【作者】 王发祥；

【导师】 夏立秋；

【作者基本信息】 湖南师范大学 ， 微生物学， 2008， 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蛋白分子之一,在农作物和森林害虫防治、蚊虫控制及水生态系统中害虫防治都具有广泛的应用,但其结构与功能的关系尚未完全明确,需要运用定点突变技术进一步阐释。本论文运用分子生物学理论和生物信息学技术,发现了Cry1Ac5蛋白结构域Ⅲ中独特的β18-β19 loop,通过定点突变技术研究了Cry1Ac5蛋白结构与功能的关系,同时还获得了杀虫活性或稳定性提高的突变子。主要创新的研究内容如下:从高毒力的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,为研究Cry1Ac5蛋白结构与功能提供了前提条件。利用Swiss-Model服务器和Swiss-pdb Viewer软件建立了Cry1Ac5蛋白的三维结构模型,并利用Clustal W和DS ViewerPro软件对比分析了其与其它Cry蛋白之间的差异,发现Cry1Ac5的结构域Ⅲ与其它Cry1蛋白的亲缘关系较远,其结构域Ⅲ上的β18-β19 loop比Cry1Aa、Cry2Aa、Cry3Aa和Cry4Aa蛋白对应的loop长,其组成的残基中色氨酸（W544）、连续的丝氨酸（S547、S548）和loop顶点的天冬酰氨（N546）在Cry蛋白中都是独特的残基分子成分。对β18-β19 loop上的7个残基进行丙氨酸扫描突变,所有突变子（N543A、W544A、G545A、N546A、S547A、S548A、I549A）都能产生菱形晶体和表达130 kDa的蛋白。室内生测表明,突变子N546A对棉铃虫和甜菜夜蛾的致死率有明显提高,而W544A的致死率则显著下降;对Cry1Ac5蛋白分子的三维结构分析表明,N546、S548的侧链伸向蛋白分子表面,可能参与蛋白与其受体分子的相互作用,而W544、G545、S547和I549的侧链伸向蛋白分子内侧,可能与维持蛋白的稳定性有关。对N546进一步突变,获得了突变子N546G、N546D、N546K和删除突变N546Δ及3个双突变和1个三突变,利用等电点沉淀法和AKTApurifier 100系统纯化了毒素蛋白,生测发现N546A(LC₅₀为1.67μg/mL)对棉铃虫毒力比野生型Cry1Ac(LC₅₀为2.98μg/mL)提高了1.78倍,而N546D、N546K和N546Δ的毒力则降低了80～90%。对野生和突变蛋白进行寡聚体形成能力和胰蛋白酶敏感性分析,发现N546不参与寡聚体的形成和维持蛋白的稳定。利用生物素标记蛋白法进行毒素蛋白与昆虫中肠BBMV的竞争和非竞争结合分析,发现N546A的初始结合能力比野生型增强了2倍;N546D初始结合能力与野生型相当,很容易被野生型Cry1Ac竞争;其它N546突变子与BBMV的初始结合能力则明显下降。因此,N546A突变蛋白毒力的强弱与其同昆虫BBMV的结合力强弱一致。进一步对W544突变获得保守的W544F、W544Y和不保守W544C突变子,生测和稳定性分析发现544位残基分子结构有无芳香环不是蛋白功能必须的,但亲水的侧链不利于蛋白的稳定;SDS-PAGE分析发现突变子W544F的原毒素明显的增加了蛋白对紫外线、胰蛋白酶和存贮的稳定性;经原子力显微镜观察,W544F产生的晶体两个顶点间的垂直距离比野生型Cry1Ac长0.6μm;W544F与野生Cry1Ac的杀虫活性相似,但经过紫外照射9 h后,其保留的杀虫活性比野生型高4倍以上,这一结果对解决Cry1Ac毒素蛋白田间应用的问题,具有重要的应用价值。本论文首次发现了Cry1Ac5蛋白结构域Ⅲ上的β18-β19 loop结构的特殊性,并第一次报道了结构域Ⅲ中的loop结构在蛋白功能中发挥的作用,为Cry1Ac的结构域Ⅲ在受体分子的识别和结合、维持蛋白的结构稳定等方面的重要性提供了新的生物学证据。更多还原

【Abstract】 Bacillus thuringiensis（Bt）is the most used insecticidal microorganism that produces one or more insecticidal crystal（Cry）proteins deposited in the form of an intracellular parasporal crystal during sporulation,and it is a current hotpot to study the structure-function relationship of the Cry proteins. Cry1Ac protein,which is one of the widely researched Cry proteins with the strongest insecticidal activities against lepidoptera insects,was extensively used in pest control of agriculture,forestry and water ecology.But the structure-function relationship of Cry1Ac protein has not been well understood and need further discussed and explained with site-directed mutagenesis.In this paper,a uniqueβ18-β19 loop in the domainⅢof Cry1Ac5 was reported,and the Structural Significance of this loop in insecticidal activity of Cry1Ac5 was studied by site-directed mutagenesis.The major contents and results are as follows: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.A homology-based Cry1Ac model of Cry1Ac5 was constructed using SWISS-MODEL and Swiss-Pdb Viewer program,and its differences from Cry1Aa,Cry2Aa,Cry3Aa and Cry4Aa structures were analyzed.The Cry1Ac5 domainⅢhas a distant relationship with other Cry1 proteins and its longβ18-β19 loop possesses a unique tryptophan（W）at residue 544 and a unique asparagine（N）at the loop apex（residue 546）,as well as two consecutive serines（S）from residue 547 to 548.Alanine scanning mutations within the loop were initially generated and all mutants could produce bipyramidal crystals and express 130 kDa protoxins. When tested in toxicity,mutant N546A showed a slight enhanced mortality to Spodoptera exigua and Heliothis armigera,and mutant W544A exhibited a remarkable reduction in mortality to the two insects.After substantial analysis of Cry1Ac5 tertiary-structure,it can be found that both the residues N546 and S548,as well as the N543,are oriented towards the solvent,in the surface of the protein,probably indicating a putative role in interaction to other macromolecules.In contrast,side chain of residues W544,G545,S547 and I549 are in close contact to residues located in the large loop linkingβ21 andβ22,playing a conceivable structural role in local stability.Four further single mutants ofN546（N546G,N546D,N546K,N546Δ）, three double mutants and a triple mutant were constructed,and toxins were purified by isoelectric precipitation and an AKTA purifier 100 system.When bioassayed against H.armigera using purified toxins,N546A(LC₅₀1.67μg/mL)showed 1.78 times increased toxicity than wide-type Cry1Ac(LC₅₀ 2.98μg/mL),and mutant N546D,N546K and N546Δshowed a great loss in toxicity.Toxin oligomerisation and proteolytic susceptibility assays revealed that this residue might not involved in toxin oligomerisation and maintaining the stability of toxin.Brush border membrane vesicles（BBMV）binding assay using biotinylated toxin revealed that the enhanced toxicity of mutant N546A was because of increased binding to BBMV,and reduction in toxicity of other mutants were caused by reduction in initial or inreversible binding to BBMV.The W544 was further conservatively substituted with phenylalanine（F） and tyrosine（Y）and nonconservatively replaced by cysteine（C）.Bioassay and protein stability analysis indicated that the aromatic ring at this position was not absolutely necessary but the hydrophilic nature of the position had unfavorable influence to protein stability.Comparative analysis by SDS-PAGE showed that the protoxin of W544F was much more stable than the wild-type Cry1Ac,when treated with ultraviolet irradiation,trypsin and preserved at room temperature.The distance between two vertexes of the crystal of W544F was 0.6μm longer than that of the wild-type Cry1Ac under an atomic force microscope.Besides,the mutation W544F had similar insecticidal activity to wild-type Cry1Ac,but when treated with ultraviolet irradiation for 9 hours,it still maintained more than 4 times higher toxicity than the wild-type Cry1Ac,which might contribute to solving the major problem of field applications of Cry1Ac toxin.In this paper,the uniqueβ18-β19 loop in domainⅢof Cry1Ac5 toxin and its structural significance in the toxin functions was reported for the first time,which provided new biological evidences for domainⅢof Cry1Ac playing important roles in maintaining the stability of protein structure,and recognizing and binding with specific receptors.更多还原