球孢白僵菌高渗适应性相关基因Bbmpd的克隆与表达分析  

Cloning and expression of the osmotic tolerance gene Bbmpd of Beauveria bassiana

作　　者：罗志兵[1] 金凯[1,2] 张永军[1] 武增强[1] 裴炎[1] 

机构地区：[1]西南大学生物技术中心,农业部生物技术与作物品质改良重点实验室,重庆400716 [2]重庆大学生物工程学院基因工程研究中心,重庆400030

出　　处：《微生物学报》2010年第6期724-728,共5页Acta Microbiologica Sinica

基　　金：国家高技术发展计划项目(2006AA10A212);重庆市自然科学基金项目(CSTC2009BB1125);西南大学博士基金项目(SWUB2008072)~~

摘　　要：【目的】克隆与球孢白僵菌(Beauveria bassiana)的高渗适应性相关基因,并对其功能进行分析,以揭示球孢白僵菌对高渗等逆境适应的分子机理。【方法】利用YADE法克隆T-DNA的侧翼序列并进行基因组步行,获得突变基因的全长及上游序列;利用RT-PCR技术分析突变基因的表达特性以及与Bbhog1的关系;采用同源重组技术敲除Bbmpd基因。【结果】克隆得到插入突变基因及其上、下游序列全长3037bp。该基因与编码球孢白僵菌的1-磷酸甘露醇脱氢酶基因相似性为98%。Bbmpd的表达受高渗环境(0.8mol/L NaCl)的诱导,受Bbhog1信号途径的激活调节,Bbhog1缺失导致Bbmpd表达下调。Bbmpd缺失突变体在高渗胁迫下的生长受到明显抑制。Bbmpd缺失不影响球孢白僵菌在查氏培养基上的生长和产孢。【结论】由T-DNA突变体克隆了编码球孢白僵菌1-磷酸甘露醇脱氢酶基因Bbmpd,该基因的表达受高渗环境的诱导和Bbhog1的调控,与球孢白僵菌高渗适应性相关。[Objective ] We isolate the gene involved in osmotolerance of Beauveria bassiana for investigation the adaptation mechanism of the fungus to adversity. [Methods]T-DNA tagging and genomic DNA walking were carried out by using Y-shaped adaptor dependent extension （YADE） method. RT-PCR was used to analyze the transcription profile of Bbmpd. [Results]A gene （Bbmpd） encoding mannitol 1-phosphate dehydrogenase in B. bassiana was identified. The transcription of Bbmpd was induced by osmostress （0. 8 mol /L NaCl） in wild type strain,while significantly decreased in Bbhog1 mutant,indicating that the transcription of Bbmpd was regulated by Hog1 pathway in B. bassiana. Bbmpd-disrupted mutants showed sensitive to osmostress on Czapek＇s medium containing 0. 8 mol /L NaCl. However,the growth and sporulation of the Bbmpd-disrupted mutants in vitro were not significantly different from that of wild type strain. [Conclusion]Mannitol 1-phosphate dehydrogenase gene was isolated by T-DNA tagging,and was found to be involved in the osmostress of B. bassiana. The transcription of Bbmpd was induced by osmostress and regulated by Hog1 pathway.

关 键 词：球孢白僵菌 1-磷酸甘露醇脱氢酶 高渗胁迫 YADE 

分 类 号：S476.12[农业科学—农业昆虫与害虫防治]