小菜蛾Cactus基因的克隆及表达模式研究

摘要

Cactus是昆虫NF-κB信号通路的核转录抑制因子(Inhibitor of nuclear transcription factor,IκB),可通过蛋白质间的相互作用与Toll信号通路中核转录因子(Nuclear transcription factor,NF-κB) Dorsal及Dif结合,从而调控抗菌肽基因的表达.为阐明Cactus在小菜蛾Toll信号通路中的功能,本研究利用RT-PCR结合RACE技术获得了小菜蛾Cactus基因的cDNA全长序列,命名为PxCactus(GenBank登录号:KY828920),其cDNA全长为1 868 bp,开放阅读框(Open reading frame,ORF)序列1 053 bp,编码350 aa.实时荧光定量PCR (qRT-PCR)对PxCactus的时空表达差异性检测表明,PxCactus基闪在小菜蛾不同的发育阶段都有表达,其中2龄幼虫表达量最低,4龄幼虫表达量最高;PxCactus在健康的4龄幼虫不同组织都有表达,其中在脂肪体中的表达量显著高于其它组织;在脂肪体中PxCactus的表达可以被Bacilhus thuringiensis和Serratia marcescens强烈诱导表达,而Metarhizium anisopliae抑制了PxCactus的表达.为了获得PxCactus的多克隆抗体,本实验构建了原核表达质粒pGEX-4T-PxCactus,并在大肠杆菌BL21 (DE3)中高效表达了融合蛋白GST-PxCactus,利用GST亲和柱纯化了融合蛋白,并免疫日本大耳兔制备了PxCactus多克隆抗体anti-PxCactus.ELISA和Western blot检测结果表明anti-PxCactus和PxCactus蛋白呈高效结合反应,表明已经成功制备的多克隆抗体;并利用Western blot检测到了小菜蛾脂肪体组织中的Cactus蛋白的表达.本研究成功克隆PxCactus基因及制备其多克隆抗体,为下一步研究PxCactus调控Toll信号传导,从而调控抗菌肽的表达奠定了分子基础.%The inhibitor of NF-κB (IκB) is an important component of NF-κB signaling pathway,which can regulate the expression of target gene by interacting with the nuclear transcription factor (NF-κB).Cactus is the homologous protein of IκB in insects.In order to illustrate the function of Cactus protein in Toll signal pathways.In this study,a full-length cDNAs of Cactus from Plutella xylostella were cloned by using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE).The full-length cDNA sequence of PxCactus (GenBank No.KY828920) is 1 868 bp,which consists of a 1 053 bp open reading frame (ORF) encoding a 350 aa.The developmental stages results of quantitative real-time reverse transcription PCR (qRT-PCR) showed that PxCactus could be expressed in the whole developmental stages,with lowest expression level in the 2nd instar larvae,and with highest expression level in the 4th instar larvae.The tissue specific results of qRT-PCR indicated that PxCactus was expressed in all tissues of P.xylostella,with the highest expression level in fatbody.The microbial induction results of qRT-PCR investigated that PxCactus was up-regulated significantly after Bacillus thuringiensis or Serratia marcescens induction,and was down-regulated after Metarhizium anisopliae induction.In order to get the polyclonal antibody of Cactus protein,the prokaryotic expression plasmid pGEX-4T-PxCactus was constructed.The fusion protein was highly expressed in BL21 (DE3) with IPTG induction.The fusion protein was purified by GST tag and then immunized with rabbit.Western blot and ELISA showed that polyclonal antibody anti-PxCactus was succeeded in rabbit.In this paper,PxCactus gene was cloned and polyclonal antibody anti-PxCactus were produced in rabbit,and the Cactus protein can be detected by western blot after 24 h induction by B.thuringiensis.All the above results will lay the foundation for further studying the function of Cactus protein which regulated in the Toll signal pathway and the expression of the downstream antimicrobial peptides.