硕士论文--杀虫蛋白基因cry8fa2的克隆及在bt无晶体突变株中的表达.doc

此篇是本人的硕士毕业论文《杀虫蛋白基因cry8Fa2的克隆及在Bt无晶体突变株中的表达
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摘 要
苏云金芽孢杆菌（Bacillus thuringiensis，简称Bt）是目前研究最深入的杀虫微生物，其杀虫蛋白基因也是应用最广泛和最有发展潜力的抗虫基因，因此，深入发掘Bt新菌株、分离克隆新型的杀虫基因对害虫的防治具有重要意义。近年来，鞘翅目害虫尤其是金龟甲科害虫的危害日趋严重，由于其幼虫（俗称蛴螬）生活在土壤中，给防治带来很大的困难，本研究从本实验室保存的菌株中筛选高毒力野生菌株，并从中分离克隆对金龟甲科害虫有效的新型杀虫蛋白基因，研究该基因的表达和杀虫活性，为构建高效广谱的Bt工程菌和培育转基因抗虫植物提供新型基因资源。本研究的主要内容和结果如下：
以本实验室分离的Bt菌株B-DLL质粒DNA为模板，利用特异性引物JJX5和JJX3扩增出3.5 kb大小的片段，将该片段插入克隆载体pMD 18-T中，筛选获得一个含新基因的克隆pMD18-cry8new。序列测定表明，该基因编码区为3525 bps，编码的蛋白质由1174个氨基酸残基组成，理论分子量为133.05 kD，等电点为pH4.69，为弱酸性蛋白。该基因核苷酸序列已在GenBank登录（Accession number：HQ174208），编码的氨基酸序列与Cry8Fa1的同源性最高达99.8%，被国际Bt δ-内毒素命名委员会正式命名为Cry8Fa2。将cry8Fa2基因插入Bt表达载体pSXY422b中，电击导入Bt无晶体突变株HD-73－中，获得重组Bt工程菌HD73-Cry8Fa2，该工程菌能形成方形的伴孢晶体，并表达130 kD的蛋白。生物测定结果表明，该基因表达产物对暗黑鳃金龟和华北大黑鳃金龟均不具有杀虫活性。
关键词：Bt；cry8Fa2基因；基因克隆；蛋白表达

Abstract
Bacillus thuringiensis（Bt）is a Gram-positive, soil-dwelling bacterium. As an important insect microbial pathogen, its insecticidal crystal proteins are the most widely and potentially used against insect pests in agriculture and forestry. Therefore, selection of new Bt strains and cloning of novel insecticidal protein genes for control of insect pests are of great significance. In recent years, the damage of coleopteran pest Scarabaeoid beetle is serious increasingly especially, because it is very difficult to control with larvae living in the soil. This study mainly includes screening of Bt strains, identification of new cry gene-type and cloning, expression and insecticidal activity of novel insecticidal protein gene that is toxic to Scarabaeoid beetle in order to provide new genetic resources for constructing genetically-engineered Bt strain and cultivatingtransgenic crops. The main contents of this study are as follows:
The 3.5 kb fragments was amplified by PCR using a pair of Bt cry8-type genes special primers, JJX5 and JJX3, and inserted into vector pMD 18-T, the new recombinant plasmid, pMD18-cry8new, was isolated and obtained. Nucleic acid sequence analysis showed that this gene was 3525 base pairs encoding 1174 amino acids, which were homolog of 99.8% compared with Cry8Fal, the molecular weight of the protein was 133.05 kD with isoelectric point pH 4.69. This gene sequence had been registered in GenBank (accession number was HQ174208), and named Cry8Fa2 as a novel gene by Bacillus thuringiensis Delta Endotoxin Nomenclature Committee. The cry8Fa2 gene could be formed cubelike crystal and expressed as a 130 kD protein in Bt acrystalliferous mutant strain HD-73－. Bioassay result showed the expression product of cry8Fa2 gene was not toxic to the larvae of Holotrichia parallela and H. oblita.
Key words：Bacillus thuringiensis; cry8Fa2 gene; Cloning; Protein expression