Bt杀虫基因cry7、cry8的鉴定及表达

发布时间：2019-04-16 11:48

【摘要】：苏云金芽胞杆菌,为芽胞杆菌属,是在世界范围内广泛分布的一种革兰氏阳性菌,作为一种环保型的杀虫剂被广泛用于害虫的防治,其产生的蛋白可以对多种害虫有高效的杀虫活性。苏云金芽胞杆菌被认为是化学农药的一个有效的替代品。随着转基因技术的发展,苏云金芽胞杆菌也成为转基因作物抗虫基因的主要来源。因此,寻找新的杀虫活性基因对害虫的防治具有重要意义。由于鞘翅目叶甲科害虫的危害日益严重,因此筛选出对其有高活性的菌株和基因具有重要意义。本研究的主要内容如下:(1)本研究从海南地区的土壤中分离得到423株苏云金芽胞杆菌,利用聚合酶链式反应(PCR)和聚丙烯酰胺凝胶电泳(SDS-PAGE)方法鉴定其菌株的基因型和杀虫晶体蛋白的成分。经PCR扩增结果显示其中23株菌中含有cry7Ab基因,片段大小为3417 bp。对含有cry7Ab基因的BJH705(GenBank登录号为KX010669)菌株进行了进一步的蛋白表达及生物活性测定。SDS-PAGE结果显示该菌株可以表达分子量约为130 k Da的杀虫晶体蛋白。对大猿叶甲进行生物活性测定,复筛结果显示,该蛋白对大猿叶甲有较高的杀虫活性其LC50为53.386μg/m L。(2)对实验室保存的菌株3-1-a进行基因型鉴定,发现其中含有cry8基因,进一步用全长引物进行扩增,测序结果显示该菌株同时含有cry8Ax和cry8Ea基因。与p EB表达载体连接,并在大肠杆菌BL21中表达,SDS-PAGE结果显示,cry8Ax和cry8Ea基因可以在大肠杆菌中分别表达分子量为80 kDa、130 kDa的杀虫蛋白。序列已提交GenBank注册,登录号分别为KU986725,KU955850。以大猿叶甲二龄幼虫为供试昆虫对两种蛋白进行生物活性初步测定,校正死亡率分别为56.89%,77.87%,利用80 k Da的蛋白进行复筛,其LC50值为83.329μg/m L。本研究针对我国海南地区和实验室保存菌株开展了菌株筛选与杀虫基因克隆的研究,获得有效新菌株BJH705和3-1-a,克隆有效杀虫基因3个,研究结果对对大猿叶甲有杀虫活性的新基因克隆及鞘翅目害虫的可持续性防治具有重要意义。
[Abstract]:Bacillus thuringiensis, a genus of Bacillus thuringiensis, is a Gram-positive bacterium widely distributed worldwide. As an environmentally friendly insecticide, Bacillus thuringiensis is widely used in pest control. The protein produced by the protein can be highly insecticidal to a variety of pests. Bacillus thuringiensis is considered to be an effective substitute for chemical pesticides. With the development of transgenic technology, Bacillus thuringiensis has become the main source of insect resistance genes in transgenic crops. Therefore, it is of great significance to search for new insecticidal genes for pest control. It is of great significance to screen out the strains and genes which have high activity because of the serious damage caused by the insect pests of the family Elaeagnus. The main contents of this study are as follows: (1) 423 strains of Bacillus thuringiensis were isolated from the soil of Hainan area. Polymerase chain reaction (PCR) and polyacrylamide gel electrophoresis (SDS-PAGE) were used to identify the genotype and the composition of insecticidal crystal protein. The results of PCR amplification showed that 23 of them contained cry7Ab gene, and the size of the fragment was 3417 bp.. The protein expression and biological activity of the strain BJH705 (GenBank accession number KX010669) containing cry7Ab gene were further studied. The results of SDS-PAGE showed that the strain could express the insecticidal crystal protein with molecular weight of 130k Da. The results of double screening showed that the protein had high insecticidal activity (LC50 was 53.386ug / m L. (2), and the genotypic analysis of the preserved strain was carried out in the laboratory, and the results showed that the protein had a high insecticidal activity to P. sibirica, and the bioactivity of the protein was 53,866 渭 g / m L. (2. The cry8 gene was found and amplified by full-length primers. Sequencing results showed that the strain contained both cry8Ax and cry8Ea genes. It was ligated with p-EB expression vector and expressed in E. coli BL21. The results of SDS-PAGE showed that cry8Ax and cry8Ea genes could express 80 kDa,130 kDa insecticidal protein in E. coli respectively. Sequence submitted for GenBank registration with login numbers of KU986725,KU955850. The biological activity of the two proteins was determined with the second instar larva of the great apes as the test insect. The corrected mortality rates were 56.89% and 77.87%, respectively. The LC _ (50) value of the two proteins was 83.329 渭 g / mL using the protein of 80 k Da for rescreening. In this study, strain screening and cloning of insecticidal genes were carried out for the preserved strains in Hainan province and laboratory in China. The effective new strains BJH705 and three effective insecticidal genes were obtained, and three effective insecticidal genes were cloned. The results of this study are of great significance for the cloning of new genes with insecticidal activity and the sustainable control of coleoptera pests.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S476

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