【摘要】：蘇云金芽孢桿菌LLP29是一株具有自主知識產(chǎn)權(quán)的高效殺蚊菌株，為進(jìn)一步了解該菌株的殺蚊毒素，本研究成功克隆分析了LLP29含有的cry11基因，結(jié)果發(fā)現(xiàn)該基因和已報(bào)道的cry11Aa同源性達(dá)99%以上，是一類殺蟲譜較廣且毒力較高的殺蚊基因。將該基因登陸NCBI，獲得登陸號XQ228567，并提交Bt命名委員會，被正式命名為cry11Aa4。原核表達(dá)并純化Cry11Aa4殺蚊蛋白，以致倦庫蚊為靶標(biāo)進(jìn)行生物測定，，其LC50為1340ng/mL。 以Cry11Aa4為抗原免疫新西蘭大白兔，獲得Cry11Aa4的多克隆抗體。免疫印跡試驗(yàn)表明，Cry11Aa4可與致倦庫蚊中腸蛋白有效結(jié)合。為了分析獲得的結(jié)合蛋白，構(gòu)建了生物素-親和素系統(tǒng)，基于生物素-親和素的沉降試驗(yàn)，成功分離到了三個(gè)Cry11Aa4的結(jié)合蛋白，其分子量分別為46、52和100KDa。經(jīng)質(zhì)譜分析，上述蛋白分別為ATP結(jié)合蛋白、熱激蛋白和未知蛋白。其中，未知蛋白可能為一種APN。本研究為深入研究毒素與受體的相互作用、毒素的作用機(jī)制及蚊子抗性產(chǎn)生機(jī)理提供重要基礎(chǔ)。
[Abstract]:Bacillus thuringiensis (LLP29) is a highly efficient mosquito killing strain with independent intellectual property rights. In order to further understand the insecticidal toxin of Bacillus thuringiensis, the cry11 gene contained in LLP29 was cloned and analyzed successfully. The results showed that the homology of this gene with the reported cry11Aa was over 99%, and it was a class of insecticidal genes with high virulence and broad insecticidal spectrum. The gene is logged in to NCBI, to obtain the landing number XQ228567, and submitted to the Bt naming committee, officially named cry11Aa4. The prokaryotic expression and purification of Cry11Aa4 insecticidal protein led to the biometric determination of Culex pipiens quinquefasciatus with a LC50 of 1340ng / mL. New Zealand white rabbits were immunized with Cry11Aa4 as antigen to obtain polyclonal antibodies against Cry11Aa4. Western blot test showed that Cry11Aa4 could effectively bind to midgut protein of Culex pipiens quinquefasciatus. In order to analyze the binding proteins, the biotin-avidin system was constructed. Three Cry11Aa4 binding proteins with molecular weight of 460.52 and 100KDawere successfully isolated by the sedimentation test based on biotin-avidin. The above proteins were identified as ATP binding proteins, heat shock proteins and unknown proteins by mass spectrometry. Among them, the unknown protein may be a kind of APN.. This study provides an important basis for the further study of the interaction between toxin and receptor, the mechanism of toxin action and the mechanism of mosquito resistance.
【學(xué)位授予單位】：福建農(nóng)林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：S476

【參考文獻(xiàn)】

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1 關(guān)雄;;蘇云金芽孢桿菌研究回顧與展望[J];中國農(nóng)業(yè)科技導(dǎo)報(bào);2006年06期

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