小菜蛾整合素integrinβ1基因的克隆及其功能分析  

Molecular cloning and functional analysis of an integrin β1 in Plutella xylostella

作　　者：郑锦龙 许小霞 余静 张展滔 兰可可 仇培 金丰良 ZHENG Jin-Long;XU Xiao-Xia;YU Jing;ZHANG Zhan-Tao;LAN Ke-Ke;QIU Pei;JIN Feng-Liang(Key Laboratory of Bio-Pesticide Innovation and Application of Guangdong Province,College of Agriculture,South China Agricultural University,Guangzhou 510642,China)

机构地区：[1]华南农业大学农学院,广东省生物农药创制与应用重点实验室,广州510642

出　　处：《环境昆虫学报》2018年第4期880-893,共14页Journal of Environmental Entomology

基　　金：国家自然科学基金(31371989);广东省科技计划项目(2015A020209128,2014A020208106)。

摘　　要：整合素(Integrin)是一种由α和β亚基组成的跨膜异二聚体蛋白,在昆虫血细胞对外物的包囊过程中起着重要的作用。为阐明integrinβ1参与小菜蛾Plutella xylostella体内细胞免疫中的功能,本研究利用RT-PCR结合RACE技术克隆了小菜蛾integrinβ1基因的cDNA全长序列,命名为PxIntβ1(Gen Bank登录号GQ178290)。小菜蛾PxIntβ1的cDNA全长序列为2 832 bp,开放阅读框为2 487 bp,编码828个氨基酸,成熟的氨基酸序列中有一个跨膜区和一个integrin亚基。系统进化树显示小菜蛾PxIntβ1与亚洲玉米螟Ostrinia furnacalis整合素的同源性最高,两者同源性达到78%。利用半定量RT-PCR技术检测PxIntβ1基因在小菜蛾不同发育历期(卵、1-4龄幼虫、蛹、成虫)、不同组织(血细胞、体壁、脂肪体、中肠、马氏管)和细菌(金黄色葡萄球菌和大肠杆菌混合菌液)诱导下的表达模式。半定量结果表明小菜蛾PxIntβ1基因在小菜蛾整个发育历期除了卵之外都有表达,4龄幼虫转录水平最高,在血细胞中特异性的高表达,混合细菌诱导2-48 h后,PxIntβ1基因在小菜蛾诱导后24 h达到诱导表达高峰。为了进一步获得重组蛋白PxIntβ1,本研究构建了原核表达质粒pET-32a-PxIntβ1,融合蛋白在大肠杆菌Eschericha coli BL21中获得高效表达,Ni-NTA一步纯化了融合蛋白,并免疫新西兰大白兔,获得了多克隆抗体anti-PxIntβ1。SDS-PAGE电泳和Western blot分析结果表明,His抗体和多克隆抗体anti-PxIntβ1能特异性识别融合蛋白PxIntβ1;进一步利用显微镜观察了小菜蛾血细胞对凝胶珠Sephadex-A25的包囊情况,结果表明抗血清anti-PxIntβ1处理过的小菜蛾血细胞对Sephadex-A25凝胶珠的包囊作用受到明显抑制,重组蛋白PxIntβ1可以提高小菜蛾血细胞对Sephadex-A25凝胶珠的包囊作用;以上研究结果表明小菜蛾PxIntβ1在小菜蛾细胞免疫中起着重要的作用。Integrin is a transmembrane protein dimers composed by alpha and beta subunits,integrin has been reported to play a vital role in hemocyte encapsulation foreign objects in insects.In order to illustrate the function of integrinβ1 involved in cellular immunity of P.xylostella.In the present study,a full-length cDNA of an integrinβ1 gene was cloned from P.xylostella by RT-PCR and rapid amplification of cDNA ends(RACE)techniques,and designated as PxIntβ1(GenBank accession no.:GQ178290).Its full-length cDNA is 2 832 bp,and the open reading frame(ORF)is 2 487 bp,encoding 828 amino acid residues.The mature amino acid sequence of PxIntβ1 is composed of a transmembrane region and an integrin subunit.Phylogenetic analysis indicated that PxIntβ1 has the highest homology to integrinβfrom O.furnacalis,the similarity between them is 78%.Semi RT-PCR was employed to analyze the expression profiles of this gene in different tissues(hemocyte,epidermis,fat body,midgut and malpighiam tubules)of the 4 th instar larvae,different developmental stages(egg,1 st-4 th instar larva,pupa,and adult)and microbial induction(the mix between Staphylococcus aureus and Escherichia coli)in P.xylostella.Semi RT-PCR results indicated that the mRNA level of PxIntβ1 was expressed during the whole developmental stages except eggs,with the highest transcript level in the 4 th instar larva.Tissue specific result showed that PxIntβ1 was specifically expressed in hemocyte.Microbial induction result showed PxIntβ1 was induced significantly by mixed bacteria from 2-48 h,with the highest expression level at 24 h post-injection mixed bacteria.In order to acquire the recombinant protein of PxIntβ1,prokaryotic expression plasmid pET-32a-PxIntβ1 was constructed and the fusion protein was highly expressed in E.coli BL21.and recombinant protein was one step purified by Ni-NTA.The polyclonal antibody anti-PxIntβ1 with high serum titer was prepared using the purified fusion protein to immunize New Zealand white rabbit.SDS-PAGE and western blot analysis inv

关 键 词：小菜蛾 PxIntβ1 RT-PCR 原核表达 包囊作用 

分 类 号：Q963[生物学—昆虫学] S433.4[农业科学—农业昆虫与害虫防治]