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

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 (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 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: BestKeeper; Enterobacter cancerogenus; NormFinder; RefFinder; geNorm; qRT-PCR; reference genes.

Figure 1

Expression levels of thirteen housekeeping genes in E. cancerogenus.

Figure 2

The stability of thirteen housekeeping genes in E. cancerogenus based on BestKeeper. The comparison was performed under two different experimental conditions including temperatures (A,B) and different OD values (C–E). The Venn diagram (F) shows the common stable genes from different conditions.

Figure 3

The stability of thirteen housekeeping genes in E. cancerogenus based on NormFinder. The comparison was performed under two different experimental conditions including temperatures (A,B) and different OD values (C–E). The Venn diagram (F) shows the common stable genes from different conditions.

Figure 4

The stability of thirteen housekeeping genes in E. cancerogenus based on geNorm. The comparison was performed under two different experimental conditions including temperatures (A,B) and different OD values (C–E). The Venn diagram (F) shows the common stable genes from different conditions.

Figure 5

Optimal number of reference genes used for normalization of gene expression by geNorm. The comparison was performed under two different experimental conditions including temperatures (A,B) and different OD values (C–E).

Figure 6

Expression stability of thirteen housekeeping genes in different samples of E. cancerogenus. The stability of the reference genes was calculated by the Geomean method of RefFinder. The comparison was performed under two different experimental conditions including temperatures (A,B), different OD values (C–E), and all samples (F).

Figure 7

Expression levels of gyrB and gyrA genes in different tissues of larvae. The fat body (FB), head capsule (H), gut (G), Malpighian tubules (MT), epidermis (EP) and hemolymph (HC) were dissected from the larvae feeding with E. cancerogenus or LB medium for 24 h. For each sample, 5 independent pools of 20–30 individuals were measured in technical triplicate using qRT-PCR. The columns represent averages, with vertical lines indicating SE. The t-test was used to analyze the results, and the asterisks (****) indicate the significant difference (p-value < 0.01).

Figure 8

The relative expression of Hcp from E. cancerogenus in the different tissues of larvae. The fat body (FB), head capsule (H), gut (G), Malpighian tubules (MT), epidermis (EP) and hemolymph (HC) were dissected from the larvae feeding with E. cancerogenus of three different OD values. For each sample, 5 independent pools of 20–30 individuals were measured in technical triplicate using qRT-PCR. The values were calculated using the 2^−∆∆Ct method, using the selected reference genes gyrB and gyrA. The relative transcripts are the ratios of copy numbers in different treatments relative to the hemolymph, which is set as 1. The columns represent averages, with vertical lines indicating SE. Different letters indicate significant differences at p value < 0.01 using analysis of variance with the Tukey–Kramer test.