通过抑制消减杂交技术分离球孢白僵菌热激响应基因  

Identification of Heat-Responsive Genes in Beauveria bassiana by Suppression Subtractive Hybridization

作　　者：李静馨[1] 应盛华[1] 冯明光[1] 

机构地区：[1]浙江大学微生物研究所,杭州310058

出　　处：《中国生物防治学报》2011年第3期308-315,共8页Chinese Journal of Biological Control

基　　金：国家自然科学基金(30971960)

摘　　要：高温等逆境因素会影响生防真菌——球孢白僵菌Beauveria bassiana的田间应用效果。为了深入了解生防真菌热胁迫响应的分子机理,本研究采用抑制消减杂交技术构建了球孢白僵菌热胁迫下的差异表达cDNA文库,筛选出28个热激响应上调基因。通过基因比对分析,大部分基因与已知功能基因如物质运输、基础代谢及胁迫响应类基因等具高度同源性。从中挑选12个基因利用荧光实时定量PCR分析了菌株在40℃下热激15、30、60、120 min后的相对表达水平,结果显示这些基因的表达模式随热激时间和基因不同而异,显示菌体在热激的不同阶段依次启动不同的应激方案来应对热激胁迫。这些热激响应基因为揭示球孢白僵菌的耐热机制和通过遗传改造提高菌株耐热力奠定基础。Beauveria bassiana,a well-known entomopathogenic fungus,plays an important role in insect control.The efficacy of B.bassiana application in the field is affected by some abiotic stresses such as high temperature during summer but mechanisms involved in the fungal tolerance to thermal stress are unknown.In this study,a suppression subtractive cDNA library consisting of the B.bassiana genes differentially expressed under a thermal stress was constructed.Twenty-eight putative heat-responsive genes were screened from the SSH library and found to be involved in the processes of substance transport,general metabolism and stress responses.Among them,12 genes were examined for their expression levels after 15,30,60,and 120 min of thermal stress at 40 ℃,respectively,by quantitative real-time PCR.All the examined genes were heat inducible and showed expression patterns differing from one to another or varying with the time length of heat stress.The results indicated that different genes are involved in the fungal responses to the heat stress of different stages.The heat-responsive genes found in this study are the targets for elucidating the fungal thermotolerance mechanism and have potential use in improving genetically the thermal stability of B.bassiana.

关 键 词：球孢白僵菌 热胁迫 抑制消减杂交 荧光实时定量PCR 

分 类 号：S476.12[农业科学—农业昆虫与害虫防治] Q785[农业科学—植物保护]