【作者】 宋荣；

【导师】 孙明；

【作者基本信息】 华中农业大学 ， 微生物学， 2008， 博士

【摘要】 本论文利用苏云金芽胞杆菌的表达调控元件及来自Cry1C、Cry1Ac和Cry7Ba1蛋白和晶体形成,蛋白稳定相关的C-半段,辅助蛋白p20等研究对营养期杀虫蛋白Vip3Aa7,杀虫晶体蛋白Cry2Aa,Cry3Aa晶体形成,表达量和杀虫活性的影响,建立了一个苏云金芽胞杆菌高效表达体系;并通过Cry1C、Cry1Ac和Cry7Ba1蛋白的N-、C-半段互换,研究了苏云金芽胞杆菌Cry1类杀虫晶体蛋白的C-半段对Cry7Ba1晶体蛋白溶解性,晶体形成及毒性的影响,主要结果如下:1.苏云金芽胞杆菌C-半段使Vip3Aa7形成包含体并且显著提高其表达量本文利用苏云金芽胞杆菌ICPs蛋白的各种表达调控元件,希望提高营养期杀虫蛋白Vip3Aa7的表达量,改善其因为分泌到胞外给生产应用带来的不便,研究芽胞依赖型BtⅠ-BtⅡ启动子,上游调控区,终止子及大分子杀虫晶体蛋白C-半段对Vip3Aa7表达量和包含体形成的影响,结果表明:1.1 Cry1C的C-半段不仅显著提高了Vip3Aa7融合蛋白的表达量,重组蛋白的表达量比出发菌株的表达量高9.3倍,而且使其形成包含体。1.2 Cry1Ac和Cry7Ba1蛋白的C-半段与Cry1C的的C-半段具有同样的功能,均能显著提高Vip3Aa7融合蛋白的表达量,重组蛋白的表达量比出发菌株的表达量分别高9.4和9.0倍,而且也能使其形成包含体。1.3生物测定结果表明,重组蛋白的杀虫毒力与出发菌株的蛋白相比普遍降低了。本实验首次报道Cry1C晶体蛋白的C-半段能使Vip3Aa7蛋白以包含体的形式存在,并且显著提高其表达量,这为Vip3Aa7蛋白以芽胞制剂的形式在生产中应用奠定了相应的理论基础,具有非常重要的理论实践意义。2.辅助蛋白p20提高Vip3Aa7融合蛋白表达的表达量将来自以色列亚种的辅助蛋白p20构建到另一穿梭载体pBE2上,与上文构建的重组质粒共转构建了一系列工程菌,发现辅助蛋白p20对以上Vip3Aa7融合蛋白表达量均有提高,几个融合蛋白在晶体形成上没有明显不同,而且p20并不影响融合蛋白的杀虫活性。说明辅助蛋白p20能提高Vip3Aa7融合蛋白的表达量。3.Cry1类蛋白的C-半段显著提高Cry2Aa和Cry3Aa蛋白的表达量将Cry1C和Cry1Ac的C-半段分别与Cry2Aa和Cry3Aa的毒力核心区融合在一起,构建融合基因,结果表明Cry1C和Cry1Ac的C-半段使不含C-半段的Cry2Aa和Cry3Aa的蛋白表达量显著提高,但仍然不能使Cry2Aa和Cry3Aa形成规则晶体,只能形成无规则形状的包含体,重组蛋白杀虫毒力与出发蛋白相比没有显著变化,说明本文建立的超量表达系统不含C-半段的苏云金芽胞杆菌的表达量也能显著,并且不影响重组蛋白的功能和活性。4.4.Cry1类蛋白的C-半段提高了Cry7Ba1蛋白的溶解性并且恢复了其毒力Cry7Ba1杀虫晶体蛋白只有在pH＞11.5的碱性溶液中才能溶解,并且晶体蛋白只有在体外溶解后才对小菜蛾表现出一定毒性,这极大影响了其在实际生产中的应用,本实验将Cry1C、Cry1Ac和Cry7Ba1的N-、C-半段互换,研究较易溶解的苏云金芽胞杆菌Cry1类的C-半段对Cry7Ba1蛋白溶解性的影响。结果表明:Cry7Ba1的难溶性是由其C-半段决定,其毒性由N-半段决定。通过替换C-半段提高了cry7Ba1晶体蛋白的溶解性从而恢复了其毒力,为解决这一类由于溶解性的丧失导致毒性丧失的苏云金芽胞杆菌的生产应用打开了一个新的思路,在理论和应用上均具重要意义。更多还原

【Abstract】 Bacillus thuringiensis has unique capability of high expression and crystal formation of these ICPs depended on various Regulated mechanisms.Here we established a high expression system to optimize the yield of Vip3Aa7 through relocating it into the mother cell of B.thuringiensis in the form of inclusion bodies using the regulated mechanism and the C-terminus,of Cry1C,Cry1Ac and Cry7Ba1.And this overexpression system has also been used to increase the yields of other B.thuringiensis proteins which have no C-terminus such as Cry2Aa and Cry3Aa.Then,In order to demonstrate the possibility of exploiting the latent insecticidal properties of crystals like Cry7Ba1,crystals vitro-solubilized were toxic to Plutella xylostella,we constructed a series of plasmids encoding recombinant forms of Cry7Ba1 in which the C-terminal domain was replaced by that of Cry1C and Cry1Ac,leading to improved solubility and effective toxicity to Pluetella xylostella.The following are the major results:1.The carboxy-terminal half of ICPs can markedly increased the yields of Vip3Aa7 through forming inclusion bodies in the mother cell of Bacillus thuringiensis.1.1 The C-terminus of the Cry1C could not markedly increase Vip3Aa7 yields 9.8-fold than that of wild type strain BMB8901 but also help it form the irregular inclusion bodies in B.thuringiensis.1.2 The C-terminal halves of Cry1Ac and Cry7Ba1 have the same function with that of Cry1C,could not largely increase Vip3Aa7 yields 9.4 and 9.0-fold recepectively than that of wild type strain BMB8901 but also help it form the irregular inclusion bodies in B. thuringiensis.1.3 Bioassays showed that all proteins produced by recombinant strains had similar toxicities with against H.armigera,S.exigua,and P.xylostella,which were evidently lower than that of wild-type Vip3Aa7 protein2.Helper protein p20 increased the yield of Vip3Aa7 fusion proteinsA searious of recombinant strains were obstained through transforming plasmid pBMB0177 containing the p20 and the expression vectors described above simultaneously.We found that the p20 could increase the yields of Vip3Aa7 protein but could not affect the forming of the inclusion bodies and the toxicities.In conclusion,we established an overexpression system to optimize the yield of Vip3Aa7 and relocated it into the mother cell of B.thuringiensis in the form of inclusion bodies.Alterations comprising BtⅠ-BtⅡpromoters,the STAB-SD sequence,the C-terminal,and the terminator of Cry1C affected the expression of Vip3Aa7.3.The C-terminus of Cry1C and Cry1Ac increased the yields of proteins of Cry2Aa and Cry3Aa In order to know whether the C-terminal of Cry1C can be used to increase the yields of other B.thuringiensis proteins without C-terminus,four recombinants,BMB0192, BMB0193,BMB0194,BMB0195,were gained by replaced the Vip code region with cry2Aa and cry3Aa.The result showed that this high expression system could increase the yields of Cry2Aa and Cry3Aa proteins without C-terminus of Cry1.However,the C-terminus of Cry1 could not make Cry2Aa and Cry3Aa form the bipyramidal(Cry1), cuboidal(Cry2Aa),flat rectangular(Cry3Aa) crystal protein,only irregular inclusion bodies,but did not affect the toxicities of recombinant protein.This result demonstrate that the overexpression system can be used to increase the yields of other B.thuringiensis proteins which cannot form crystals or other low expression level proteins and could not affect the activity of recombinants.The expression strategy would facilitate the development of a suitable formulation for the application of this class of insecticidal proteins in the field,and offers an additional method for potentially improving the efficacy of insecticides based on B.thuringiensis.4.The C-terminus of Cry1C and Cry1Ac increase the solubility of protein of Cry7Ba1After exchanging the C-terminal halves of Cry7Ba1 and Cry1,we found that the limited solubility of Cry7Ba1 was due to its C-terminal half,and that recombinant Cry7Ba1 possessing the C-terminal half of Cry1 showed good solubility and toxicity to P. xylostella but could not form the bypimid crystal the same as that of the parent wild type strain Cry1C,Cry1Ac and Cry7Ba1.Our research opens a new way to release hidden biological function from "non-insecticidal" B.thuringiensis strains resulting from limited solubility by simply replacing their C-terminal halves.更多还原