利用枯草芽胞杆菌表达系统进行马铃薯抗细菌基因的筛选

Screening of potato antimicrobial genes using a Bacillus subtilis expression system

抗菌肽是植物先天免疫系统的重要组成部分,具有良好的广谱抗菌活性,因此作为抗生素的替代品有较好的应用前景。马铃薯野生种(Solanum demissum)作为重要的种质资源,含有丰富的抗性基因。本研究利用指示菌内置文库抗性基因筛选技术,将马铃薯野生种cDNA文库转入指示菌枯草芽胞杆菌内,筛选出两种具有广谱抗菌活性的抗菌肽SdR2332和SdR2770。课题组分别测定了它们的热稳定性、酶稳定性、紫外线稳定性、最低抑菌浓度(minimum inhibitory concentration,MIC)和溶血性,旨在明确这两种新抗菌肽的实际可应用性;同时通过测定抗菌肽处理后病原菌活性氧(reactive oxygen species,ROS)迸发量、病原菌细胞形态观察和基因表达分析来探究抗菌肽的抑菌机制及防效。结果表明,抗菌肽SdR2332和SdR2770均具有良好的广谱抗细菌活性,且在低浓度下即可致死病原菌;它们还具有较为稳定的理化性质和较低的溶血性。经抗菌肽SdR2332和SdR2770处理后的青枯菌(Ralstonia solancearum)出现了明显的活性氧迸发现象,电镜下观察发现其细胞有一定程度的皱缩和破损,这表明抗菌肽对病原菌有杀伤作用。在qRT-PCR分析中,接种马铃薯的植株中SdR2770表达量明显上调,推测可能与植物抗病相关。本研究结果为新抗菌肽的研发提供了一定的理论基础和参考价值。

Antimicrobial peptides(AMPs)are an important part of the innate immune system of plants,which have strong broad-spectrum antimicrobial activities.As an alternative of antibiotics,AMPs have good application prospects.As a major germplasm resource,Solanum demissum is abundant in resistance genes.In this study,the S.demissum cDNA library was transferred into the indicator bacterium Bacillus subtilis cells,and two antimicrobial peptides SdR2332 and SdR2770 with broad antibacterial activities were identified by resistance gene screening technology in the indicator bacteria built-in library.The thermal stability,enzyme stability,UV stability,minimum inhibitory concentration(MIC),hemolysis,reactive oxygen species(ROS)production of pathogens treated with antimicrobial peptide,and the control effect of the antimicrobial peptides on plants were determined,respectively.Our findings showed that the antimicrobial peptides SdR2332 and SdR2770 had excellent broad-spectrum antibacterial activities,and could kill pathogens at low concentrations;they also had stable physical and chemical properties and low hemolysis activity.After treatment with antimicrobial peptide,Ralstonia solancearum showed obvious ROS burst and the cells had a certain degree of shrinkage and damage under electron microscope observation,which indicated that the antimicrobial peptide had a toxic effect on the pathogen.Revealed by qRT-PCR analysis,the expression of SdR2770 in plants inoculated with R.solancearum was significantly up-regulated,which may be related to plant disease resistance.The results of this study provide a basis for the development of new antimicrobial peptides.