小菜蛾中肠Polycalin蛋白的基因克隆、表达及与Cry1Ac毒素的结合特性分析

摘要

[目的]Polycalin蛋白是一种新发现的Bt毒素受体,可能与昆虫对Bt的抗性有关.本研究旨在明确小菜蛾Plutella xylosteUa Polycalin蛋白与Bt Cry1 Ac毒素的关系.[方法]本研究通过PCR结合RACE技术从小菜蛾3龄幼虫中肠克隆获得Polycalin蛋白基因的全长cDNA序列,采用实时定量PCR技术研究Polycalin蛋白在小菜蛾不同发育阶段、4龄幼虫不同组织及用不同浓度Cry1 Ac毒素饲喂3龄幼虫后的表达量.提取小菜蛾幼虫中肠的刷状缘膜囊泡(BBMV),运用合成多肽的方法制备Polycalin蛋白抗体,利用Western blot和Ligand blot技术对小菜蛾Polycalin蛋白进行鉴定,分析其与Cry1Ac毒素的结合特性.[结果]克隆得到的小菜蛾Polycalin蛋白基因的cDNA序列全长为9 102 bp(GenBank登录号:MF138149),其中,开放阅读框为8 778 bp,编码2 925个氨基酸;预测蛋白质分子量为326.38 kD,等电点为4.39;在推导的氨基酸序列中,前20个氨基酸为N-末端信号肽序列,含有14个N-糖基化位点,67个O-糖基化位点,6个脂质运载蛋白特征序列,6个脂质运载蛋白位点和13个类脂质运载蛋白结构域,并且在第20和21位(TSG-QVV)氨基酸之间存在一个裂解位点,在C-末端存在2个GPI结合位点,具有Bt毒素受体的特征.该蛋白在幼虫期的表达量较高,尤其在3龄幼虫体内表达量最高,在蛹和成虫中的表达量最低;在4龄幼虫的头、胸、腹中均有表达,在幼虫腹部中的表达量最高;3龄幼虫取食不同浓度的Cry1 Ac毒素后,Polycalin蛋白的表达量下降,且Cry1 Ac毒素浓度越高,下降越明显.通过Western blot检测到小菜蛾中肠刷状缘膜囊泡(BBMV)上存在大约300 kD的Polycalin蛋白条带;Ligand blot实验证明了小菜蛾Polycalin蛋白能与Cry1 Ac毒素结合.[结论]本研究首次初步明确了小菜蛾Polycalin蛋白具有与Bt毒素结合的特性,为研究Bt对昆虫的作用机理和利用Bt防治害虫提供一定的理论基础和参考价值.%[Aim] As a newly found receptor of Bt toxin,polycalin is considered to be related to the resistance of insects to Bt toxin.This study aims to clarify the relationship between polycalin and the Bt Cry1Ac toxin in Plutella xylostella.[Methods] The full-length cDNA sequence of polycalin gene was cloned from the midgut of the 3rd instar larvae of P.xylostella by PCR and RACE techniques.The expression levels of the polycalin gene in different developmental stages and different tissues of the 4th instar larvae were determind by quantitative real-time PCR.The expression levels of the polycalin gene in the 3rd instar larvae fed with different concentrations of Cry1Ac toxin were also compared.The brush border membrane vesicles (BBMV) proteins in larval midgut were separated,and monoclonal antibodies were prepared by peptide synthesis technique.The polycalin and its binding characteristics to CrylAc toxin were identified by using Western blot and Ligand blot.[Results] The full-length cDNA (GenBank accession no.:MF138149) of polycalin gene obtained from P.xylostella is 9 102 bp in length,with the open reading frame of 8 778 bp,encoding 2 925 amino acids.The predicted molecular weight and isoelectric point of the encoded protein are 326.38 kD and 4.39,respectively.The putative protein sequence contains an N-terminal signal peptide of 20 amino acid residues,14 N-linked and 67 O-linked glycosylation sites,with six lipocalin signatures,six lipocalin hits and 13 lipocalin like structures.There are a glycoprotein cleavage-activation site between the 20th and 21th (TSG-QVV) amino acid residues,and a GPI anchor signal peptide with two amino acid residues at the C-terminus.The polycalin has the feature of Bt toxin receptor.Developmental expression profiles revealed that the polycalin gene had higher expression level in larva than in pupa and adult,with the highest expression level in the 3rd instar larva.Tissue expression profiles revealed that the polycalin gene was expressed in the head,thorax and abdomen of the 4th instar larvae,with the highest expression level in the abdomen.The expression of the polycalin gene in the 3rd instar larvae fed with different concentrations of Cryl Ac toxin was dramatically decreased as compared with that of the control (fed with PBS).The higher the concentration of Cry1 Ac toxin,the lower the expression level of the polycalin gene.The polycalin in the brush border membrane vesicles (BBMV) of the midgut of P.xylostella was approximately 300 kD by Western blot analysis.The Ligand blot result confirmed that this polycalin protein could bind to Cry1 Ac toxin.[Conclusion] This study verified that the polycalin from P.xylostella could bind to Bt toxin,and provides useful information for further study on the action mechanisms of Bt against insects and pest control by utilization of Bt.