【作者】 王艳敏；

【导师】 宋水山；

【作者基本信息】 河北工业大学 ， 生物化工， 2005， 硕士

【摘要】 以马铃薯为实验材料,对病原菌萝卜E.C.C的致病能力进行验证分析。确定实验用量为50μl菌悬液。具体分析相同吸光度下每毫升病原菌和拮抗菌芽孢杆菌、苏云金芽孢杆菌的菌液中所含菌落数。结果表明,相同吸光度下不同菌种的每毫升菌液中含大致相同的菌数。然后利用马铃薯作为试验材料对现有的菌种进行筛选,得到结果是对病原菌抑制作用较好的是苏云金芽孢杆菌鲇泽变种ACCC10016,想利用农杆菌试验进一步证明所选菌种,结果没能如愿。抑病试验证明芽孢杆菌Bt4和苏云金芽孢杆菌山东亚种ACCC10314中含有水解AHLs的水解酶——AHLs水解酶。其中,Bt4和病原菌萝卜E.C.C的菌落个数比为2:15,苏云金芽孢杆菌山东亚种ACCC10314和病原菌萝卜E.C.C的菌落个数比为3:15时,就能很好的抑制病原菌对马铃薯的侵染。 分别提取芽孢杆菌的基因组DNA,和载体的质粒DNA。 根据文献发表的基因序列设计特异性引物,以芽孢杆菌和苏云金芽孢杆菌的基因组DNA为模板进行PCR扩增,得到两个大小约为800bp的PCR产物,它们分别来自于Bt4和苏云金芽孢杆菌山东亚种ACCC10314。回收PCR产物,命名为SS1和SS10。 核苷酸序列分析表明,所扩增的两个基因片段大小均为753bp,和基因库中已知的AHLs酶 N-酰基高丝氨酸内酯酶基因aiiA的同源性达96%以上,其活性部位为“¹⁰⁶HFDH～¹⁹⁹H～²²¹D”。和其他一些苏云金芽孢杆菌比较,同源性高达98%,其中的活性催化中心为His119,His188和Asp210。构建融合表达载体pGEX-SS1和pGEX-SS10,用IPTG对融合蛋白进行诱导表达,琼脂糖凝胶电泳结果表明,其大小为55KDa。然后利用亲和柱层析法纯化融合蛋白。更多还原

【Abstract】 Using potato to analysis the pathogeny. And knew that the dosage of the experiment is 50μl. Determine the number of the Bucillus sp. and Bacillus thuringiensis subsp. in the same OD, and the result is that in the same OD there are about same mubeT bacteria of that all. Then, use the potato to filter the bacterium, and we know that the Bacillus thuringienis var. aisawai Heimel ACCC10016 can control the pathogeny best. Using the Agrobacterium tumefaciens to test the Bucillus sp. and Bacillus thuringiensis subsp., but the result cannot be expressed due to all kinds of conductions. Mixed to culture the different bacteria in the same condition, then at last knew there are AHLase in the both Bucillus sp. Bt4 and Bacillus thuringiensis subsp. Shandongiensis ACCC10314. And when the ratio is 2:15 of the Bucillus sp. Bt4 and pathogeny, the pathogeny cannot cause the potato to rot. The ratio of Bacillus thuringiensis subsp. Shandongiensis ACCC10314 and the pathogeny is 3:15.Extracting the genome DNA of the Bucillus sp. and the vector isolatedly.The primers were designed based on the thesises that had be published. And PCR amplifications were performed with the genomic DNA of Bucillus sp. and Bacillus thuringiensis subsp., PCR fragments of about 800bp were obtained and named SSl and SS10, which was from Bt4 and Bacillus thuringiensis subsp. Shandongiensis ACCC10314. Reclamation the production of PCR fragment.Analysised the gene fragments, results show that the complete sequence legthe of corresponding PCR product from Bucillus sp. Bt4 and Bacillus thuringiensis subsp. Shandongiensis ACCC10314 are 753bp. Comparison of the report homologue genes showed high homologies of over 96% in the nucleotide sequence, and alive part is"106HFDH~199H~221D". Otherwise, comparison of the other Bacillus thuringiensis subsp. showed more high homologies of 98%. The center of alive catalyse is His119, His 188 and Asp210. Constrict the fusing gene pGEX-SS1 and pGEX-SS10, and inducement it with IPTG, purification, electrophoresis by agar, result showed that the size were 55kDa.更多还原