Reference Genes for Expression Analyses by qRT-PCR in .

The strain EcHa1 was isolated from the dead larvae of , 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 (C, 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 and was recommended as the best reference gene combination at 28 °C, while and 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 and , and , and and , respectively. The most suitable reference genes were and under all experimental conditions. Using and as the reference genes for qRT-PCR, EcHa1 was found to invade all tissues of the larvae, and expressed a candidate pathogenic factor 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 .