RESUMEN
The coffee berry borer, Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae), is a major destructive insect pest of coffee, which impacts the coffee crops negatively. As a draft genome has been completed for this insect, most molecular studies on gene transcriptional levels under different experimental conditions will be conducted using real-time reverse-transcription quantitative polymerase chain reactions (RT-qPCR). However, the lack of suitable internal reference genes will affect the accuracy of RT-qPCR results. In this study, the expression stability of nine candidate reference genes was evaluated under different developmental stages, temperature stress, and Beauveria bassiana infection. Data analyses were completed by four commonly used programs, BestKeeper, NormFinder, geNorm, and RefFinder. The result showed that RPL3 and EF1α combination were recommended as the most stable reference genes for developmental stages. EF1α and RPS3a combination were the top two stable reference genes for B. bassiana infection. RPS3a and RPL3 combination performed as the optimal reference genes both in temperature stress and all samples. Our results should provide a good foundation for the expression profile analyses of target genes in the future, especially for molecular studies on insect genetic development, temperature adaptability, and immune mechanism to entomogenous fungi in H. hampei.
Asunto(s)
Beauveria , Coffea , Escarabajos , Gorgojos , Animales , Coffea/genética , TemperaturaRESUMEN
The coffee white stem borer, Xylotrechus quadripes Chevrolat (Coleoptera: Cerambycidae), is a major destructive pest of Coffea arabica L. (Gentianales: Rubiaceae), widely planted in many Asian countries, including China. Quantitative real-time polymerase chain reaction (qRT-PCR) is a common method for quantitative analysis of gene transcription levels. To obtain accurate and reliable qRT-PCR results, it is necessary to select suitable reference genes to different experimental conditions for normalizing the target gene expression. However, the stability of the expression of reference genes in X. quadripes has rarely been studied. In this study, the expression stability of nine candidate reference genes were investigated under biotic and abiotic conditions for use in qRT-PCR's normalization. By integrating the results of four algorithms of NormFinder, BestKeeper, geNorm, and RefFinder, the optimal reference gene combinations in different experimental conditions were performed as follows: RPL10a and EIF3D were the optimal reference genes for developmental stage samples, EIF4E, RPL10a, and RPS27a for tissue samples, V-ATP and EF1α for the sex samples, EIF3D and V-ATP for temperature treatment, RPS27a and RPL10a for insecticide stress, and RPL10a, RPS27a, and EF1α for all the samples. This study will help to obtain the stable internal reference genes under biotic and abiotic conditions and lay the foundation for in-depth functional research of target genes or genomics on olfactory molecular mechanisms, temperature adaptability, and insecticide resistance in X. quadripes.