本文選題：蘇云金芽胞桿菌 + σ54因子��； 參考：《東北農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：蘇云金芽胞桿菌(Bacillus thuringiensis,簡(jiǎn)稱(chēng)Bt)是蠟樣芽胞桿菌族的一個(gè)種,與其它芽胞桿菌相比,Bt的顯著特點(diǎn)是在形成芽胞的同時(shí),還能產(chǎn)生由殺蟲(chóng)蛋白組成的伴胞晶體。因具有殺蟲(chóng)效果好,對(duì)人類(lèi)和環(huán)境友好等優(yōu)點(diǎn)而成為廣泛應(yīng)用的殺蟲(chóng)微生物。Bt中芽胞的形成與伴胞晶體的產(chǎn)量與菌體不同生長(zhǎng)時(shí)期的代謝模式之間存在一定的關(guān)系。本實(shí)驗(yàn)室前期研究發(fā)現(xiàn)Bt HD73菌株中sig L基因(編碼σ54因子)的缺失降低了芽胞形成率和Cry蛋白的產(chǎn)量,說(shuō)明σ54調(diào)控的代謝途徑可能在芽胞形成及晶體合成上具有重要作用。依賴(lài)于σ54的轉(zhuǎn)錄需要增強(qiáng)子結(jié)合蛋白(bacteria Enhancer Binding Proteins,b EBPs)的激活,Bt HD73全基因組中,已發(fā)現(xiàn)8種b EBP,本實(shí)驗(yàn)室目前已經(jīng)構(gòu)建并表達(dá)純化了Gab R和Sox R兩種蛋白,并驗(yàn)證了它們?cè)贖D73中的功能。Gab R和Sox R兩種蛋白分別調(diào)控γ-氨基丁酸(GABA)代謝途徑和肌氨酸代謝途徑。本研究對(duì)其它6種b EBP(Aco R、Bkd R、Kam R、Lev R、Prd R和Roc R)進(jìn)行了克隆表達(dá)。構(gòu)建了這6種b EBP基因帶有組氨酸(His)標(biāo)簽的表達(dá)載體,并且都能夠在大腸桿菌BL21菌株中進(jìn)行表達(dá)。本研究通過(guò)鎳親和層析柱純化得到了Bkd R和Lev R兩種蛋白,為進(jìn)一步研究EBP的分子調(diào)控機(jī)制奠定了基礎(chǔ)。本研究集中在HD73_1024(編碼脯氨酸消旋酶)、HD73_2025(編碼支鏈氨基酸轉(zhuǎn)氨酶)、HD73_4161(編碼脯氨酸二肽酶)、HD73_4943(編碼乙酸輔酶A連接酶)和HD73_5327(編碼依賴(lài)于NADPH的脫氫酶),5個(gè)基因啟動(dòng)子的轉(zhuǎn)錄活性研究,測(cè)定了它們?cè)?個(gè)EBP缺失突變體中的轉(zhuǎn)錄活性。β-半乳糖苷酶活性測(cè)定結(jié)果表明只有HD73_4161的啟動(dòng)子在gab R突變體中的活性降低,而其它啟動(dòng)子在所有b EBP突變體中的活性與HD73野生菌株相比無(wú)明顯差異,說(shuō)明:Aco R、Bkd R、Gab R、Kam R、Lev R、Prd R、Rco R和Sox R不是HD73_1024、HD73_2025、HD73_4943和HD73_5327的轉(zhuǎn)錄調(diào)控因子,Gab R可能是HD73_4161的轉(zhuǎn)錄調(diào)控因子。本研究對(duì)Aco R參與的3-羥基丁酮代謝途徑的轉(zhuǎn)錄調(diào)控進(jìn)行了深入的研究。3-羥基丁酮(acetoin)是許多微生物糖代謝的中間產(chǎn)物,對(duì)微生物本身具有重要的生理意義:如抵御環(huán)境的酸化、參與NAD+/NADH2比率的調(diào)節(jié)和作為儲(chǔ)存碳源等。在枯草芽胞桿菌、富氧產(chǎn)堿菌(Alcaligenes eutrophus)和肺炎桿菌(Klebsiella pneumoniae)等微生物中,3-羥基丁酮是通過(guò)氧化裂解降解的,即通過(guò)aco基因簇編碼的3-羥基丁酮脫氫酶系統(tǒng)降解成乙醛和乙酸。Bt HD73菌株的3-羥基丁酮脫氫酶系統(tǒng)也由aco基因簇編碼,生物信息學(xué)和RT-PCR分析表明Bt HD73的aco基因簇由aco ABCL 4個(gè)基因組成,形成一個(gè)轉(zhuǎn)錄單元,aco基因簇的啟動(dòng)子含有依賴(lài)于σ54轉(zhuǎn)錄的-12/-24保守序列。β-半乳糖苷酶活性分析表明,aco基因簇的啟動(dòng)子Paco A轉(zhuǎn)錄活性在sig L和aco R突變體中均明顯降低。對(duì)Δaco R突變體進(jìn)行表型測(cè)定,發(fā)現(xiàn)aco R基因的缺失對(duì)菌體生長(zhǎng)和Cry1Ac蛋白產(chǎn)量無(wú)顯著影響,但使菌體運(yùn)動(dòng)能力減弱,使芽胞產(chǎn)量略有下降,并且不能利用3-羥基丁酮,說(shuō)明Aco R調(diào)控的代謝途徑與3-羥基丁酮的利用及菌體的運(yùn)動(dòng)能力和芽胞產(chǎn)量相關(guān)。
[Abstract]:Bacillus thuringiensis (Bt) is one of the species of Bacillus cereus. Compared with other Bacillus, the significant characteristic of Bt is that it can produce the cell crystal composed of insecticidal protein at the same time, and it has been widely used because of its good insecticidal effect and good environmental friendliness. There is a certain relationship between the formation of the bud in the insect microbe.Bt and the yield of the companion cell crystal and the metabolic pattern of the different growth period. In the previous study, we found that the deletion of the sig L gene (coding sigma 54 factor) in the Bt HD73 strain reduced the formation rate of the bud and the yield of the Cry protein. Bacteria Enhancer Binding Proteins (B EBPs) is activated by the transcription of sigma 54, and 8 B EBP have been found in the whole genome of Bt HD73. This laboratory has now constructed and expressed the purification of Gab R and two proteins. Functional.Gab R and Sox R two proteins regulate the metabolic pathway of gamma aminobutyric acid (GABA) and the pathway of muscle ammonia metabolism respectively. This study has carried out cloning and expression of the other 6 B EBP (Aco R, Bkd R, Kam definitions, etc.). Two proteins of Bkd R and Lev R were purified by nickel affinity chromatography column, which laid the foundation for further research on the molecular regulation mechanism of EBP. This study was focused on HD73_1024 (encoded proline racemase), HD73_2025 (encoded branched amino acid aminotransferase), HD73_4161 (encoding proline two peptidase), HD73_4943 (encoding B). The transcriptional activity of 5 gene promoters, acid coenzyme A ligase and HD73_5327 (encoded by NADPH dehydrogenase), was used to determine their transcriptional activity in 8 EBP deletion mutants. Beta galactosidase activity assay showed that only the promoter of HD73_4161 decreased in the gab R mutants, while other promoters were all in the gab R mutant. The activity of B EBP mutants is not significantly different from that of the wild strains of HD73, indicating that Aco R, Bkd R, Gab R, Kam R are transcriptional regulatory factors that may be the transcriptional regulatory factors. An in-depth study of.3- hydroxy butanone (acetoin) is a intermediate product of many microbial glycometabolism, which has important physiological significance for microorganism itself, such as resisting acidification of the environment, regulating the ratio of NAD+/NADH2 and storing carbon sources, etc. in Bacillus subtilis, Alcaligenes eutrophus (Alcaligenes eutrophus) and Bacillus pneumoniae In (Klebsiella pneumoniae) and other microorganisms, 3- hydroxy butanone is degraded by oxidative cracking, that is, the 3- hydroxy butanone dehydrogenase system degraded into acetaldehyde and.Bt HD73 by ACO gene cluster encoded 3- hydroxy butanone dehydrogenase system is also encoded by the ACO gene cluster. Bioinformatics and RT-PCR analysis show that Bt HD73 is the cluster of genes. The 4 genes of ACO ABCL form a transcriptional unit, and the promoter of the ACO gene cluster contains the -12/-24 conservative sequence dependent on the sigma 54 transcription. The analysis of beta galactosidase activity analysis shows that the Paco A transcriptional activity of the promoter of the ACO gene cluster is obviously reduced in sig L and ACO R mutants. The loss of the cause had no significant effect on the growth of the mycelium and the yield of Cry1Ac protein, but weakened the activity of the mycelium, reduced the yield of the bud slightly, and did not use 3- hydroxybutanone. It showed that the metabolic pathways regulated by Aco R were related to the use of 3- hydroxy butanone and the exercise ability of the mycelium and the yield of the bud.
【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：S476.1

【共引文獻(xiàn)】

相關(guān)期刊論文 前1條

1 劉曉霏;付晶;霍廣鑫;章博;王智文;陳濤;;生物法制備平臺(tái)化合物乙偶姻的最新研究進(jìn)展[J];中國(guó)生物工程雜志;2015年10期

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1 張顯;高產(chǎn)乙偶姻枯草芽孢桿菌的代謝工程改造[D];江南大學(xué);2013年

2 楊靜妮;蘇云金芽胞桿菌芽胞期母細(xì)胞裂解機(jī)制的研究[D];東北農(nóng)業(yè)大學(xué);2013年

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1 李穎;新型vip3A基因的克隆、表達(dá)與殺蟲(chóng)活性研究[D];中國(guó)農(nóng)業(yè)科學(xué)院;2014年

2 張U，

本文編號(hào)：2019266