mlnB基因敲除对解淀粉芽孢杆菌W1杀螨活性的影响

Effect of mlnB gene knockout on the acaricidal activity of Bacillus amyloliquefaciens W1

【目的】研究拟通过构建W1菌株突变体及其杀螨抑菌活性检测以从分子水平探究并验证此菌株的杀螨机理,为二斑叶螨的防治提供一种新的微生物资源和防治策略,为后续开发利用更多微生物资源奠定基础,也为新型微生物杀螨剂的研发与应用提供理论基础。【方法】前期对解淀粉芽孢杆菌W1的次级代谢产物的活性化合物进行研究,已确定其对二斑叶螨的杀虫活性物质为二酮哌嗪类,大环内酯类及巨杆菌素类等化合物。大环内酯类化合物中以大环内酯A为主要杀螨活性物质,而大环内酯A是由mln基因簇调控合成。通过构建敲除整合载体pBS-mlnB-kan^(R)转化导入W1菌株自然感受态细胞等操作,获得了其杀螨活性化合物——大环内酯生物合成缺陷突变体W1△mlnB::kan^(R),利用PCR扩增及产物测序和HPLC-MS/MS技术证实敲除突变体的成功构建,并检测其杀螨抑菌活性。【结果】mlnB基因的敲除不仅导致W1菌株无法合成大环内酯A,还削弱了其对大白菜软腐病菌、柑橘溃疡病菌、大白菜黑腐病菌及茄科青枯菌的拮抗活性,相比W1野生型菌株二斑叶螨的致死率73.33%~77.22%,大环内酯合成缺陷突变体的杀螨活性也下降了46.66%~50.55%。【结论】解淀粉芽孢杆菌W1菌株合成的大环内酯A具有杀二斑叶螨活性,同时还能抑制大白菜软腐病菌、柑橘溃疡病菌、大白菜黑腐病菌以及茄科青枯病菌。大环内酯A合成受阻后,W1菌株的活性均明显地被削弱。

【Objective】This study intended to explore and verify the acaricidal mechanism of the strain W1 at the molecular level by constructing the mutant of strain W1 and detecting its acaricidal and antibacterial activities,so as to provide a new microbial resource and control strategy for the prevention and control of two-spotted spider mites,which would lay a foundation for the subsequent development and utilization of more microbial resources,and also provide a theoretical basis for the development and application of new microbial acaricides.【Method】The active compounds of secondary metabolites of B.amyloliquefaciens W1 were studied in the early stage,and it was confirmed that the acaricidal active substances were diketopiperazines,macrolides,bacimethrins and other compounds.Macrolactin A was the main acaricidal active substance in macrolide compounds,and synthesized by the mln gene cluster.By constructing knockout integration vector pBS-mlnB-kan^(R) and transforming it into natural competent cells of the W1 strain,the macrolactin A biosynthesis-defective mutant W1ΔmlnB::kan^(R) was obtained.The successful construction of the knockout mutant was confirmed by PCR amplification,product sequencing and HPLC-MS/MS technology,and its acaricidal and bacteriostatic activities were detected.【Result】The knockout of the mlnB gene not only resulted in the inability of stain W1 to biosynthesize macrolactin A,but also weakened its antagonistic activity against pathogens causing soft rot of cabbage,citrus canker,black rot of cabbage,and bacterial wilt of Solanaceae.Compared with 73.33%-77.22% of the leathality of the W1 wild-type strain,the acaricidal activity of the macrolactin A biosynthesis defective mutant was decreased by 46.66%-50.55%.【Conclusion】Macrolactin A synthesized by B.amyloliquefaciens W1 strain had the activity of killing two-spotted mites,and can inhibit the activity of pathogenic bacteria of Erwinia carotovora subsp.carotovora,Xanthomonas citri subsp.citri,Xanthomonas campestris pv.campestri and Ralstonia solanacearum.After the synthesis of macrolactin A was blocked,the activity of W1 strain was obviously weakened.