假眼小绿叶蝉中肠受体结合短肽的融合表达及其与叶蝉中肠刷状缘膜囊泡(BBMV)互作  

Fusion Expression of Peptide Sequences Bound to Midgut Receptors of Empoasca vitis and the Interaction with Brash Border Membrane Vesicle(BBMV)

作　　者：林秋秋[1] 姚祖杰[1] 吴松青[1,2] 刘昭霞[1] 朱棚丽 黄志鹏[1] 关雄[1] 张灵玲[1] 

机构地区：[1]福建农林大学教育部生物农药与化学生物学重点实验室,福州350002 [2]福建农林大学林学院,福州350002

出　　处：《农业生物技术学报》2015年第11期1494-1500,共7页Journal of Agricultural Biotechnology

基　　金：国家高技术研究发展计划(863计划)(No.2011AA10A203);教育厅高校领军人才(No.k8012012a);国家粮食局粮食公益性行业科研专项(No.201313002-3)

摘　　要：茶假眼小绿叶蝉(Empoasca vitis Gothe)是危害我国经济作物茶叶(Camellia sinensis)的优势种,其发生最广、危害最重。为寻找利用生物农药解决虫害问题,提高茶叶产量质量,同时初步探索苏云金芽胞杆菌(Bacillus thuringiensis,Bt)Crystal(Cry)毒素与叶蝉中肠的互作机制,本研究以前期噬菌体文库筛选得到的能与靶标昆虫中肠Cry潜在受体结合的短肽序列设计引物,通过PCR扩增大小为750 bp的短肽序列,与p MD-18T克隆载体连接,构建重组表达载体p T-egfp-32a并进行诱导表达,采用His-tag亲和层析技术对融合蛋白进行纯化,最后通过免疫印迹实验验证表达的短肽与叶蝉中肠刷状缘膜囊泡(brash border membrane vesicle,BBMV)的结合活性。本研究成功克隆了短肽序列,并诱导纯化目的蛋白,成功获得具有结合活性的短肽片段,为活性片段定向改造Cry domain获得对叶蝉有毒性作用的新型毒素提供了工作基础,为进一步了解毒素与受体间互作关系提供依据。Empoasca vitis（Gothe）, one of the most serious tea plant（Camellia sinensis） insects, occurs throughout the Chinese tea growing areas and causes significant losses in both production and quality of tea. It is of important significance to solve pest problems by biological pesticide and initially explore the intoxication mechanisms of Bacillus thuringiensis Cry toxins to E. vitis midgut cells. Using specific primers, peptide sequence bound to midgut receptors by screening phage library was amplified. The 750 bp PCR product was purified and cloned into the cloning vector p MD-18 T, then the recombinant plamid DNA was used for Eco RⅠ/Xho Ⅰ digested identification and gene sequencing. The targeted fragment was subcloned into expressionvector, and then the strain with recombinant plasmid of p T-egfp-32 a was induced and expressed. Moreover, the fusion protein was purified by His affinity chromatography and its binding activity to E. vitis midgut was verified by Western blot. Results indicated that the relative molecular weight of T-EGFP was about 46 k D in supernatant after purification step. After that, it also achieved good effect that Western blot had been identified its binding activity to E. vitis midgut brash border membrane vesicle（BBMV）. Successful projects for expression, purification and verification of T-EGFP bound to midgut receptors of E. vitis would provide basic data for further research on understanding the interaction mechanism between Cry toxins and E. vitis midgut cells, and also contribute to further study of directed modification for Bt Cry toxin against E. vitis.

关 键 词：茶假眼小绿叶蝉 刷状缘膜囊泡(BBMV) 克隆 表达 免疫印迹 

分 类 号：S435.711[农业科学—农业昆虫与害虫防治]