【作者】 郑大胜；

【导师】 袁志明；

【作者基本信息】 中国科学院研究生院（武汉病毒研究所） ， 微生物学， 2007， 博士

【摘要】 苏云金芽胞杆菌（Bacillus thuringiensis，Bt）是一种自然界中广泛分布的好气芽胞杆菌，由于对昆虫具有特异性毒杀作用，不污染环境和对人畜无害，成为目前应用最广、产量最大的生物杀虫剂菌种。其杀虫活性主要依赖于在芽胞期产生的杀虫晶体蛋白（Insecticidal Crystal Proteins，ICPs）或称δ-内毒素。本文研究了Bt杀蚊毒素蛋白在水生革兰氏阴性菌中的异源表达及表达调控分析，并进行了Bt杀虫毒素的遗传改良，分别获得了几株杀虫活性和持效性得到改良的杀蚊和杀虫工程菌株。在蚊幼虫天然食物之一的离中不粘柄菌（Asticcacaulis excentricus，Ae）中已成功表达Bti的杀蚊毒素Cry11Aa的基础上，将另一个具有协同杀蚊作用和阻抑蚊虫抗性产生的Bti杀蚊毒素的基因cyt1Aa转入Ae中表达。分别构建了含有cyt1Aa基因和同时含有cry11Aa基因的重组质粒pSODCyt20和pSODCryCyt20，获得相应的Ae重组子Ae-Cyt20和Ae-CC20。尽管重组质粒pSODCryCyt20中插入的cyt1Aa基因和cry11Aa的DNA序列正确，而且在重组菌株Ae-CC20中能检测到cry11Aa和cyt1Aa基因的mRNA，然而Ae-Cyt20和Ae-CC20只能分别表达Cyt1Aa和Cry11Aa蛋白，Ae-CC20菌株不能共表达两类毒素蛋白，因此认为Ae-CC20中cyt1Aa的表达受转录后水平因子调控。为了进行cyt1Aa和cry11Aa的共表达，在pSODCryCyt20中cyt1Aa基因的上游插入一个单独的启动子，构建了同时含有两基因的重组质粒pSODCryPCyt20，获得重组子Ae-CPC20，结果两基因都获得表达，表达产物对目标蚊幼虫具有协同毒杀作用。因此，认为cry11Aa和cyt1Aa在Ae中的共表达需要两个独立的启动子，这可能与其有着特殊的RNA系统、修饰限制系统有关。虽然表达的Cyt1Aa蛋白影响了Ae-CPC20的生长和发育，导致该重组菌株生长缓慢、终培养液的菌密度较低，但由于Cyt1A与Cry11A的协同杀蚊作用，Ae-CPC20对敏感和抗性致倦库蚊（Culex quinquefasciatus）的毒力分别是Ae-CC20的2.21倍和12.6倍。此外，Ae-CPC20菌株抗紫外线的能力比野生型Bti菌株强，因此该工程菌株在蚊虫的生物防治中具有潜在的应用价值。同时，本研究通过Bt不同Cry蛋白之间毒性区域的结构域交换而获得具有特异性杀虫活性的杂交晶体蛋白。通过Cry1Ca和Cry1Ab、Cry11Aa和Cry11Bb的结构域Ⅱ-Ⅲ的互换，在Bt无晶体突变菌株中进行了杂交毒素蛋白Cry1CAA和Cry11ABB的表达，得到了形态、蛋白质分子量大小和免疫原性都与野生型蛋白相符合的伴胞晶体。生物测定表明，杂交晶体Cry1CAA对棉铃虫的毒力(按LC₅₀计算)分别是Cry1Ab和Cry1Ca的55.8％和3.26倍，对甜菜夜蛾的毒力分别是Cry1Ab和Cry1Ca的4.08倍和35.5％；杂交晶体Cry11ABB对敏感和抗性库蚊的LC₅₀值分别为339.2和245.1 ng／ml、其毒力分别比野生型的Cry11Aa分别高40％和25％。证明晶体蛋白不同结构域的片段交换可以用于晶体蛋白的遗传改良，此研究将为探究Cry蛋白的不同结构域的功能及不同结构域相互作用的关系和筛选高效的毒素蛋白奠定基础。更多还原

【Abstract】 Bacillus thuringiensis, a widely distributed Gram-positive, spore-forming,aerobic bacterium, has been the most used biological pesticide due to its specificactivity, which is mainly contributed from the crystalline inclusion body formedduring sporulation composed of one or more insecticidal crystal proteins (ICPs, orδ-endotoxins), against target insects with environmental safety.In this paper, the heterogenous expression of ICPs from B. thuringiensis as wellas the regulation analysis in an aquatic recipient strain and the genetical modificationof ICPs were studied to improve the activity and persistence of the toxins againstlepidopteran and dipteran larvae.The cyt1Aa gene from Bti, whose product synergizes other mosquitocidal toxinsand functions as repressor of resistance developed by mosquitoes against Bacilliinsecticides, solely and alongside the cry11Aa gene from Bti as well, were introducedinto the aquatic Gram-negative bacterium Asticcacaulis excentricus, living in upperwaters as a natural food resource for mosquito larvae, who has been proved as asuccessful host for Bacilli mosquitocidal toxins. The genes were introduced as anoperon but, although DNA sequencing verified correct and mRNA was detected forboth genes, no Cyt1Aa toxin was detected even though cyt1Aa solely was expressedwell in another operon. Both Cyt1Aa and Cry11Aa were expressed using a constructin which a promoter was inserted upstream of each gene. The necessity of twopromoters for co-expression of cyt1Aa and cry11Aa in A. excentricus could becorrelated with the special RNA and restriction-modification system of A. excentricus.Recombinant A. excentricus expressing both toxins was found with nearly the samepace in toxicity to 3rd instar larvae of Culex quinquefasciatus but with slower growingpace, ultimately to be 2.21- and 12.6-fold as toxic as the recombinant expressing just Cry11Aa against susceptible and resistant C. quinquefasciatus larvae, respectively,suggesting that the expressed Cyt1Aa burdens the growth of, but synergizes Cry11Aaand thus increases the toxicity of recombinant A. excentricus cells. Furthermore, thisrecombinant A. excentricus co-expressing cry11Aa and cyt1Aa was less sensitive toultra-violet radiation than wild Bti stain was, potentially acting as a candidate forfurther mosquito control.In addition, hybrid toxins Cry1CAA and Cry11ABB, which were constructed byreplacing domainsⅡ-Ⅲof Cry1Ca and Cry11Aa by the corresponding domains ofCry1Ab and Cry11Bb, respectively, were expressed in Bt acrystalliferous strain.SDS-PAGE and Western blot showed that the recombinant strains could express twohybrid proteins in recombinant strains during sporulation, with molecule weight andimmunogenicity identical to the original wide-type toxins. Importantly, the twoproteins has improved activity to the tested insects than their origins. ProteinCry1CAA was 0.558- and 3.26-fold toxic to neonate Helicoverpa armigera larvaethan Cry1Ab and Cry1Ca respectively, and 4.08- and 0.355-fold toxic to neonateSpodoptera exigua than the two origins, respectively. The toxicity of hybrid toxinCry11ABB was not significantly higher than wide-type proteins, with LC50 values of339.2 and 245.1 ng/ml to susceptible and resistant third-instar larvae of C.quinquefasciatus, respectively, corresponding to 40 % and 25 % increase of toxicitythan Cry11Aa. It implies that domain swapping technology might be applied for thegenetical modification ofB. thuringiensis toxins.更多还原