龟裂链霉菌M527荧光定量PCR内参基因的选择及其稳定性评估  

Selection of Suitable Internal Reference Genes for the Real-Time Quantitative PCR in Streptomyces rimosus M527 and Evaluation of Their Stabilities

作　　者：徐洁 胡叶锋 廖芷君 马正 俞晓平 XU Jie;HU Yefeng;LIAO Zhijun;MA Zheng;YU Xiaoping(Zhejiang Provincial Key Laboratory of Biometrology and Inspection&Quarantine/College of Life Sciences,China Jiliang University,Hangzhou 310018,China)

机构地区：[1]中国计量大学生命科学学院/浙江省生物计量及检验检疫技术重点实验室,杭州310018

出　　处：《中国生物防治学报》2020年第3期429-436,共8页Chinese Journal of Biological Control

基　　金：国家自然科学基金(31772213,31972320);浙江省杰出青年基金(LR17C140002)。

摘　　要：龟裂链霉菌Streptomyces rimosus M527是龟裂霉素(rimocidin)的生产菌,其对多种植物病原真菌具有较强的拮抗作用。为从转录水平分析菌株M527以及其抗性突变株中龟裂霉素的合成调控机制,荧光定量PCR内参基因的选择与稳定性评估显得尤为重要。本研究选用16SrRNAsr、hrd Bsr、rpoAsr、sigFsr、sigBsr、gyrBsr这6个基因作为候选内参基因,使用geNorm、NormFinder、BestKeeper三个软件分析在野生型龟裂链霉菌M527、高产龟裂霉素抗性突变株M527-GR7和低产龟裂霉素抗性突变株M527-GR21中候选内参基因的稳定性,筛选出稳定性最高的内参基因。结果显示,sigBsr基因在菌株M527及抗性突变株M527-GR7和M527-GR21中表达稳定性最好。通过以基因sigBsr、16S rRNAsr、rpoAsr为内参基因,检测龟裂霉素生物合成基因簇中的结构基因rim Gsr在菌株M527-GR7中不同时间的相对表达量,发现基因sigBsr作为内参基因时能得到更准确的结果。Streptomyces rimosus M527, a rimocidin producer, has a strong antagonistic effect against a variety of plant pathogenic fungi. In order to analyze the biosynthetic and regulatory mechanism of rimocidin in strain M527 and its resistant mutant at transcriptional level, it is very important to select the most stable internal reference gene for the strain M527 and its resistant mutant. In this study, 16S rRNAsr, hrdBsr, rpoAsr, sigFsr, sig Bsr, gyr Bsr were selected as candidate reference genes for real-time quantitative PCR, and their stabilities were analyzed in wild-type strain M527 and its resistant strains M527-GR7(higher-rimocidin producer) and M527-GR21(lower-rimocidin producer) by using software geNorm, NormFinder, Best Keeper, so as to select the internal reference gene with the highest stability. The results showed that sig Bsr gene was the most stable internal reference gene among all tested internal reference genes either in the wild-type M527 or in resistant mutants M527-GR7 and M527-GR21. Subsequently, the relative expression of the structural gene rim Gsr in strain M527-GR7 was detected at different time during fermentation process by using the genes sigBsr, 16 S rRNAsr and rpo Asr as internal reference genes. The results revealed that analysis at transcriptional level could be more accurate when the gene sigBsr was used as an internal reference gene.

关 键 词：龟裂链霉菌M527 荧光定量PCR 内参基因 稳定性 

分 类 号：S467[农业科学—植物保护]