皱环球盖菇实时荧光定量PCR内参基因的筛选  

Screening of the reference genes for quantitative real-time PCR analysis of gene expression in Stropharia rugosoannulata

作　　者：闫慧文 关体坤 张国庆[1] 陈青君[1] YAN Huiwen;GUAN Tikun;ZHANG Guoqing;CHEN Qingjun(School of Plant Science and Technology,Beijing University of Agriculture,Bejing 100096,China)

机构地区：[1]北京农学院植物科学技术学院,北京100096

出　　处：《菌物学报》2023年第6期1298-1310,共13页Mycosystema

基　　金：北京市食用菌创新团队(BAIC03-05)。

摘　　要：本研究以皱环球盖菇Stropharia rugosoannulata不同发育时期(菌丝期、原基期、幼菇期、小菇期、成熟期)、不同组织部位(菌盖、菌柄、菌褶)和不同颜色菌盖(红色、黄色、白色)为试验材料,选择10个内参基因(ACT、GAPDH、TEF、RPL4、PGI、PGM、RPB2、β-TUB、α-TUB、UBQ)并设计跨内含子的引物,采用实时荧光定量PCR(RT-qPCR)技术进行扩增,利用geNorm、NormFinder、BestKeeper和ΔCt算法进行表达稳定性分析以及综合评价算法ReFinder进行加权评比,最终筛选适宜各类样本的内参基因。根据内参基因稳定性的最终排名,最适宜作为不同颜色菌盖内参基因的组合是UBQ和GAPDH,最不适宜的是ACT、PGI和TEF;最适宜作为子实体不同组织部位内参基因的组合是UBQ和RPB2,最不适宜的是ACT、RPL4和TEF;最适宜作为不同发育时期内参基因的组合是ACT和RPB2,最不适宜的是GAPDH、α-TUB和β-TUB。Vegetative mycelia,primordium,button and pilei(red,yellow and white),stipes and gills of young and mature fruiting bodies of Stropharia rugosoannulata were used as test materials.Ten reference genes(ACT,GAPDH,TEF,RPL4,PGI,PGM,RPB2,β-Tub,α-Tub,UBQ) were selected and primers across introns were designed.Quantitative real-time PCR(RT-qPCR) was used for amplification.GeNorm,NormFinder,BestKeeper and ΔCT algorithms were used for expression stability analysis,and ReFinder was used for weighted evaluation.Finally,reference genes suitable for various samples were screened.According to the final ranking of the stability of reference genes,UBQ and GAPDH were the most suitable combination of reference genes for different color pileus,while ACT,PGI and TEF were the least suitable.UBQ and RPB2 were the most suitable reference genes for different parts of fruiting bodies,while ACT,RPL4 and TEF were the least suitable.ACT and RPB2 were the most suitable combinations for reference genes for different developmental stages,while GAPDH,α-TUB and β-TUB were the least suitable.

关 键 词：皱环球盖菇 内参基因 RT-QPCR 发育时期 组织部位 菌盖颜色 

分 类 号：S646[农业科学—蔬菜学]