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

doi:10.16409/j.cnki.2095-039x.2020.03.020

摘要/Abstract

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16S rRNA_sr、hrdB_sr、rpoA_sr、sigF_sr、sigB_sr、gyrB_sr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，sigB_sr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sigB_sr、16S rRNA_sr、rpoA_sr为内参基因，检测龟裂霉素生物合成基因簇中的结构基因rimG_sr在菌株M527-GR7中不同时间的相对表达量，发现基因sigB_sr作为内参基因时能得到更准确的结果。

关键词: 龟裂链霉菌M527, 荧光定量PCR, 内参基因, 稳定性

Abstract: 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 rRNA_sr, hrdB_sr, rpoA_sr, sigF_sr, sigB_sr, gyrB_sr 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, BestKeeper, so as to select the internal reference gene with the highest stability. The results showed that sigB_sr 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 rimG_sr in strain M527-GR7 was detected at different time during fermentation process by using the genes sigB_sr, 16S rRNA_sr and rpoA_sr as internal reference genes. The results revealed that analysis at transcriptional level could be more accurate when the gene sigB_sr was used as an internal reference gene.

Key words: Streptomyces rimosus M527, real-time quantitative PCR, internal reference genes, stability

中图分类号:

S467

徐洁, 胡叶锋, 廖芷君, 马正, 俞晓平. 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[J]. 中国生物防治学报, 2020, 36(3): 429-436.

XU Jie, HU Yefeng, LIAO Zhijun, MA Zheng, YU Xiaoping. Selection of Suitable Internal Reference Genes for the Real-Time Quantitative PCR in Streptomyces rimosus M527 and Evaluation of Their Stabilities[J]. Chinese Journal Of Biological Control, 2020, 36(3): 429-436.

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