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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 is the dominant species that threatens Camellia sinensis, the most widely occurring and hardest crop in China. In order to find out the solution of pest problems by using biological pesticides and improve the yield and quality of tea, we also explored the interaction mechanism between Bacillus thuringiensis (Ct) Crystal (Cry) toxin and leafhopper midgut. In this study, Primers were designed for the short peptide sequence that binds to the potential Cry in the target insect midgut. A 750 bp short peptide sequence was amplified by PCR and ligated with the pMD-18T cloning vector to construct the recombinant expression vector pT-egfp-32a The fusion protein was purified by His-tag affinity chromatography. Finally, the expression of the short peptide was verified by immunoblotting in the midgut of brach border membrane vesicle (BBMV) Binding activity. In this study, we successfully cloned the short peptide sequence and induced the purification of the target protein. We successfully obtained the short peptide fragment with binding activity, and provided a basis for the directional transformation of Cry domain for the active fragment to obtain a novel toxin that has toxic effects on the leafhopper. In order to further understand Toxins and receptors to provide the basis for the interaction between.