基于转录组测序的姬松茸镉胁迫下内参基因筛选  

Screening of reference genes under cadmium stress in Agaricus brasiliensis based on transcriptome sequencing

作　　者：黄在兴 陈华[3] 翁伯琦 刘斌[2] 王义祥 刘朋虎[1,4] Zaixing Huang;Hua Chen;Boqi Weng;Bin Liu;Yixiang Wang;Penghu Liu(China National Engineering Research Center of JUNCAO Technology,Fujian Agriculture and Forestry University,Fuzhou 350002,Fujian Province,China;College of Agriculture,Guangxi University,Institute of Mushroom,Nanning 530004,Guangxi Province,China;Scientific Research Office of Fujian Academy of Agricultural Sciences,Fuzhou 350003,Fujian Province,China;Fujian Province Key Laboratory of Agro-Ecological Processes in Hilly Red Soil,Fuzhou 350003,Fujian Province,China)

机构地区：[1]福建农林大学国家菌草工程技术研究中心,福建福州350002 [2]广西大学农学院,广西大学食用菌研究所,广西南宁530004 [3]福建省农业科学院科研处,福建福州350003 [4]福建省红壤山地农业生态过程重点实验室,福建福州350003

出　　处：《微生物学报》2021年第11期3458-3470,共13页Acta Microbiologica Sinica

基　　金：中央引导地方科技发展专项(2020L3030);福建省科技厅农业引导性(重点)项目(2020N0007);福建省红壤山地农业生态过程重点实验室开放课题(Aephrs-202001);福建农林大学学科交叉融合项目(XKJC-71202103C)。

摘　　要：【目的】筛选不同实验条件下,尤其是镉胁迫下的姬松茸内参基因,为研究姬松茸镉富集相关基因功能研究奠定基础。【方法】根据不同浓度Cd(0、2、5 mg/L)胁迫下菌丝中转录组表达量数据,筛选出18个候选基因,设计了30对引物;根据引物扩增的特异性、扩增效率初步筛选出来自不同基因的5对引物;运用qRT-PCR检测了5个基因在不同浓度Cd胁迫下的菌丝样品、原基、菌柄和菌盖中的Ct值,利用geNorm、NormFinder和BestKeeper对这些基因的表达稳定性进行评价。【结果】SGT2和STK是菌丝阶段Cd胁迫下最稳定的2个候选内参基因,GAPDH是不同组织中最稳定的内参基因,SGT2、STK和GAPDH是所有实验条件下最稳定的3个内参基因。【结论】在姬松茸镉胁迫条件下,本研究筛选出的SGT2和STK比常用的内参基因表现出更强的稳定性。[Objective]The aim of this study was to screen the reference genes of Agaricus brasiliensis under different experimental conditions,especially under cadmium(Cd)stress,and to lay the foundation for studying the functional studies of cadmium enrichment-related genes in A.brasiliensis.[Methods]We screened 18 candidate genes and designed 30 primer pairs based on the transcriptomes of mycelia under the stress of different concentrations of Cd(0,2,5 mg/L).According to the specificity and amplification efficiency of primers,5 pairs of primers from different genes were selected.The Ct values of mycelia,primordium,stipe,and pileus of 5 genes under different concentrations of cadmium stress were detected by QRT-PCR,and the expression stability of these genes was evaluated by geNorm,NormFinder,and BestKeeper.[Results]The results showed that Small glutamine-rich tetratricopeptide repeat-containing protein 2(SGT2)and Serine/threonine-protein kinase(STK)were the two most stable candidate reference genes under Cd stress in the mycelial stage,GAPDH was the most stable reference gene in different tissues and SGT2,STK and GAPDH were the three most stable reference genes under all experimental conditions.[Conclusion]Under Cd stress conditions in A.brasiliensis,SGT2 and STK screened in this study were more stable compared to the commonly used reference genes.

关 键 词：巴西蘑菇 镉胁迫 不同组织 荧光定量PCR 

分 类 号：X173[环境科学与工程—环境科学] S646[农业科学—蔬菜学]