids.Subcellu lar localization prediction showed that JsMVD protein may be located in the cytoplasm.The JsMVD contained GHMP_kinases_N domain and GHMP_kinases_C domain,many conserved amino acid residues and ATP binding sites.JsMVD was most homologous to Olea europaea based on phylogenetic tree,with an identity up to 88%.Plantcare analysis indicated that the upstream 5′sequence of JsMVD was 893 bp in length.It contained multiple light responsive cis regulatory elements and hormones responsive cis regulatory elements such as auxin(IAA),gibberellin(GA)and abscisic acid(ABA).RT qPCR analysis showed that the expression of JsMVD was highest in mature flower,followed by bud,stem,leaf and root,and it was induced by GA,IAA,ABA to some extent.