茶尺蠖类免疫球蛋白基因EoHML的克隆和表达分析

Molecular Cloning and Expression Analysis of Hemolin Gene in Tea Geometrid(Ectropis obliqua)

类免疫球蛋白(Hemolin)是一种鳞翅目昆虫特有的免疫相关蛋白,也是唯一的无脊椎动物免疫球蛋白家族成员。本实验应用RACE技术获得了茶尺蠖(Ectropis obliqua Prout)类免疫球蛋白基因的全长c DNA序列,命名为Eo HML(Gen Bank登录号:KM885983),并分析了相关生物信息学特性,检测了病毒感染后基因的表达水平。结果表明,Eo HML基因序列全长1 772 bp,包含1 239 bp的开放阅读框,编码412个氨基酸,预测蛋白分子量大小为45.8 k D,等电点(p I)为8.297,属于典型的分泌型蛋白,且具有昆虫Hemolin基因保守的4个Ig功能区和2个N-glycosylation位点。系统进化分析表明,该蛋白与目前已知的类免疫球蛋白氨基酸序列的亲缘关系都较远,与烟草天蛾(Manduca sexta)类免疫球蛋白氨基酸序列相似度最高,为53%。荧光定量检测结果表明,茶尺蠖核型多角体病毒(Eo NPV)感染茶尺蠖幼虫后该基因表达量显著升高,最高表达量是正常对照的36.5倍,表明茶尺蠖Eo HML基因可能参与了茶尺蠖对Eo NPV的免疫代谢反应。

Hemolin, a unique immune protein, belongs to the immunoglobulin superfamily of Lepidoptera insects. A full length c DNA sequence of hemolin gene was cloned by RACE-PCR from tea geometrid(Ectropis obliqua Prout), which was named as Eo HML(Gen Bank Accession No. KM885983). The results of bioinformatics characterization showed that the full length c DNA of Eo HML was 1 772 bp, which contained an intact CDS of 1 239 bp and encoded 412 amino acid residues with a putative molecular mass of 45.8 k D and an isoionic point of 8.297. The deduced amino acid sequence demonstrates it belongs to a typical secretory protein that contains 4 Ig functional conservative areas and 2 N-glycosylation sites. Phylogenetic analysis showed that Eo HML share the far genetic distance with published hemolin gene and the highest similarity with that of Manduca sexta was only 53%. The results of quantitative real-time PCR analysis further revealed a significant up-regulated expression about 36.5 times in the larvae infected E.obliqua Nucleopolyhedrovirus(Eo NPV) than the controls. The study indicated that the Eo HML gene may participate in the immune response to Eo NPV in tea geometrid.