小菜蛾气味受体蛋白PlxyOr83b基因的克隆及表达

Cloning and expression of odorant receptor gene PlxyOr83b from Plutella xyostella(Lepidoptera:Plutellidae)

【目的】克隆小菜蛾Plutella xyostella气味受体Or83b基因,并进行原核表达,为研究小菜蛾寄主选择行为的分子机理,开发昆虫行为调节剂提供基础。【方法】提取小菜蛾的总RNA,反转录获得总cDNA,采用RT-PCR方法扩增目的基因,将其克隆至T载体并测序,然后将目的基因克隆到大肠杆菌Escherichia coli表达载体pET-32a(+)中表达。经酶切、PCR及测序鉴定正确后转化BL21(DE3)菌株,用IPTG诱导表达,通过SDS-PAGE,Western印迹鉴定表达蛋白。【结果】获得了编码小菜蛾Or83b的cDNA序列,该基因阅读框长1 413 bp,编码471个氨基酸,预测的等电点为7.19,命名为PlxyOr83b(GenBank登录号为GQ923610);成功构建了pET-PlxyOr83b原核表达重组质粒,目的基因获得高效表达,其融合蛋白分子量为32.0 kD,Western blot检测结果进一步表明PlxyOr83b在大肠杆菌DE3中得到正确表达。【结论】成功克隆和表达了小菜蛾气味受体基因PlxyOr83b,该基因与其他昆虫Or83b基因基本一致。

【Aim】 cDNA cloning and prokaryotic expression of Plutella xylostella odorant receptor Or83b were conducted in order to provide the basis for studying the molecular mechanism of selection behaviour between P.xylostella and host plants,and developing insect behavior regulators.【Methods】 cDNA encoding Or83b of P.xylostella was amplified by RT-PCR and cloned into pMD18-T vector for sequence analysis,the target gene was subcloned into pET-32a（＋） for prokaryotic expression and then expressed under the induction of IPTG.The expressed product was identified by SDS-PAGE and Western blot.【Results】 A gene encoding odorant receptor 83b from P.xylostella was cloned and named PlxyOr83b（GenBank accession number： GQ923610）.The ORF of PlxyOr83b was 1 413 bp in length,encoding 471 amino acid residues,with the predicted pI of 7.19.Prokaryotic expression recombinant vector,pET-PlxyOr83b,was successfully constructed.SDS-PAGE assay showed that the target gene was highly expressed in BL21（DE3） and the molecular weight of fusion protein was about 32.0 kD.Western blot indicated that PlxyOr83b was expressed correctly in DE3.【Conclusion】 PlxyOr83b was successfully cloned and expressed,and its sequence and protein structure are consistent with those of Or83b genes in Drosophila and other insects.