【作者】 吴峰；

【导师】 夏立秋；

【作者基本信息】 湖南师范大学 ， 微生物学， 2009， 硕士

【摘要】 苏云金芽胞杆菌(Bacillus thuringiensis,简称Bt)在芽胞形成过程中能产生对目标害虫具有特异性毒杀作用的伴胞晶体。研究表明伴胞晶体中存在与原毒素相互作用的20kb DNA片段,该片段对晶体的形成及杀虫晶体蛋白在敏感害虫体内的激活过程具有重要的作用。但是,与毒素蛋白结合的20kb DNA的序列信息仍未得到阐明。本论文旨在利用基因文库的技术手段,挖掘20kb DNA更多的序列信息,进一步对Bt野生菌株晶体中DNA的序列与来源进行研究,为探讨晶体中原毒素与20 kb DNA的结合机制以及20kb DNA的功能提供基础。在本研究中,以本室选育的Bt野生菌株4.0718为材料,提取与晶体蛋白Cry1Ac相结合的20kb DNA,以其Sau3AI部分酶切片段为供体、pbluescriptⅡ(SK+)为载体构建基因文库。基因文库建立后,通过检验文库的随机性、插入片段的平均长度、载体自连率等对文库质量进行鉴定。根据部分阳性转化子的测序结果,运用NCBI的BLASTn工具,通过网络数据库(http://www.ncbi.nlm.nih.gov/BLAST/)进行比对,根据NCBI输出的比对结果分析Bt基因组和20kb DNA之间的同源性。比对结果发现20kb DNA中存在来源于Bt染色体的基因,如16S rRNA基因、23S rRNA基因、编码RNA聚合酶亚单位的基因ropB,编码金属氨肽酶的基因pepQ编码细菌毒力因子硫酸透明质酸酶的基因sulP,以及编码与转座相关的解离酶基因tnpR。同源性分析结果证明,Bt染色体上的基因参与20kb DNA的序列构成,即20kb DNA来源于宿主菌的染色体。研究结果证实了20kb DNA来源于Bt染色体,并新发现了来自染色体的基因。本研究为进一步阐明Bt伴胞晶体中20kb DNA分子的来源、结构、功能及其与原毒素的相互作用机制奠定了重要基础。更多还原

【Abstract】 Bacillus thuringiensis has been widely used for insect control because of the specific larvicidal activity of parasporal crystals produced during sporulation.It was proved that the parasporal crystals contained 20kb DNA fragments intimately associated with protoxins,which play a important role in the assembling and stability of parasporal crystals as well as the activation of crystal toxin in the midgut of target larvae.However,the whole sequence of 20kb DNA was unclear to date.The present investigation constructed 20kb DNA gene library to discover more sequence information and further determined the origin of DNA.The data will be helpful for further understanding the mechanism of interaction between Bt protoxin and DNA as well as the role of the DNA in the structure and fuction of Bt crystal protein.In this research,we took Bt strain 4.0718 as material and isolated 20kb DNA from its parasporal crystals.We constructed gene library using DNA fragments of Sau3AI partial digestion and pbluescriptⅡ(SK+),vector.The number of recombinants which were screened out was 245,yielding a genomic coverage of five and concluding 99%sequence of DNA which achieved the requested theoretical value to construct gene library.Then, we checked randomicity,average length of insert fragments and empty vector rate of gene library.By NCBI Blast,the.sequencing results proved that the DNA came from genome of Bt.Moreover, we found a series of genes located in 20kb DNA for the frist time.This research laid a basis for future understanding the mechanism.of interaction between Bt protoxin and DNA as well as the the origin,structure and fuction of DNA.更多还原