胶孢炭疽菌热休克蛋白基因Cghsp90的RNAi突变体获得与功能分析  

Acquisition and Functional Analysis of RNAi Mutants of Heat Shock Protein Gene Cghsp90 from Colletotrichum gloeosporioides

作　　者：苏初连 蒲金基 康浩[2] 杨石有[1] 刘晓妹 张贺 Su Chulian;Pu Jinji;Kang Hao;Yang Shiyou;Liu Xiaomei;Zhang He(Institute of Tropical Agriculture and Forestry,Hainan University,Haikou,570228;Environment and Plant Protection Institute,Chinese Academy of Tropical Agricultural Sciences,Haikou,571101)

机构地区：[1]海南大学热带农林学院,海口570228 [2]中国热带农业科学院环境与植物保护研究所,海口571101

出　　处：《基因组学与应用生物学》2019年第12期5450-5457,共8页Genomics and Applied Biology

基　　金：国家重点研发计划(2017YFD0202107);国家自然科学基金(31460455);中国热带农业科学院基本科研业务费专项资金(1630042017019);海南省重大科技计划项目(ZDKJ2017003)共同资助

摘　　要：为揭示胶孢炭疽菌(Colletotrichum gloeosporioides)热休克蛋白基因Cghsp90在抗逆境胁迫中的功能,利用RNAi技术结合PEG介导的原生质体转化方法,获得了Cghsp90的RNAi突变体菌株,经实时荧光定量PCR(qRT-PCR)对野生型菌株和突变体菌株间Cghsp90基因差异表达分析以及NaCl、H2O2、SDS、Congo Red、18℃(Cold)和33℃(Hot)等非生物胁迫条件下野生型菌株和突变体菌株的胁迫耐受性分析。通过特异性引物检测鉴定、qRT-PCR分析,获得Cghsp90的RNAi突变体pSilent-1:Cghsp90-1和pSilent-1:Cghsp90-2;与野生型菌株相比,侵染过程中Cghsp90基因的表达量显著低于野生型菌株,8个致病相关基因显著下调表达;突变体菌株的产孢量下降、分生孢子畸形、萌发率下降;在非生物胁迫条件下,突变体菌株较野生型菌株更为敏感;在胶孢炭疽菌的分生孢子中研究Cghsp90基因的形态建成与萌发,对应变逆境胁迫以及调控致病相关基因等方面发挥重要的作用。To reveal the function of heat shock protein gene Cghsp90 of Colletotrichum gloeosporioides in stress resistance. The RNAi mutant isolate of Cghsp90 was obtained by using RNAi technique combined with PEG-mediated transformation. Wild-type isolate and mutant-isolates, in which the differential expression of Cghsp90 gene was analyzed by real-time quantitative PCR(q RT-PCR), were analyzed their tolerance under abiotic stress conditions such as NaCl, H2O2, SDS, Congo red, 18℃(Cold) and 33℃(Hot). The RNAi mutants(p Silent-1:Cghsp90-1 and p Silent-1:Cghsp90-2) of Cghsp90 were obtained with specific primer detection and qRT-PCR analysis. The wildtype isolation was the control, the results are as follows. The expression leves of Cghsp90 gene decreased significantly during the process of infecting the host. eight pathogenicity related genes decreased significantly in mutant-isolates.Compared with the wild-type isolate, the number of conidia producted and conidia germination rate by mutan-isolates, mutan-isolates were decreased, and abnormal conidia. The mutan-isolates was more sensitive than the wild-type isolates in abiotic stress. It was found that Cghsp90 gene with conidium of Colletotrichum gloeosporioides plays an important role in conidia morphogenesis, conidia germination, response to stress, and regulation of pathogenicity related genes.

关 键 词：胶孢炭疽菌 Cghsp90基因 RNAi突变体 q RT-PCR 

分 类 号：S432.4[农业科学—植物病理学]