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

摘要:

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3037 bp。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（0.8 mol/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。

Abstract:

Abstact: [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.