金龟子绿僵菌fluG基因的敲除及对产孢的影响

Effects of fluG Gene Knockout on Sporulation in Metarhizium anisopliae

丝状真菌的fluG基因参与调控分生孢子的生成,然而在昆虫病原真菌金龟子绿僵菌中fluG的作用鲜有研究报道。本研究利用DNA同源重组方法,构建敲除fluG的金龟子绿僵菌突变株,分析突变株的产孢特性。以苯菌灵抗性基因ben作为筛选标记,fluG基因侧翼序列作为同源臂,构建了打靶载体pDHt/sk-fluG-Ben。利用PEG介导将打靶载体转化金龟子绿僵菌的原生质体,获得了苯菌灵抗性转化子。根据靶基因和标记基因检验,确定获得了敲除fluG的金龟子绿僵菌突变株。表型分析显示,fluG突变株继代培养5代仍保持苯菌灵抗性,菌落呈疏松毛絮状,生长相较野生型明显缓慢,不能或者仅能产生极少量分生孢子。说明fluG基因的敲除影响了菌株生长发育,并阻止了分生孢子形成,是金龟子绿僵菌产孢调节的重要基因。本研究为阐述金龟子绿僵菌产孢调控机制提供依据。

The fungal gene fluG is involved in the regulation of conidial generation.However,the role of fluG in the entomopathogenic fungus Metarhizium anisopliae is rarely reported.To investigate the role of fluG in M.anisopliae,we constructed a mutant strain of M.anisopliae with fluG knocked out by DNA homologous recombination to analyze its effect on the sporulation characteristics.The target vector pDHt/sk-fluG-Ben was constructed by using benomyl resistance gene ben as selective marker and sequence of fluG gene flanks as homologous arms.The target vector was transformed into the protoplasts of M.anisopliae by PEG mediating,and the resistant transformants of benomyl were obtained.According to the test of target genes and marker genes,the mutant strains of M.anisopliae with fluG knocked out were determined.Phenotypic analysis showed that the fluG mutant still maintained benomyl resistance after 5 generations of subculture.The mutant colonies looked loose and flocculent,grew significantly slower than the wild type,and could not or only produce a very small number of conidia.These results indicate that the knockout of fluG gene affect the development of the strain and prevent conidia formation,showing fluG an important gene in the regulation of spore-producing of M.anisopliae.This study establishes the basis for further elaborating the regulation mechanism of M.anisopliae conidiation.