深绿木霉(Trichoderma atroviride)T23降解有机磷农药敌敌畏转运蛋白TaPdr2基因的克隆与功能预测分析
Cloning,Function Prediction of ABC Transporter (TaPdr2) Related to the Degradation of Dichlorvos in Trichoderma atroviride T23

根据前期研究,进行了对比分析后明确了深绿木霉中7个敌敌畏耐受相关ABC转运蛋白,其中TaPdr2基因是在短时间内对敌敌畏胁迫应答最为明显的一个基因。本研究通过简并引物同源克隆,在深绿木霉T23中克隆出TaPdr2基因,并对其全长序列进行了测序;明确了其外显子序列和内含子序列;构建了TaPdr2的系统发生树,发现深绿木霉TaPdr2与木霉属其他种PDR5亚家族ABC转运蛋白有很高的同源性,通过生物信息学分析后预测TaPdr2蛋白的ATPase及部分跨膜结构域序列相对保守且具有11个跨膜结构域;通过构建TaPdr2的敲除载体pC1300qh-F,为后续研究TaPdr2蛋白对麦角甾醇和鞘脂类物质含量影响的研究奠定了基础。
Dichlorvos is one of the most widely used organophosphate pesticides.The tolerance to dichlorvos is essential for the application of Trichoderma for both biological remediation and integrated pest management.Based on former RNA-seq transcriptome analysis and RT-qPCR analysis of Trichoderma atroviride T23,7 ABC transporter genes related to dichlorvos tolerance were confirmed,and particularly,the expression of one PDR-type ABC transporter gene,designated TaPdr2,was up-regulated over 3000 fold after 6 h of dichlorvos stress.In this paper,ABC transporter gene TaPdr2 in T.atroviride,was cloned and sequenced,after which phylogenetic analysis and prediction of the ABC transporter protein was conducted.The homology between the TaPar2 and ABC transporter proteins of PDR5 sub-family in other Trichoderma species is up to 91%.Through bioinformatics analysis,the ATPase of TaPdr2 and sectional sequence of transmembrane domain are conserved,and it has 11 transmembrane domains.Moreover,TaPdr2-deleted mutant was obtained by ATMT transformation and gene knockout.This research presented a sound foundation for the consequent study of the effect of TaPdr2 on onergosterol and sphingolipid contents.