【作者】 赵新民；

【导师】 夏立秋；

【作者基本信息】 湖南师范大学 ， 微生物学， 2009， 博士

【摘要】 苏云金芽胞杆菌(Bacillus thuringiensis,简称Bt)是世界上应用范围最广的杀虫微生物,其杀虫活性主要来源于芽胞形成过程中产生的杀虫晶体蛋白,研究杀虫晶体蛋白的结构与功能的关系已成为当前的一个热点。Cry1Ac蛋白是目前所知的对鳞翅目昆虫毒性最高和研究最多的Bt毒素之一。研究表明伴胞晶体中存在与原毒素相互作用的20-kb DNA片段,同时在活化毒素蛋白中仍然存在。该片段对晶体的形成及晶体蛋白在敏感昆虫体内的激活过程可能具有重要的作用,有关晶体蛋白与20-kbDNA相互作用的机制目前还不清楚。本文运用生物信息学理论和分子生物学实验技术手段研究了Cry1Ac5蛋白理论三维结构,分离和纯化了.Cry1Ac5和与之结合的20-kb DNA,研究了Cry1Ac5蛋白与20-kb DNA相互作用的分子机制,探讨了20-kb DNA对Cry1Ac5蛋白的稳定性和杀虫毒力的影响。主要创新的研究内容如下:构建了Cry1Ac5蛋白理论三维结构,并对模型进行了验证。通过与已经解析结构的毒素比较,探讨各结构域的生物学功能。Cry1Ac5蛋白由3个不同的结构域构成,结构域Ⅰ位于肽链的N端,为一组由7个两亲的α螺旋围绕着一个疏水的α螺旋形成的α螺旋束,主要参与细胞膜的穿孔。结构域Ⅱ位于肽链的中间,为反平行的β折叠片层,其顶端的突环参与毒素与受体蛋白的结合。位于C端的结构域Ⅲ是由两组反平行的β折叠片层组成的夹心结构,以β果酱卷(jelly roll)拓扑结构排列。其功能是防止蛋白酶对毒素分子的过度降解。从本实验室筛选的高毒力的Bt4.0718菌株中分离克隆了cry1Ac5全长基因,利用穿梭载体pHT315构建了表达载体pHTAc35,电转入无晶体突变株cry~-B中,高效表达了130 kDa的Cry1Ac5蛋白,采取凝胶层析和阴离子交换层析分离纯化了Cry1Ac5蛋白和20-kb DNA,同时为研究Cry1Ac5蛋白与20-kb DNA的相互作用提供了条件。利用荧光光谱法研究了Cry1Ac5蛋白与20-kb DNA相互作用荧光淬灭光谱。结果表明:DNA与蛋白存在特异结合,Cry1Ac5与20-kbDNA相互作用不仅与DNA序列有一定关系,而且还与晶体蛋白本身结构有关系。结合常数K=2.5×10~9,位点数n=1.72。这种结合具有很强的稳定性。对Cry1Ac5与20-kb DNA分子中一段20-bp DNA核苷酸结合的分子模拟研究表明Cry1Ac5蛋白有多个位点与DNA结合。位点Ser283-Ala284-Gln285、位点Ser440-Ser441-Ser442和位点Ser555-Leu556-Asn557深入位点DNA螺旋的沟中,主要通过疏水作用识别DNA的特定序列,与碱基直接接触。位点Ser555-Leu556-Asn557因为含有非极性的Leu556,相对而言具有更强的疏水作用。结构域Ⅰα6螺旋上位点Arg209和Asn212与B链磷酸骨架发生作用形成氢键,结构域Ⅱ上的Gly339也和与B链磷酸骨架发生作用形成氢键。Cry1Ac5和20-kb DNA相互作用力包括疏水作用力、范德华力。静电作用力,其中主要作用力为疏水作用力。研究表明20-kb DNA存在于晶体蛋白中,而不是吸附在晶体表面,它与晶体蛋白的核心片段结合。稳定性实验表明活化Cry1Ac5蛋白与20-kb DNA的体外结合能够增强Cry1Ac5蛋白对胰蛋白酶处理的稳定性和在紫外光下的稳定性。室内生测表明,体外活化Cry1Ac5蛋白和20-kb DNA结合没有明显地改变蛋白对棉铃虫的毒力。说明棉铃虫中肠道可以对含20-kb DNA的Cry1Ac5蛋白产生合理的降解。研究结果不仅有助于了解Bt晶体蛋白的结构与生物学功能,同时在分子水平上对20-kb DNA与晶体蛋白的相互作用机制以及20-kbDNA对晶体蛋白稳定性和杀虫毒力的影响有了新的认识。此外研究结果还对提高晶体蛋白田间应用的稳定性具有重要的实践指导意义。更多还原

【Abstract】 Bacillus thuringiensis(Bt) is the most widely used insecticidal microorganism that produces one or more insecticidal crystal(Cry) proteins deposited in the form of an parasporal crystal during sporulation, Cry1Ac,which is one of the deeply researched Cry proteins with the strongest insecticidal activities against lepidoptera insects,was extensively used in pest control of agriculture.But the structure-function relationship of Cry1Ac protein has not been well understood and need further investigated.It was reported that the parasporal crystals contained 20-kb heterofogous DNA fragments intimately associated with protoxin, which may play a key 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 nature of the interaction of the Bt protoxin with DNA,the sequences and origin of 20kbDN remained to be investigated.The theoretical model of the three-dimensional structure of Cry1Ac was predicted by homology modeling on the structures of the Cry1Aa, and its differences from Cry1Aa,Cry2Aa,Cry3Aa and Cry4Aa structures were analyzed.It is composed of three structurally conserved domains. DomainⅠconsists of seven alpha helices in which helix 5 is surrounded by the others,forming a helical bundle.DomainⅡconsists of three antiparallel b-sheets joined in a Greek key topology,arranged in a b-prism,DomainⅢis formed by two antiparallel b-sheets forming aβ-sandwich in a jelly roll topology.The full-length cry1Ac5 gene was isolated and cloned from Bt 4.0718 strain,and the expression plasmid pHTAc35 was generated by cloning cry1Ac5 gene to shuttle vector pHT315.Acrystalliferous Bacillus thuringiensis cry~-B was transformed with pHTAc35 and well expressed a 130-kDa Cry1Ac5 protoxin,which offered the precondition for our latter research.20-kbDNA and the Cry1Ac5 were purified,and their interaction was analyzed using and fluorescence titration.The binding regions were in all the three domainsThe fluorescence titration curves of the calf thymus DNA and 20-kbDNA additions to the DNA-free Cry1A toxins were different.The binding of 20-kb was almost infinitely tight that was quite different from that of calf thymus DNA.Besides the structural features common to all DNA molecules,the toxins may recognizes specific nucleotide sequences feature present in the various 20-kb DNA fragments.It was confirmed that the mechanism of the fluorescence quenching of Cry1Ac5 induced by 20-kbDNA was static qunechnig.The binding constant K was 2.5×10~9,and the binding sites was 1.72.Molecular simulation using Cry1Ac5 and a piece of 20-bp DNA from 20-kb DNA showed that there were several binding sites on the three domains of Cry1Ac5.The binding site Ser283-Ala284-Gln285, Ser440-Ser441-Ser442 and Ser555-Leu556-Asn557 were deep in the grooves of the DNA to form the hydrophobic contacts.There were hydrogen bond interactions between the phosphorate backbone of DNA and three amino acids(Arg209,Asn212 and Gly339) of Cry1Ac5.The addition of 20-kb DNA to the activated Cry1Ac 5 solution in vitro reduced the degradation of the toxins under trypsin and ultraviolet irradiation that indicated the original existence of 20-kb DNA in toxins had the role of protection.In this paper,the mechanism of the interaction 20-kb DNA and Cry1Ac5 was reported for the first time,which provided new biological evidences for the interaction playing important roles in maintaining the stability of the toxin.更多还原