松墨天牛包囊蛋白基因的克隆及表达特性分析

Cloning and expression of encapsulation-relating protein gene of Monochamus alternatus

松墨天牛Monochamus alternatus是危害马尾松树重大蛀干害虫,也是松材线虫的主要传播媒介昆虫,对我国松林资源构成严重威胁。用c DNA末端快速扩增(RACE)方法克隆了松墨天牛ERP基因(Mal ERP),Gen Bank登录号为KF357881.1。该基因含有一个长为864 bp的开放阅读框,编码287个氨基。同源性分析表明,Mal ERP与光肩星天牛Anoplophora glabripennis ERP的同源性最高,达到79%;与赤拟谷盗Tribolium castaneum的同源性为45%。实时荧光定量PCR分析显示,Ma ERP在松墨天牛3龄幼虫的脂肪体、马氏管、中肠、体壁、血淋巴和头部中均有表达,在脂肪体中的表达量最高;Ma ERP经溴氰菊酯、磷化铝、B t、印楝素、绿僵菌胁迫后,上调表达;但经阿维菌素胁迫后,下调表达。本研究为进一步研究Mal ERP基因在松墨天牛防御过程中的作用奠定基础,为探索松墨天牛免疫系统与分子毒理互作关系提供参考。

Pine sawyer, Monochamus alternatus Hope （Coleoptera： Cerambyeidae）, a serious trunk borer in pines stands, also is a major media insects of pine wood nematode, posed a serious threat to the forest resources in China. It is the key vector of the exotic pine wood nematode Bursaphelenchus xylophilus throughout Asia. The ERP gene from M.. alternates, called MaERP （GenBank accession number is KF357881.1）, was cloned and sequenced by rapid amplification of eDNA ends. Sequence analysis revealed that the open reading frame of MaERP had 864 bp in full length encoding a 287 predicted amino acids residues. Blastx showed that MaERP and Anoplophora glabripennis had EPR homology, up to 79%, and had EPR homology to Tribolium castaneum, being 45%. The Real-time PCR quantitative analysis showed that MaERP was expressed in body wall, head, midgut, Malpighian tubule and hemolymph from the 3rd instar larvae of M.. alternatus, and expressed in fat body with the highest expression. The RT-qPCR also indicated that MaERP was upregulated when exposed to Metarrhizium anisopliae, azadirachtin, aluminum phosphide, Bt and deltamethrin, and downregulated when exposed to avermectin.The findings of the study lay a foundation for further research on MalERP＇s defensive reaction to M. alternatus, provide a reference for studying the interaction between immune system and molecular toxicology of M. alternates.