脐孢木霉菌Trichoderma brevicompactum 0248 tri5基因片段的克隆及表达分析  

Cloning and Expression Analysis of tri5 Gene Fragment from Trichoderma brevicompactum 0248

作　　者：詹晓奂[1] 申屠旭萍[1] 刘卫平[1] 马正[1] 俞晓平[1] 

机构地区：[1]中国计量学院生命科学学院/浙江省生物计量及检验检疫技术重点实验室,杭州310018

出　　处：《中国生物防治学报》2013年第4期555-560,共6页Chinese Journal of Biological Control

基　　金：浙江省自然科学基金项目(LY12C14012);浙江省公益技术研究项目(2010C32063);浙江省科技创新团队项目(2009R500273);杭州市科技计划项目(20120232B17)

摘　　要：脐孢木霉菌Trichoderma brevicompactum能合成木霉素等单端孢霉烯类化合物,而基因tri5编码的单端孢霉二烯合酶是合成途径中的关键酶,它催化反式法呢基焦磷酸(tFPP)合成单端孢霉二烯。为分析基因tri5与木霉素合成的关系,本研究从脐孢木霉菌0248中克隆了基因tri5片段,并运用实时荧光定量PCR技术分析了基因tri5在不产木霉素培养基和产木霉素培养基中的差异表达。在相同培养时间下,基因tri5在不同产木霉素状态间的表达量差异显著;在培养96 h时基因tri5在2种培养基中均达到最大值,基因tri5在产木霉素状态下的表达量是不产木霉素状态下的107.18倍。通过检测产木霉素培养基中基因tri5表达量与木霉素含量的变化,结果显示木霉素的含量随着基因tri5表达量的增加而不断增加,在培养96 h时达到最大值118.71 mg L 1,推测基因tri5表达量与木霉素合成相关。该研究为基因tri5功能的进一步研究和对脐孢木霉菌0248进行基因工程菌改造奠定了理论基础。The filamentous fungus Trichoderma brevicompactum was proved to produce the trichodermin, one of trichothecenes. Trichodiene synthase, which is encoded by the gene tri5, catalyses the conversion of famesyl pyrophosphate to trichodiene in Fusarium spp. and is the key enzyme in trichothecenes biosynthesis. However, the biosynthetic pathway for trichothecene metabolism is keeping unclear in T. brevicompactum. In order to understand the functions of gene tri5 cloned from T. brevicompactum 0248, which was isolated from garlic Allium sativum in our previous work, the relative expression levels of this gene in two different cultural media were evaluated by the real-time quantitative PCR. The results showed that significantly different expression of gene tri5 occurred in trichodermin producing and non-producing media. The relative expression level of gene tri5 in the trichodermin producing medium was 107.18 times higher than that in non-producing medium after 96 hours incubation, and the production of trichodermin was enhanced with the increasing expression level of gene tri5, which reached to the maximum of 118.71mg-L-1. The results indicated that tri5 gene may have a close relationship with trichodermin metabolism in T. brevicompactum.

关 键 词：脐孢木霉菌 基因tri5 木霉素 基因克隆 实时荧光定量PCR 

分 类 号：S476[农业科学—农业昆虫与害虫防治] Q344.13[农业科学—植物保护]