苏云金芽胞杆菌增强子结合蛋白的表达纯化及功能研究

发布时间：2018-06-14 22:47

本文选题：苏云金芽胞杆菌 + σ54因子　；参考：《东北农业大学》2015年硕士论文

【摘要】：苏云金芽胞杆菌(Bacillus thuringiensis,简称Bt)是蜡样芽胞杆菌族的一个种,与其它芽胞杆菌相比,Bt的显著特点是在形成芽胞的同时,还能产生由杀虫蛋白组成的伴胞晶体。因具有杀虫效果好,对人类和环境友好等优点而成为广泛应用的杀虫微生物。Bt中芽胞的形成与伴胞晶体的产量与菌体不同生长时期的代谢模式之间存在一定的关系。本实验室前期研究发现Bt HD73菌株中sig L基因(编码σ54因子)的缺失降低了芽胞形成率和Cry蛋白的产量,说明σ54调控的代谢途径可能在芽胞形成及晶体合成上具有重要作用。依赖于σ54的转录需要增强子结合蛋白(bacteria Enhancer Binding Proteins,b EBPs)的激活,Bt HD73全基因组中,已发现8种b EBP,本实验室目前已经构建并表达纯化了Gab R和Sox R两种蛋白,并验证了它们在HD73中的功能。Gab R和Sox R两种蛋白分别调控γ-氨基丁酸(GABA)代谢途径和肌氨酸代谢途径。本研究对其它6种b EBP(Aco R、Bkd R、Kam R、Lev R、Prd R和Roc R)进行了克隆表达。构建了这6种b EBP基因带有组氨酸(His)标签的表达载体,并且都能够在大肠杆菌BL21菌株中进行表达。本研究通过镍亲和层析柱纯化得到了Bkd R和Lev R两种蛋白,为进一步研究EBP的分子调控机制奠定了基础。本研究集中在HD73_1024(编码脯氨酸消旋酶)、HD73_2025(编码支链氨基酸转氨酶)、HD73_4161(编码脯氨酸二肽酶)、HD73_4943(编码乙酸辅酶A连接酶)和HD73_5327(编码依赖于NADPH的脱氢酶),5个基因启动子的转录活性研究,测定了它们在8个EBP缺失突变体中的转录活性。β-半乳糖苷酶活性测定结果表明只有HD73_4161的启动子在gab R突变体中的活性降低,而其它启动子在所有b EBP突变体中的活性与HD73野生菌株相比无明显差异,说明: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的转录调控因子,Gab R可能是HD73_4161的转录调控因子。本研究对Aco R参与的3-羟基丁酮代谢途径的转录调控进行了深入的研究。3-羟基丁酮(acetoin)是许多微生物糖代谢的中间产物,对微生物本身具有重要的生理意义:如抵御环境的酸化、参与NAD+/NADH2比率的调节和作为储存碳源等。在枯草芽胞杆菌、富氧产碱菌(Alcaligenes eutrophus)和肺炎杆菌(Klebsiella pneumoniae)等微生物中,3-羟基丁酮是通过氧化裂解降解的,即通过aco基因簇编码的3-羟基丁酮脱氢酶系统降解成乙醛和乙酸。Bt HD73菌株的3-羟基丁酮脱氢酶系统也由aco基因簇编码,生物信息学和RT-PCR分析表明Bt HD73的aco基因簇由aco ABCL 4个基因组成,形成一个转录单元,aco基因簇的启动子含有依赖于σ54转录的-12/-24保守序列。β-半乳糖苷酶活性分析表明,aco基因簇的启动子Paco A转录活性在sig L和aco R突变体中均明显降低。对Δaco R突变体进行表型测定,发现aco R基因的缺失对菌体生长和Cry1Ac蛋白产量无显著影响,但使菌体运动能力减弱,使芽胞产量略有下降,并且不能利用3-羟基丁酮,说明Aco R调控的代谢途径与3-羟基丁酮的利用及菌体的运动能力和芽胞产量相关。
[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.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S476.1

【共引文献】