本文選題：蘇云金芽胞桿菌 + SigmaK因子��； 參考：《中國農(nóng)業(yè)科學院》2015年碩士論文

【摘要】：蘇云金芽胞桿菌(Bacillus thuringiensis,簡稱Bt)屬于芽胞桿菌屬的蠟樣芽胞桿菌群(B.cereus group),是一種特殊革蘭氏陽性細菌,在產(chǎn)生芽胞的同時產(chǎn)生由殺蟲蛋白組成的具有規(guī)則形狀的伴胞晶體(主要由cry和cyt基因編碼)。因其對靶標生物高效、對環(huán)境安全友好等特點而成為目前應用最廣泛的微生物殺蟲劑。在芽胞形成過程中,不同的Sigma(σ)因子在時空上級聯(lián)起始基因轉錄,σK由sig K基因編碼,是Bt芽胞晚期重要的Sigma因子,芽胞晚期主要涉及芽胞外壁形成、芽胞的成熟和釋放、母細胞裂解、晶體的形成等重要細胞發(fā)育過程,目前對這些細胞過程所知仍然很少。因此本研究主要明確Bt中σK因子的調(diào)節(jié)子,為解析母細胞生長過程的晚期調(diào)控網(wǎng)絡進而為闡明芽胞形成晚期細胞過程的分子機制奠定基礎。以芽胞形成晚期全局調(diào)控因子ger E突變體作為對照,分別提取sig K和ger E突變體在T7、T10時期的總RNA進行DNA芯片分析,明確受σK控制的基因或操縱子。結果表明T7時期有158個受σK下調(diào)的基因和138個上調(diào)的基因。T10時期有878個受σK下調(diào)的基因和1317個上調(diào)的基因。利用σK因子結合位點的一致序列,分析T7和T10下調(diào)基因的啟動子區(qū)域,發(fā)現(xiàn)264個基因的啟動子具有σK因子結合位點,包括10個操縱子和242個基因。其功能包括氨基酸運輸和代謝、碳水化合物運輸和代謝、無機鹽離子運輸和代謝、信號轉導、蛋白轉譯后修飾等其他功能和未知功能。表達并純化了具有His標簽的Sigma K蛋白,并對上述σK因子的調(diào)節(jié)子進行驗證。前人研究已明確Sigma K控制cry1Ac基因的啟動子轉錄,凝膠阻滯實驗EMSA結果表明純化的Sigma K蛋白可以與cry1Ac基因啟動子結合,說明純化的Sigma K具有體外與受其控制的啟動子結合的功能。因此進一步通過EMSA實驗驗證了6個基因(芽胞萌發(fā)相關的基因HD73_3496,HD73_335水解酶基因HD73_3410和芽胞外壁相關的基因bxp B,exs B,bcl B)和一個操縱子(HD73_2493,HD73_2494)的啟動子能與Sigma K因子結合,證明了這6個基因和1個操縱子受Sigma K因子控制。此外構建了水解酶基因HD73_3156的啟動子和lac Z基因的融合載體,將載體轉入HD73菌株及sig K突變體中,測定β-半乳糖苷酶活性發(fā)現(xiàn)HD73_3156的啟動子活性在突變體中完全喪失,進一步證明了HD73_3156的轉錄受sK控制。
[Abstract]:Bacillus thuringiensis (BT) belongs to B.cereus group of Bacillus, which is a special Gram-positive bacteria. In addition to the production of spores, regular paracellular crystals composed of insecticidal proteins (mainly encoded by cry and cyt genes) were produced. It has become the most widely used microbial insecticide because of its high efficiency to target organisms and environmental safety. In the process of spore formation, different Sigma (蟽) factors are cascade initiation gene transcription in time and space. 蟽 K is encoded by sig K gene and is an important Sigma factor in the late stage of BT spores. The late stage of spores is mainly involved in the formation of the outer wall of the spores, the maturation and release of the spores. Some important cellular processes, such as mother cell cleavage and crystal formation, are still poorly understood. Therefore, in this study, the regulator of factor 蟽 K in BT was clarified, which laid a foundation for the elucidation of the molecular mechanism of late cell formation in the process of spores formation by analyzing the late regulatory network of the growth process of the mother cell. The total RNAs of ger K and ger E mutants at T7 / T10 stage were extracted from spores to identify the genes or operons controlled by 蟽 K, respectively. The results showed that there were 158 genes down-regulated by 蟽 K and 138 up-regulated genes at T7. At T10, there were 878 genes down-regulated by 蟽 K and 1317 genes up-regulated. By using the consistent sequence of 蟽 K factor binding sites, the promoter regions of T7 and T10 down-regulated genes were analyzed. It was found that 264 gene promoters had 蟽 K factor binding sites, including 10 operons and 242 genes. Its functions include amino acid transport and metabolism, carbohydrate transport and metabolism, inorganic salt ion transport and metabolism, signal transduction, protein post-translational modification and other functions and unknown functions. Sigma K protein with his label was expressed and purified, and the regulator of 蟽 K factor was verified. Previous studies have shown that Sigma K controls the promoter transcription of cry1Ac gene, and the results of gel block assay show that the purified Sigma K protein can bind to the promoter of cry1Ac gene, indicating that the purified Sigma K protein has the function of binding to the promoter controlled by Sigma K in vitro. Therefore, the promoters of six genes (HD733496 / HD73335 hydrolase gene HD733410 and bxp Bnexs Bbcl BCL BCL) and one operon HD732493 HD73SII 2494 were further verified by EMSA experiments to bind to Sigma K factor. It was proved that the six genes and one operon were controlled by Sigma K factor. In addition, a fusion vector of the promoter of the hydrolase gene HD73S3156 and the lac Z gene was constructed. The vector was transferred into the strain HD73 and sig K mutants. The activity of 尾 -galactosidase was determined and the promoter activity of HD733156 was completely lost in the mutant. It is further proved that the transcription of HD733156 is controlled by SK.
【學位授予單位】：中國農(nóng)業(yè)科學院
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S476.1

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