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Microorganisms
Volume 12
Issue 5
10.3390/microorganisms12051024
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Article

Reference Genes for Expression Analyses by qRT-PCR in Enterobacter cancerogenus

by
Yang Pan
,
Yue Zhao
,
Hua-Rui Zeng
,
Jia-Qi Wu
,
Ying-Ying Song
,
Ya-Hao Rao
,
Guo-Qing Li
and
Lin Jin
*
Education Ministry Key Laboratory of Integrated Management of Crop Diseases and Pests/State & Local Joint Engineering Research Center of Green Pesticide Invention and Application, Department of Entomology, College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China
*
Author to whom correspondence should be addressed.
Microorganisms 2024, 12(5), 1024; https://doi.org/10.3390/microorganisms12051024
Submission received: 25 March 2024 / Revised: 14 May 2024 / Accepted: 15 May 2024 / Published: 19 May 2024
(This article belongs to the Section Microbiomes)
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Abstract

The Enterobacter cancerogenus strain EcHa1 was isolated from the dead larvae of Helicoverpa armigera, and has the potential for biocontrol of some Lepidoptera insects. In order to screen insecticidal-related genes by qRT-PCR, stable endogenous reference genes used for normalizing qRT-PCR data were selected and evaluated from 13 housekeeping genes (HKGs). The expression levels of the HKGs were determined using qRT-PCR under different experimental conditions, including two culture temperatures and three bacterial OD values. Five stability analysis methods (Ct, BestKeeper, NormFinder, geNorm, and RefFinder) were used to comprehensively rank the candidate genes. The results showed that the optimal reference genes varied under different experimental conditions. The combination of gyrA and gyrB was recommended as the best reference gene combination at 28 °C, while gyrA and rpoB was the best combination at 37 °C. When the OD values were 0.5, 1.0 and 2.0, the recommended reference gene combinations were ftsZ and gyrA, rpoB and gyrB, and gyrA and pyk, respectively. The most suitable reference genes were gyrA and gyrB under all experimental conditions. Using gyrA and gyrB as the reference genes for qRT-PCR, EcHa1 was found to invade all tissues of the H. armigera larvae, and expressed a candidate pathogenic factor Hcp at high levels in gut, Malpighian tubules, and epidermis tissues. This study not only establishes an accurate and reliable normalization for qRT-PCR in entomopathogenic bacteria but also lays a solid foundation for further study of functional genes in E. cancerogenus.
Keywords: Enterobacter cancerogenus; qRT-PCR; reference genes; BestKeeper; NormFinder; geNorm; RefFinder Enterobacter cancerogenus; qRT-PCR; reference genes; BestKeeper; NormFinder; geNorm; RefFinder

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MDPI and ACS Style

Pan, Y.; Zhao, Y.; Zeng, H.-R.; Wu, J.-Q.; Song, Y.-Y.; Rao, Y.-H.; Li, G.-Q.; Jin, L. Reference Genes for Expression Analyses by qRT-PCR in Enterobacter cancerogenus. Microorganisms 2024, 12, 1024. https://doi.org/10.3390/microorganisms12051024

AMA Style

Pan Y, Zhao Y, Zeng H-R, Wu J-Q, Song Y-Y, Rao Y-H, Li G-Q, Jin L. Reference Genes for Expression Analyses by qRT-PCR in Enterobacter cancerogenus. Microorganisms. 2024; 12(5):1024. https://doi.org/10.3390/microorganisms12051024

Chicago/Turabian Style

Pan, Yang, Yue Zhao, Hua-Rui Zeng, Jia-Qi Wu, Ying-Ying Song, Ya-Hao Rao, Guo-Qing Li, and Lin Jin. 2024. "Reference Genes for Expression Analyses by qRT-PCR in Enterobacter cancerogenus" Microorganisms 12, no. 5: 1024. https://doi.org/10.3390/microorganisms12051024

APA Style

Pan, Y., Zhao, Y., Zeng, H.-R., Wu, J.-Q., Song, Y.-Y., Rao, Y.-H., Li, G.-Q., & Jin, L. (2024). Reference Genes for Expression Analyses by qRT-PCR in Enterobacter cancerogenus. Microorganisms, 12(5), 1024. https://doi.org/10.3390/microorganisms12051024

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