棉铃虫中肠蛋白氨肽酶N1的克

发布时间：2018-04-12 06:59

本文选题：苏云金芽胞杆菌 + Cry1Ah蛋白　；参考：《东北农业大学》2014年硕士论文

【摘要】：苏云金芽孢杆菌(Bacillus thuringiensis,简称Bt)由于其对靶标害虫具有杀虫特异性,而对人畜无害,不污染环境,因而被作为微生物杀虫剂应用已有60年的历史,并且cry基因也已经应用于转基因作物,同时苏云金芽胞杆菌也成为目前全球应用最广泛的微生物杀虫剂。Cry毒素对靶标害虫所具有的杀虫特异性取决于毒素与昆虫中肠上皮细胞特异性受体之间的相互作用,因此对于昆虫中肠受体结构与功能的研究不仅可以为毒素与受体间的相互作用提供材料,同时也为研究Cry毒素的作用机制奠定基础。氨肽酶N(animopeptidase N,简称APN)是公认的Cry蛋白的受体之一,也是研究得最成熟的受体,属于肽链端解酶,在鳞翅目昆虫中可以水解蛋白质或多肽N末端的氨基酸。 crylAhl基因是由本实验室从天然菌株BT8中分离并克隆的,并且是具有自主知识产权的新型杀虫晶体蛋白基因,表达133kDa的Cry1Ah蛋白,并对棉铃虫(Helicoverpa armigera)、亚洲玉米螟(Ostrinia furnacalis)和水稻二化螟(Chilo suppressalis)等多种鳞翅目害虫表现出高于商业化crylAc基因的杀虫活性。同源性比较结果表明：crylAh全长基因与cry1Ac全长基因所编码的氨基酸序列相似性为86%,并且两者表现出相似的生物活性,即对棉铃虫高毒力,对家蚕安全低毒。本实验室周子珊博士利用配体垂钓平台,发现Cry1Ah和CrylAc毒素在棉铃虫的BBMV上虽然存在着不同的结合蛋白,但都有一条特异性较强的结合蛋白条带,而在家蚕的BBMV上并没有检测到这种高亲和力的蛋白,经过质谱鉴定,结果表明这种蛋白是棉铃虫中肠蛋白APN1,因此推测这种结合蛋白可能是CrylAh和Cry1Ac蛋白对棉铃虫产生杀虫特异性与高毒力的原因。本研究通过设计全长引物克隆得到棉铃虫中肠受体apnl基因,并利用原核系统进行表达,同时在体外通过Western blotting杂交实验,验证了APN1可以与CrylAh和CrylAc毒素结合。生物信息学分析表明：本研究克隆并表达的APN1含有鳞翅目昆虫中肠氨肽酶的结构特点,即具有高度保守的GAMEN区域和HEXXH锌指结构,经预测其氨基酸序列中存在潜在的N-连接和O-连接的糖基化位点。利用Western blotting检测APN1是否可以与毒素结合时,我们发现100kDa和70kDa左右的APN1短片段也可以与Cry1Ah和Cry1Ac蛋白结合,经质谱鉴定发现它们都含有APN1,因此我们认为这些短片段是APN1全长蛋白降解的结果。为了进一步确定棉铃虫中肠受体APN1与Cry1Ah蛋白的结合区,根据APN1的结构特点将其分成四段进行分段克隆、表达及与CrylAh蛋白的体外结合实验。结果发现：片段H1、H2和H3在大肠杆菌中成功表达蛋白。结合实验发现只有片段H3与Cry1Ah蛋白有比较明显的结合。本研究所构建的原核表达体系可以为其它昆虫中肠受体的克隆与表达奠定基础,同时也为Cry毒素与受体互作机制的研究提供思路。体外结合实验和Western杂交可以作为体外验证昆虫中肠受体与毒素结合能力的一种可行性方案,初步确定受体与毒素之间相互作用的关系。后续研究是对片段H3蛋白进行纯化并与Cry1Ah毒素结合,最终确定棉铃虫APN1与CrylAh蛋白的结合区。
[Abstract]:Bacillus thuringiensis (Bacillus thuringiensis, referred to as Bt) because of its insecticidal specificity to the target pest, and is harmless to human beings and animals, do not pollute the environment, which is used as a microbial insecticide has a history of 60 years, and the cry gene has also been applied to transgenic crops, while Bacillus thuringiensis has become each other effect on specific microbial pesticide insecticidal.Cry toxin is currently the world's most widely used of target pests in between the toxin and the insect midgut epithelial cell specific receptor, therefore research on the structure and function of insect midgut receptors not only can provide materials for interaction between toxin and receptor, but also laid the foundation for the mechanism of action study of Cry toxin. Aminopeptidase N (animopeptidase N, referred to as APN) is one of the most recognized Cry receptor protein, is studied by the most mature peptide, which belongs to Chain end hydrolysate, an amino acid that can hydrolyze the N terminal of protein or polypeptide in Lepidoptera.
The crylAhl gene is from the laboratory from natural strain BT8 isolated and cloned, and novel insecticidal crystal protein gene with independent intellectual property rights, the expression of 133kDa Cry1Ah protein, and the cotton bollworm (Helicoverpa armigera), the Asian corn borer (Ostrinia furnacalis) and Chilo suppressalis (Chilo suppressalis) and other Lepidoptera pests show higher than the commercial insecticidal activity of crylAc gene. The homology comparison showed that the amino acid sequence of the full-length crylAh gene and cry1Ac gene encoding the similarity of 86%, and both showed similar biological activity of cotton bollworm with high toxicity to silkworm, safety and low toxicity.
The laboratory of Dr. Zhou Zishan using ligand fishing platform, found that Cry1Ah and CrylAc toxin in cotton bollworm BBMV binding protein, although there are different, but have a strong specific binding protein bands in the silkworm BBMV did not detect this high affinity protein identification by mass spectrometry. Results show that this protein is the midgut protein APN1, suggesting that this may be the CrylAh binding protein and Cry1Ac protein have insecticidal specificity and high virulence on the cotton bollworm.
This study obtained the midgut receptor apnl gene by cloning full-length primer design, and expressed by prokaryotic system, at the same time, through the experiment of Western blotting hybridization in vitro, proved that APN1 could combine with CrylAh and CrylAc toxin. Bioinformatics analysis showed that: the structure of the research on cloning and expression of APN1 containing the lepidopteran midgut the aminopeptidase, which is the GAMEN and HEXXH regions of highly conserved zinc fingers, the predicted the existence of potential glycosylation sites N- and O- linked its amino acid sequence.
Whether the use of Western blotting APN1 could be detected with toxin binding, we found that APN1 and 100kDa short fragments of about 70kDa can be combined with Cry1Ah and Cry1Ac proteins were identified by mass spectrometry revealed that they all contain APN1, so we think that these short fragments are APN1 full-length protein degradation results.
In order to further determine the binding region of the midgut receptors APN1 and Cry1Ah protein, according to the structure characteristics of APN1 will be divided into four sections section cloning, expression and in vitro binding experiments with CrylAh protein. The results showed that the fragment of H1, H2 and H3 successfully expressed protein in Escherichia coli. Combined with the experiment found that only fragments of H3 and Cry1Ah a combination of protein obviously.
The prokaryotic expression system constructed in this research for cloning and expression of other insect midgut receptors to lay the foundation, but also provide ideas for the research on the interaction mechanism of Cry toxin receptor. A feasible scheme of in vitro binding assay and Western hybridization can be used as in vitro validation of insect midgut receptors and toxin binding ability, preliminary to determine the relationship between each other interactions between the receptor and the toxin. Further research is the fragment of H3 protein was purified and combined with Cry1Ah toxin, and ultimately determine the binding region of cotton bollworm APN1 and CrylAh protein.

【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：S435.622

【参考文献】