腺苷酸核糖基化因子BbarfA介导白僵菌孢子萌发和毒力

摘要

[目的]探究腺苷酸糖基化因子ARF在球孢白僵菌(Beauveria bassiana)中存在种类及生物学功能.[方法]利用BLASTp搜索球孢白僵菌非冗余蛋白数据库,鉴定ARF并进行聚类分析,结合表达分析、反义抑制、超量表达野生型基因和GTP解离位点与结合位点突变的基因,解析其中1个ARF与白僵菌发育分化、逆境胁迫反应和毒力的关系.[结果]球孢白僵菌中存在至少6个ARF或类似蛋白,分别聚类于酵母、人类ARF及其类似蛋白的不同类群.其中BBA 01574与人类的ARF3、ARF4和ARF5聚为一类,命名为BbarfA.BbarfA在成熟的分生孢子和球形膨大时期表达明显高于芽管伸长期.反义抑制BbarfA加速了孢子萌发,提高了菌株毒力,而超量表达BbarfA和点突变GTP解离区域的BbarfA则延迟了孢子萌发速度,降低了菌株毒力.尽管BbarfA转录受高盐、高渗、氧化和高温胁迫的诱导,但遗传修饰的转化子与野生菌株对上述胁迫反应的敏感性无明显差异.[结论]BbarfA介导分生孢子萌发和毒力.%[Objective] To understand the number of ADP-ribosylation factor (ARFs) and their roles in Beauveria bassiana.[Methods] ARFs were identified by BLASTp searching against the database of B.bassiana proteins and analyzed using molecular phylogenetics.Role of an ARF in fungal growth,stress responses and virulence was characterized by analysis of the gene transcription pattem,and investigation of the genetically modified B.bassiana strains.[Results] At least six ARFs and ARF-like proteins (ARFLs) were identified in B.bassiana,which were distributed in different groups of yeast Saccharomyces cerevisiae and human Homo sapiens ARFs and ARFLs.One of ARFs,BBA_01574,designated BbarfA,was clustered in the group of human ARF3,ARF4 and ARF5.Transcription levels of BbarfA were obviously higher in the mature conidia or during the isotropic growth (swelling) phase of conidia than those during germ tube elongation phase.Antisense inhibition of BbarfA accelerated conidial germination and resulted in an increase in fungal virulence,whereas overexpression of BbarfA and the gene with site-mutation in GTP-binding sequences (BbarfAQ711) caused the opposite phenotypes.Although expression of the gene was induced by high salt,osmotic,oxidative and high temperature stresses,no obvious difference was noted in sensitivities to these adverse stresses in all the genetically modified transformants,which included strains suppressing (by antisense inhibition) or overexpressing BbarfA,overexpressing the genes with site-mutation in GTP-dissociating (BbarfAD26G) or GTP-binding (BbarfAtQ711) sequences,and the wild type strains.[Conclusion] ADP-ribosylation factor,BbarfA,was involved in conidial germination and virulence in B.bassiana.