金针菇实时荧光定量PCR内参基因的筛选  

Screening of Reference Genes for qRT-PCR Amplification in Flammulina filiformis

作　　者：武晨剑 袁学文 宋淋浩 常明昌[1,2] 刘靖宇 邓冰[1,2] 孟俊龙 WU Chenjian;YUAN Xuewen;SONG Linhao;CHANG Mingchang;LIU Jingyu;DENG Bing;MENG Junlong(College of Food Science and Engineering,Shanxi Agricultural Universty,Taigu 030801,Shanxi,China;Shanxi Engineering Research Center of Edible Fungi,Taigu 030801,Shanxi,China)

机构地区：[1]山西农业大学食品科学与工程学院,山西太谷030801 [2]山西省食用菌工程技术研究中心,山西太谷030801

出　　处：《食用菌学报》2021年第1期30-39,共10页Acta Edulis Fungi

基　　金：国家自然科学基金(32072647);2019年山西省研究生教育创新资助项目(2019SY207);山西省煤基重点科技攻关项目(FT2014-03-01)。

摘　　要：基于金针菇(Flammulina filiformis)基因组和转录组的测序组装结果,选取13个内参基因(α-TUB、β-TUB、PGM、RPL19C、RPL6、CYC、18S rRNA、Vha68、ACT、GPD、PAS、TEF和VSN),通过设计跨内含子引物,分别对金针菇不同菌株(Fy331、Fw12、川金10、Fv296、金黄11和福建白金)菌丝和Fy331菌株不同发育阶段(菌丝、原基、幼嫩子实体和成熟子实体)样品进行实时荧光定量PCR(quantitative real time polymerase chain reaction,qRT-PCR)检测,并采用ΔCt算法和NormFinder、BestKeeper、GeNorm等软件对检测结果进行表达稳定性分析,以期筛选出在不同菌株菌丝和不同发育阶段中稳定表达的内参基因。结果表明:Vha68、TEF和β-TUB基因在不同菌株菌丝中表达稳定;Vha68、ACT和GPD基因在不同发育阶段样品中表达稳定。综合评价与qRT-PCR验证分析结果显示,Vha68和TEF、Vha68和ACT分别适合作为金针菇不同菌株菌丝、不同发育阶段qRT-PCR检测基因表达的最佳内参基因。Based on the genome and transcriptome data of Flammulina filiformis,13 genes,includingα-TUB,β-TUB,PGM,RPL19C,RPL6,CYC,18 S rRNA,Vha68,ACT,GPD,PAS,TEF and VSN were selected as candidate reference genes.Then the cross-intron primers of these genes were designed for real-time quantitative PCR(qRT-PCR)on different F.filiformis strains(Fy331,Fw12,Chuanjin10,Fv296,Jinhuang11 and Fujianbaijin)and different developmental stages of Fy331,respectively.The expression stability of each candidate reference gene was further analyzed byΔCt,NormFinder,BestKeeper and GeNorm.The results showed that Vha68,TEF andβ-TUB were stably expressed in mycelia of different strains;and Vha68,ACT and GPD were stably expressed at different developmental stages.Through comprehensive evaluation and qRT-PCR verification,Vha68 and TEF were selected as reference genes for expression in mycelia,and Vha68 and ACT were selected as reference genes for expression at different developmental stages.

关 键 词：金针菇 △Ct算法 NORMFINDER BestKeeper GENORM 

分 类 号：S646.15[农业科学—蔬菜学]