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The <i>Enterobacter cancerogenus</i> strain EcHa1 was isolated from the dead larvae of <i>Helicoverpa armigera</i>, 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_t, 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 <i>gyrA</i> and <i>gyrB</i> was recommended as the best reference gene combination at 28 °C, while <i>gyrA</i> and <i>rpoB</i> 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 <i>ftsZ</i> and <i>gyrA</i>, <i>rpoB</i> and <i>gyrB</i>, and <i>gyrA</i> and <i>pyk</i>, respectively. The most suitable reference genes were <i>gyrA</i> and <i>gyrB</i> under all experimental conditions. Using <i>gyrA</i> and <i>gyrB</i> as the reference genes for qRT-PCR, EcHa1 was found to invade all tissues of the <i>H. armigera</i> larvae, and expressed a candidate pathogenic factor <i>Hcp</i> 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 <i>E. cancerogenus</i>.