欧洲山芥皂苷合成关键酶基因Bv-beta-AS 克隆及表达分析

 以能够产生抗虫皂苷的高抗小菜蛾资源G 型欧洲山芥（Barbarea vulgaris R. Br.）B44 为材料，利用RACE 技术克隆出皂苷合成关键酶beta–香树脂合成酶的基因（Barbarea vulgaris beta-amyrin synthase，Bv-beta-AS）。该基因编码区序列长为2 289 bp（GenBank 登录号JQ172795），推导其编码762个氨基酸；在基因组水平上长度为4 107 bp （GenBank 登录号JQ172796），含有15 个内含子。Bv-beta-AS编码的氨基酸具有beta–香树脂合成酶基因家族的保守序列，即DCTAE 序列和QW 特征序列，氨基酸多序列比对和进化树分析表明，该基因与拟南芥beta–香树脂合成酶基因的相似性最高，为74%。利用荧光定量PCR 对欧洲山芥在小菜蛾诱导下该基因的表达进行研究，结果表明，该基因受虫害诱导时上调表达，但是上升到12 h 达顶峰后随时间推移呈回归的趋势。从序列特征和表达模式上推测，Bv-beta-AS 可能是抗虫皂苷合成途径的一个关键酶的基因。

Cloning,Characterization and Real-time RT-PCR Analysis of a Key Gene beta-Amyrin synthase for Saponin Biosynthesis in Barbarea vulgaris

G-type Barbarea vulgaris R.Br. which produce saponins is a rare insect resistant vegetable in Cruciferae. A beta-amyrin synthase gene which codes a key enzyme of saponin biosynthesis pathway was cloned from Barbarea vulgaris R.Br. using RT-PCR and RACE(Rapid Amplification of cDNA Ends) method,and named as Bv-beta-AS. The Bv-beta-AS contained a coding sequence of 2 289 bp(GenBank accession number:JQ172795),which encoded 762 amino acids. The gene of 4 107 bp from genomic DNA was further cloned,which contained 15 introns embedded in its coding sequences. The deduced protein of this gene contained the conserved characteristic motif of beta-amyrin synthase -DCTAE and QW. And thehigh similarity of this gene with the beta-amyrin synthase from other plants indicated it was a homologue of beta-amyrin synthase. Multiple sequence alignments and phylogenetic tree analyses showed that Bv-beta-AS was clustered with Arabidopsis beta-amyrin synthase and showed the highly similarity(74%) in amino acid sequences. The expression of Bv-beta-AS under the diamondback moth(Plutella xylostella) attack was determined by real-time RT-PCR. The results showed that Bv-beta-AS gene expression was up-regulated within 12 h of diamondback moth attack,but decline tendency appeared after 12 hours. Both the sequences analysis results and the real-time RT-PCR data indicated that Bv-beta-AS may be an important candidate gene of saponin biosynthesis pathway.