rpoB and efp are stable candidate reference genes for quantitative real-time PCR analysis in Saccharopolyspora spinosa

Abstract

Spinosad (spinosyn A and spinosyn D), the secondary metabolite produced by Saccharopolyspora spinosa, is a potent insecticide with low effects on the environment and mammals. Strategies such as metabolic engineering, mutagenesis and fermentation process optimization have been employed for its production enhancement. Quantitative real-time polymerase chain reaction(qRT-PCR) is one of the preferred methods for the evaluation of gene transcript levels, whose accuracy and sensitivity depend on the normalization using optimal reference genes. However, no single reference gene is universally appropriate for all strains under various conditions. In this study, the transcriptional stability of 35 candidate reference genes, including 23 traditionally used reference genes and 12 novel reference genes identified in S. spinosa homologous species, was analysed in S. spinosa ATCC 49460 and spinosad high-yield strain S. spinosa S3-3 under three fermentation phases. The transcriptional stability of these genes was assessed by three statistical algorithms, geNorm, NormFinder and BestKeeper. The overall rankings suggested that rpoB and efp were the most stable candidate reference genes, and they may be the most promising reference genes for further study on the measurement of expression levels of target genes involved in the biosynthetic process of spinosad.

Keywords:

• Saccharopolyspora spinosa
• spinosad
• quantitative real-time PCR
• reference genes
• fermentation periods

Data availability

The data that support the findings reported in this study are available from the corresponding author upon reasonable request.

Disclosure statement

The authors declare that they have no conflict of interest. Xiaomeng Liu, Yunpeng Zhang, Tie Yin, Qi Li, Qiulong Zou and Xiaolin Zhang are affiliated with COFCO, and conceived the experiments, designed the experiments, performed the experiments, analysed the data, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft; Kexue Huang is affiliated with Qilu Pharmaceutical, and authored or reviewed drafts of the paper.

Additional information

Funding

This work was supported by the National Nature Science Foundation of China [Grant No. 31670055].