布莱克韦尔虫草实时荧光定量逆转录PCR内参基因的筛选  

Screening of reference genes for real-time quantitative reversetranscription PCR in Cordyceps blackwelliae

作　　者：李佳妮 张姝[1,2] 张永杰 LI Jiani;ZHANG Shu;ZHANG Yongjie(School of Life Science,Shanxi University,Taiyuan 030006,Shanxi,China;Key Laboratory of Microbial Resources Collection and Preservation,Ministry of Agriculture and Rural Affairs,Beijing 100081,China)

机构地区：[1]山西大学生命科学学院,山西太原030006 [2]农业农村部农业微生物资源收集与保藏重点实验室,北京100081

出　　处：《微生物学通报》2025年第1期219-229,共11页Microbiology China

基　　金：农业农村部农业微生物资源收集与保藏重点实验室开放基金(KLMRCP2023-01);山西省基础研究计划(202203021221036)。

摘　　要：【背景】实时荧光定量逆转录PCR(real-time quantitative reverse transcription PCR,RT-qPCR)常用于分析基因表达,但选择合适的内参基因,对分析结果的稳定性非常重要。布莱克韦尔虫草是一种具有开发价值的虫草,研究其功能基因表达具有重要意义。【目的】筛选获得布莱克韦尔虫草不同生长阶段(液体发酵菌丝体、小麦粒培养基菌丝体及子实体)最稳定的内参基因。【方法】选取8个候选内参基因(18S rRNA、gapdh、β-tubulin、cyclophilin A、γ-actin、rpl10、tef1α和ubiquitin),采用RT-qPCR技术进行扩增,结合geNorm、NormFinder、BestKeeper、ΔC_(t)等算法,评估这些基因在布莱克韦尔虫草不同生长发育阶段的表达稳定性。【结果】相比其他基因,γ-actin和18S rRNA在不同生长发育阶段的表达稳定性最好,可作为布莱克韦尔虫草基因表达分析的内参基因。【结论】本研究筛选出了布莱克韦尔虫草RT-qPCR的内参基因,为深入研究生长发育过程中的基因表达规律奠定了基础。[Background]Gene expression is often studied by real-time quantitative reverse transcription PCR(RT-qPCR),the stability of which largely depends on the choice of reference genes.Cordyceps blackwelliae is a fungus of development potential,and it is thus vital to understand the expression patterns of functional genes in this fungus.[Objective]To identify the optimal reference genes that express stably across different developmental stages(mycelia from liquid culture,mycelia from wheat grain media,and fruiting bodies)of C.blackwelliae.[Methods]We utilized RT-qPCR to investigate the expression of eight reference genes(18S rRNA,gapdh,β-tubulin,cyclophilin A,γ-actin,rpl10,tef1α,and ubiquitin).The expression stability of these genes across different developmental stages was evaluated using geNorm,NormFinder,BestKeeper,and∆C_(t) algorithms.[Results]The results indicated thatγ-actin and 18S rRNA exhibited more stable expression than the other genes,making them suitable reference genes for the transcriptional analysis of functional genes in C.blackwelliae.[Conclusion]This study successfully identified appropriate reference genes for RT-qPCR in C.blackwelliae,aiding future research on gene expression dynamics.

关 键 词：布莱克韦尔虫草 内参基因 RT-QPCR 

分 类 号：S567.3[农业科学—中草药栽培]