关键词: 棉花粉蚧, 温度, 热胁迫, 内参基因, 表达稳定性, 实时荧光定量PCR

Abstract: 【Aim】 To select the suitable reference genes in Phenacoccus solenopsis after exposure to different temperature stress by using quantitative real-time PCR (RT-qPCR). 【Methods】 In this study, we investigated the mRNA expression stability of six candidate reference genes (α-tub, GAPDH, rpl32, β-tub, SDHA and TBP) in different developmental stages (2nd instar nymph, 3rd instar nymph and female adult) of P. solenopsis under seven different temperature regimes (constant temperature 18℃ and 32℃, and temporary heat shock (1 h) at 37℃, 39℃, 41℃, 43℃ and 45℃ and then recovery at 26℃ for 1 h) by RT-qPCR method. Three software-based approaches (geNorm, Bestkeeper and Normfinder) and one web-based comprehensive tool (RefFinder) were used to analyze and rank the tested genes, and evaluate the expression stability of these genes. 【Results】 In the 2nd instar nymphs, the M values of six reference genes stability in the ascending order were α-tub (0.579)<GAPDH (0.654)<TBP (0.663)<β-tub (0.668)<rpl32 (0.675)<SDHA (0.755), and the SD values were TBP (0.31)<α-tub (0.37)< rpl32 (0.38)<SDHA (0.52)<β-tub (0.53)<GAPDH (0.57). In the 3rd instar nymphs, the M values in the ascending order were α-tub (0.542)<β-tub (0.596)<TBP (0.618)< GAPDH (0.655)<SDHA (0.668)<rpl32(0.749), while the SD values were α-tub (0.29)<β-tub (0.40)<rpl32 (0.45)<TBP (0.49)<GAPDH (0.50)<SDHA (0.55). The M values in female adults in the ascending order were rpl32 (0.554)<α-tub (0.635)<GAPDH (0.662)<β-tub (0.666)<SDHA (0.704)<TBP (0.837), and the SD values were rpl32 (0.31)<α-tub (0.35)<GAPDH (0.44)<SDHA (0.44)<β-tub (0.48)<TBP (0.71). According to the comprehensive rank of RefFinder, α-tub was the most stable for the 2nd and 3rd instar nymph, while rpl32 for female adult under seven different temperature regimes. 【Conclusion】α-tub can be used as a reference gene for the 2nd and 3rd instar nymph, and rpl32 could be used as the reference gene for female adult. This study provides an accurate reference for future study of the heat shock protein gene expression in P. solenopsis under heat stress.

Key words: Phenacoccus solenopsis, temperature, heat stress, reference genes, expression stability, real-time fluorescence quantitative PCR (RT-qPCR)