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

Area of Science:

• Microbiology
• Molecular Biology
• Insect Pathology

Background:

• Enterobacter cancerogenus EcHa1 shows potential for biocontrol of Lepidoptera insects like Helicoverpa armigera.
• Accurate gene expression analysis using quantitative real-time PCR (qRT-PCR) requires stable endogenous reference genes (housekeeping genes, HKGs).
• Identifying optimal HKGs for E. cancerogenus under various conditions is crucial for reliable gene expression studies.

Purpose of the Study:

• To select and evaluate stable housekeeping genes for qRT-PCR normalization in E. cancerogenus strain EcHa1.
• To determine the most suitable reference genes under different culture temperatures and bacterial optical density (OD) values.
• To validate the utility of selected reference genes for studying gene expression in E. cancerogenus during insect infection.

Main Methods:

• Thirteen candidate housekeeping genes were screened in E. cancerogenus EcHa1.
• Gene expression levels were quantified using qRT-PCR across varying temperatures (28°C, 37°C) and OD values (0.5, 1.0, 2.0).
• Five statistical algorithms (Ct, BestKeeper, NormFinder, geNorm, RefFinder) were employed to assess gene stability.

Main Results:

• Reference gene stability varied significantly with experimental conditions.
• Optimal reference gene pairs included gyrA/gyrB at 28°C, gyrA/rpoB at 37°C, ftsZ/gyrA at OD 0.5, rpoB/gyrB at OD 1.0, and gyrA/pyk at OD 2.0.
• The combination of gyrA and gyrB demonstrated the highest stability across all tested conditions.
• Using gyrA/gyrB for normalization, E. cancerogenus EcHa1 was observed to invade H. armigera tissues and highly express the Hcp factor in the gut, Malpighian tubules, and epidermis.

Conclusions:

• The study provides validated reference gene sets for accurate qRT-PCR in E. cancerogenus under diverse conditions.
• gyrA and gyrB are recommended as the most reliable reference genes for E. cancerogenus functional gene expression studies.
• This research facilitates deeper investigation into the functional genes and pathogenicity mechanisms of entomopathogenic bacteria like E. cancerogenus.