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Chinese Journal Of Biological Control ›› 2020, Vol. 36 ›› Issue (3): 429-436.DOI: 10.16409/j.cnki.2095-039x.2020.03.020

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

XU Jie, HU Yefeng, LIAO Zhijun, MA Zheng, YU Xiaoping   

  1. Zhejiang Provincial Key Laboratory of Biometrology and Inspection & Quarantine/College of Life Sciences, China Jiliang University, Hangzhou, 310018, China
  • Received:2019-11-19 Online:2020-06-08 Published:2020-06-12

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 rRNAsr, hrdBsr, rpoAsr, sigFsr, sigBsr, gyrBsr 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 sigBsr 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 rimGsr in strain M527-GR7 was detected at different time during fermentation process by using the genes sigBsr, 16S rRNAsr and rpoAsr 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.

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

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