影响球孢白僵菌穿透昆虫体壁的BbMPK1 MAPK下游基因研究

项目名称： 影响球孢白僵菌穿透昆虫体壁的BbMPK1 MAPK下游基因研究

项目编号： No.30871668

项目类型： 面上项目

立项/批准年度： 2009

项目学科： 轻工业、手工业

项目作者： 张永军

作者单位： 西南大学

项目金额： 32万元

中文摘要： Bbmpk1信号途径是调节球孢白僵菌附着胞形成和穿透昆虫体壁等侵染过程的重要途径。为揭示该信号途径调节病原菌侵染昆虫的分子基础，课题组分别建立了野生菌株和Bbmpk1突变体在侵染和附着胞形成时期的SSH文库，筛选了13个可能与昆虫免疫相关的基因，31个在附着胞形成时期可能受Bbmpk1调控的下游基因；比较分析了野生菌株和Bbmpk1突变体穿出昆虫体壁过程中的转录组，筛选了1440个表达差异的contig。利用基因破坏和回复互补等方法分析了2个附着胞SSH文库中筛选的差异克隆，其中Bbrpdp编码蛋白与金龟子绿僵菌Regulatory P domain-containing protein有一定相似性(identities=67%)，该基因依赖于氮源调控球孢白僵菌生长和分生孢子产生；Bbarf1编码基因与真菌ARF (ADP-Ribosyation Factor)高度同源，该基因在不同营养、胁迫条件以及分生孢子萌发的不同阶段均表达，但在昆虫血淋巴诱导、高渗、氧化及高温胁迫条件下以及孢子膨大时期的表达水平明显升高，推测该基因可能与侵染致病、逆境胁迫以及与细胞膜的迅速扩张及曲度有关。

中文关键词： 昆虫病原真菌；球孢白僵菌；MAPK；侵染；附着胞

英文摘要： Our previous results showed that Bbmpk1 MAPK signal pathway play a crucial role in redulation of appressorium differentation and penetration of insect cuticle in the insect fungal patgogen Beauveria bassiana. To understand the molecular mechanism of invasion controlled by Bbmpk1 pathway,two suppressive subtractive hybridization (SSH) libraries were generated comparing mutant and wild-type transcripts isolated during appressorium formation and invasion processes.Thirteen genes and 31 genes, which are involved in insect immune responses and appressorium differentation,respectively, were determined to be expressed in the wild-type treatments but either significantly reduced or not expressed in the mutant treatments.Comparative transcriptome analysis indicated that expression values of 1440 contigs were significantly different in the wild-type and Bbmpk1 mutant strains during penetration of insect cuticle from the inside-out.Two target genes,Bbrpdp1 and Bbarf1,screened from the SSH library of appressorium formation,were characterized using gene disruption and reverse complementation methods.The putative amino sequence of Bbrpd1 displayed 67% of identity with a regulartory P domain-containing protein from Metarhizium anisopliae.This gene controlled growth and conidiation depending on nitrogen sources.Bbarf1 showed high homologous to fungal ARFs (ADP-ribosylation factors).qRT-PCR analysis revealed that Bbarf1 were expressed under various nutritional and stress conditions,and at different development stages of conidia.But expression levels of Bbarf1 were significantly increased when the insect hemolymph was as sole nutrition source, or fungal culture were exposed to osmotic or oxdative stresses or high temprature,and during conidial swelling of germination. This result suggested that Bbafr1 might be involved in pathogenecity, abiotic stress response, and membrane fast expansion and curvature of cell membrane.In addition, no Bbarf1 disruption mutant was screened from over 800 transforants using homologous recombination strategy,suggesting that Bbarf1 might be essential for the fungal survival.

英文关键词： entomopathogenic fungi; Beauveria bassiana; MAPK; invasion; appressorium