绿豆象实时荧光定量PCR内参基因的筛选

Selection and validation of candidate reference genes for gene expression analysis in Callosobruchus chinensis(Coleoptera∶Bruchidae)by qRT-PCR

绿豆象Callosobruchus chinensis是一种世界性分布的害虫,对绿豆、豇豆等豆科作物的产量与品质造成严重影响。本研究旨在筛选出适合分析绿豆象不同发育阶段以及成虫不同组织中稳定表达的内参基因,根据绿豆象转录组测序以及相关参考文献报道,筛选出8个候选内参基因(β-Actin、β-Tubulin、α-Tubulin、AK、GAPDH、RPL40、Hsc70、eEF1-α),采用qRT-PCR技术分析在绿豆象不同发育阶段(幼虫、蛹、雌雄成虫)以及成虫不同组织中的(触角、头、腹、足、翅)各候选基因的表达量;利用GeNorm、NormFinder、BestKeeper和在线网站RefFinder并结合△Ct值评估8个候选内参基因的稳定性。结果显示,在绿豆象不同发育阶段以及成虫不同组织中,各候选内参基因Ct值和跨度均不同,表明各候选内参基因表达量存在差异。不同算法综合比较内参基因稳定性以及GeNorm软件最佳内参基因数目的分析显示,在绿豆象虫不同发育阶段中推荐采用β-Act、β-Tub作为内参基因,而在绿豆象成虫不同组织中推荐采用β-Tub、α-Tub作为内参基因,这有利于在绿豆象的基因表达研究中取得更为可靠、准确的数据。

Callosobruchus chinensis is a worldwide storage pest,which has a serious impact on the yield and quality of legume crops such as mung bean and adzuki bean.This paper aims to selection and validation of endogenous reference genes for analyzing the stable expression of C.chinensis in different development stages and tissues of male and female adults and different sex tissues.According to literature reports and the results of C.chinensis transcriptome sequencing,eight candidate endogenous reference genes(β-Actin,β-Tubulin,α-Tubulin,AK,GAPDH,RPL40,Hsc70,eEF1-α)were screened,and the expression of each candidate gene in C.chinensis different developmental stages(larvae,pupae,female and male adults)and adults different tissues(antennae,head,abdomen,foot,and wings)was analyzed by qRT-PCR.These candidate genes expression stabilities were evaluated by using four software-based(△Ct,geNorm,NormFinder and BestKeeper)and one web-based(RefFinder)comprehensive analysis tools.The results indicated that the Ct values of the 8 candidate endogenous reference genes were between 15~30 in different stages of development of C.chinensis and different tissues of adult.Through the comprehensive analysis of the stability of the endogenous reference gene by various software,the endogenous reference genes in different stages development and different tissues of adults in C.chinensis are recommended to useβ-Act,β-Tub andβ-Tub,α-Tub.The results of this study are conducive to obtaining more reliable and accurate data in the expression of C.chinensis gene of interest.