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摘要：生防枯草芽孢杆菌能产生多类抑菌物质，研究这类物质的合成和调控机理是重要内容之一。本课题组前期研究发现，枯草芽孢杆菌模式菌株BS168的抑菌物质泛革素（fengycin）的合成受sfp和degQ 2个功能基因的调控。其中，sfp 编码的4'-磷酸泛酰巯基乙胺基转移酶负责修饰fengycin合成酶，使其成为有活性的全酶，而deg Q的调控机理未知。在本研究中，我们利用突变了BS168菌株中的双组份系统degS/degU中的调节子degU，得到了BS168△degU突变体。该突变体中分别转入sfp和sfp+degQ，得到了菌株BS168△degU(sfp)和BS168△degU(sfp+degQ)。活菌和脂肽类提取物的抑菌活性检测结果表明，只有BS168(sfp+degQ)具有抑制油菜菌核病菌的活性，而BS168(sfp)、BS168△degU(sfp)和BS168△degU(sfp+degQ)则不具有该抑菌活性。质谱检测结果表明，只有BS168(sfp+degQ)能同时产生fengycin和surfactin，而BS168(sfp)、BS168△degU(sfp)和BS168△degU(sfp+degQ)都只能产生surfactin。进一步利用EMSA的研究表明，DegU蛋白能结合到fengycin的启动子部分。这说明degQ能通过双组份系统degS/degU调控fengycin的合成。25647
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毕业论文关键词：枯草芽孢杆菌；脂肽化合物；泛革素；degQ；degU
Study on regulatory mechanism of Fengycin by degQ
Abstract：It is one of the important contents to study the biosynthesis and regulation mechanism of antibiotics produced by biocontrol Bacillus subtilis strains. In our previous study, it was found that the synthesis of fengycin, an antibacterial substance of Bacillus subtilis BS168 strain, was regulated by sfp and degQ genes. Among them, the sfp gene encoding the 4'-phosphopantetheinyl transferase, which is responsible for the modification of fengycin synthases, and makes it acting as an active holoenzyme. The regulation mechanism of degQ is unknown. In this study, the degU gene from the two-component system degS/degU was mutated using CRISPR/Cas9 tool, then the BS168△degU was gotten. When this mutant was respectively introduced with sfp and sfp+degQ, the corresponding strains BS168△degU(sfp) and BS168△degU(sfp+degQ) were gotten. The results of antifungal activity of the strains and their lipopeptides extact showed that only BS168 (sfp+degQ) can inhibit the growth of Sclerotinia sclerotiorum activity. But the BS168(sfp), BS168△degU(sfp) and BS168△degU(sfp+degQ) did not have such activity. The results of Mass Spectrometry indicated that only BS168 (sfp+degQ) can produce fengycin and surfactin, but the other three stains only synthesized surfactin. These results indicated the production of fengycin need sfp, degQ and degS/degU two-component system. The further result of EMSA showed that the DegU can bind to the promotor region of fengycin. These study indicated that degQ can regulate the synthesis of fengycin through two component system degS/degU.
Key words: Bacillus subtilis; lipopeptide;fengycin；degQ；degU
目 录
摘要1
关键词1
Abstract1
Key words1
引言1
1 材料与方法2
1.1 材料 2
1.2 方法 3
1.2.1 BS168 degU基因突变菌株的构建3
1.2.2 BS168相关突变型菌株的脂肽类化合物的提取及质谱分析6
1.2.3 BS168相关突变型菌株的脂肽化合物粗提取物平板对峙实验7
1.2.4 利用EMSA实验检测DegU蛋白结合Fengycin启动子的实验7
2 结果与分析8
2.1 BS168△degU突变型菌株的验证结果8
2.2 相关菌株抑菌活性的检测9
2.3 脂肽类化合物粗提物的质谱分析10
2.4 EMSA结果证明degU蛋白能结合到fengycin的启动子部位11
3 讨论 12
致谢12
参考文献13
degQ调控Fengycin合成机理研究
芽孢杆菌属（Bacillus）中的某些种，例如枯草芽孢杆菌（B. subtilis）和解淀粉芽孢杆菌（B. amyloliquefaciens）等等，是研究植物根围促生细菌（Plant growth-promoting rhizobacteria，PGPR）的典型代表。这类有益细菌通过成功定殖到植物根际、体表或体内，与病原菌竞争生存空间和营养物质，而且可以分泌抗菌物质以抑制病原菌生长，并且同时诱导植物产生系统抗性抵御病原菌入侵，从而起到防治植物病害的作用。由于芽孢杆菌属细菌既能产生丰富的抗菌物质，还能形成适应性和抗逆性强的芽孢，所以成为了最具防病潜力与应用价值的一类生防菌。目前，许多芽孢杆菌菌株都被开发成商品化的生防制剂，比如GB03和MBI600等。拜耳公司开发的两种芽孢杆菌类生物杀菌剂产品Kodiak®Concentrate和Yield Shield®Concentrate Biological Fungicides，对镰刀菌属（Fusarium spp.）和丝核菌属（Rhizoctonia spp.）等植物病原真菌具有良好的防治效果。