【作者】 朱莉；

【导师】 黄大昉；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2007， 博士

【摘要】 苏云金芽胞杆菌（Bacillus thuringiensis，简称Bt）因其杀虫效果好、安全、高效等优点而成为农业生物防治上应用最为广泛的杀虫微生物。Bt与其它芽胞杆菌相比，一个显著特点就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。有关Bt伴胞晶体形成机制方面的研究主要是集中在杀虫晶体蛋白基因的表达调控上，而从代谢调控的角度研究晶体蛋白形成的分子机制目前尚未见报道。本论文围绕苏云金芽胞杆菌中与γ-氨基丁酸代谢旁路相关基因簇的结构和功能进行了一系列的研究，主要内容包括：本研究从国内分离得到的苏云金芽胞杆菌G03菌株中克隆了gabT和gabD基因，并分别在大肠杆菌中进行了表达和纯化。酶活测定结果表明，GabT和GabD蛋白分别呈现出γ-氨基丁酸转氨酶和琥珀酸半醛脱氢酶的活性。氨基酸序列同源性比对分析发现，这两个蛋白质在蜡样芽胞杆菌群（B. cereus group）中具有较高的相似性，而与枯草芽胞杆菌的相似性较低，分别为58％、51％。利用基因敲除技术分别构建了Bt HD-73菌株的gabT、gabD基因缺失突变株，同时也构建了相应的回复突变株，对gabT、gabD基因的功能进行了研究，结果表明：gabT、gabD基因敲除不会影响突变株在丰富培养基中的正常生长，但在基础培养基（含0.2％GABA）中gabT基因缺失突变株的生长会受到抑制；gabT、gabD基因敲除对晶体蛋白的表达量无显著影响，但gabT的缺失会推迟晶体蛋白的表达；gabT、gabD基因敲除均会导致芽胞产量的降低；GABA代谢旁路的功能还有待于进一步了解。通过对Bt HD-73菌株gab基因簇的克隆及序列分析发现，gab基因簇的结构与大肠杆菌和枯草芽胞杆菌的明显不同，而在蜡样芽胞杆菌群中基本上是一致的，说明γ-氨基丁酸代谢途径相关的基因簇结构在蜡样芽胞杆菌群中是比较保守的。通过RT-PCR转录分析发现gabD和gabT基因并未同时进行转录，其中gabT单独转录，而gabD与其上游的转录激活因子编码基因reg共转录。根据克隆并测序的gab基因簇序列，分别克隆了gabT和reg基因的启动子，并构建了相应的lacZ融合表达载体。通过β-半乳糖苷酶活性的定性和定量分析，表明gabT和reg基因的转录都是由自身的启动子调控的。以上研究结果表明gabT和gabD基因属于不同的转录单元，而gabD和reg基因组成了一个操纵子。利用敲除载体pMADDreg通过同源重组方法敲除reg基因后获得缺失突变株HD-73（Δreg）。发现突变菌株的生长、产芽胞和晶体蛋白的能力与出发菌株相比并无明显差异。但reg基因的缺失严重地影响了SSADH酶活性，并通过其回复突变株恢复了SSADH酶活性，说明reg基因对gabD基因的表达起正调控作用。同样地，发现reg基因对gabT基因的表达也起正调控作用。通过对reg基因的表达产物Reg蛋白的结构域分析，推测它可能是一种依赖于σ⁵⁴因子的激活蛋白，在蜡样芽胞杆菌群中比较保守，有关其调控作用需进一步的验证。总之，本研究为深入研究苏云金芽胞杆菌γ-氨基丁酸代谢旁路相关基因簇的结构和功能奠定了基础，也为进一步研究芽胞和伴胞晶体形成机制提供了线索。更多还原

【Abstract】 Bacillus thuringiensis （Bt） has been widely used as bioinsecticidal microbe for its high toxicity, safety and the other merits. Comparing with other Bacillus species, Bt can produce large crystalline parasporal inclusions during sporulation. The study on Insecticidal Crystal Proteins （ICPs） is mainly focused on the expression and regulation of cry gene encoding ICPs. However, the research about the molecular mechanism of metabolism regulation involved in crystal protein formation is less. In this report, the structure and function of gene cluster involved inγ-aminobutyric acid （GABA） shunt in Bt were discussed.We characterized gabT and gabD genes cloned from B. thuringiensis strain G03 isolated in China. Both genes were expressed in E. coli BL21 （DE3） strain, and their products were purified by affinity chromatography respectively. By enzymatic assay, GabT protein showed the activity of GABA transaminase, while GabD protein exhibited SSADH activity. The amino acid sequences of two proteins, GABASE and SSADH, showed significant identity with those in the B. cereus group, but their similarity score between G03 and B. subtilis 168 was lower, only 58% and 51%, respectively.To determine the function of gabT and gabD of Bt HD-73 strain, the gabT, gabD gene deleted mutants were obtained by means of gene knock-out respectively, and their corresponding complementary strains were also constructed. The deletions of gabT and gabD gene could not affect the growth of mutant strains in rich culture medium, but the growth of gabT deletion mutant strain was repressed in basic medium （containing 0.2% GABA）. The gabT deletion mutant strain could retard the formation of crystal protein Cry1 Ac, comparing with the host strain HD-73. The amounts of active spore both in gabT and gabD deletion mutant strains were all less than that in host strain HD-73.The structure of gab gene cluster in Bt HD-73 is distinctly different from that in E. coli and B. subtilis, but is common in B. cereus group by genomic sequence alignment. It means that the gene cluster involved in GABA shunt is very special in B. cereus group. The result of RT-PCR showed that gabT and gabD were not co-transcribed, in which gabT seperately was transcribed, while gabD and its upstream reg gene were co-transcribed. We had cloned the promoters of gabT and reg gene respectively, and constructed their corresponding lacZ fusion expression vectors. Assay ofβ-galactosidase activity showed that both gabT and reg gene was transcribed by its own promoter. These results indicated that gabT and gabD gene belonged to different transcription units, while gabD and reg gene formed an operon.The plasmid pMADDreg was constructed for knock-out of reg gene, and then introduced into HD-73 strain. Both the growth and the formation of crystal and spore were not affected in the relevant mutant strain HD-73 （Δreg） comparing with the host strain HD-73. However the activity of SSADH was almost lost in the reg deletion mutant strain, and restored in complementary strain HDHFreg. These results indicated that reg gene played a positive regulation role in gabD expression. The reg gene was also confirmed to positively regulate the gabT expression. Reg protein encoded by reg gene maybe a potentialσ⁵⁴-dependent transcriptional activator according to the conserved domian search in NCBI, and it is very conservative in B. cereus group by animo acid sequence alignment. Its function in transcriptional regulation need to be further confirmed.In a word, this study will be helpful to better understand the regulation mechanism and physiological function of GABA shunt, and to further study the mechanism of sporulation and crystal formation in B. thuringiensis.更多还原