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【目的】本研究拟选择适合用于分析松墨天牛Monochamus alternatus化学感受组织中基因表达的内参基因。【方法】依据转录组测序结果进行内参基因鉴定,利用RT-q PCR技术分析内参基因在松墨天牛不同发育阶段和不同性别化学感受组织间的表达差异,并利用软件ge Norm,Norm Finder和Best Keeper比较其表达的稳定性。【结果】松墨天牛转录组中鉴定出9个候选内参基因(Actin,TUB,18S rRNA,RPS27A,RPS3,RPL10,AK,GAPDH和EF1A),其中后7个候选内参基因在松墨天牛中被首次鉴定,松墨天牛候选内参基因和其他昆虫相应基因的同源性很高。9个候选内参基因引物均具有良好的扩增效率,18S rRNA的表达水平最高,EF1A的表达水平最低;18S rRNA和Actin在不同样品间的表达水平差异最大,GAPDH和TUB表达水平在不同样品间差异最小。ge Norm和Norm Finder软件分析认为,GAPDH是最稳定的内参基因,TUB是较为稳定的内参基因,18S rRNA和Actin是最不稳定的内参基因;Best Keeper软件分析认为,GAPDH和TUB是合适的内参基因,18S rRNA和Actin是不适合的内参基因。最适合校正松墨天牛化学感受组织中基因表达数据的内参基因数量为2个,即GAPDH和TUB,并且这样的内参基因组合可以用于不同发育阶段和不同性别的不同化学感受组织。【结论】本研究结果为利用RT-q PCR技术准确分析松墨天牛和其他天牛基因包括化学感受组织基因相对表达量的内参基因选择提供了重要参考。
【Objective】 The aim of this study was to select a suitable reference gene for the analysis of the gene expression in Monachamus alternatus chemosensitive tissues of Monochamus alternatus. 【Method】 According to the transcriptome sequencing results, the internal reference genes were identified. RT-q PCR was used to analyze the expression differences of internal reference genes in different developmental stages and genotypes in different genus Monochamus alternatus. The software ge Norm, Norm Finder and Best Keeper compared the stability of his expression. 【Result】 Nine candidate reference genes (Actin, TUB, 18S rRNA, RPS27A, RPS3, RPL10, AK, GAPDH and EF1A) were identified in the transcriptome of Monochamus alternatus. Was identified for the first time, Monochamus alternatus candidate reference genes and other insects corresponding gene homology is high. Nine candidate primers had good amplification efficiency, 18S rRNA had the highest expression level, EF1A had the lowest expression level, 18S rRNA and Actin had the highest difference among different samples, and the expression levels of GAPDH and TUB among different samples The least difference. Ge Norm and Norm Finder software analysis, GAPDH is the most stable internal control gene, TUB is more stable internal control gene, 18S rRNA and Actin is the most unstable internal control gene; Best Keeper software analysis, GAPDH and TUB is a suitable internal control Genes, 18S rRNA and Actin are unsuitable internal reference genes. The most suitable number of internal reference genes for correcting gene expression data in chemically sensitized tissues of Monochamus alternatus is 2, ie, GAPDH and TUB, and such internal control gene combinations can be used in different chemosensitive tissues of different developmental stages and different genders. 【Conclusion】 This study provides an important reference for the accurate analysis of the expression of reference genes in Monochamus alternatus and other seasons, including the relative expression level of chemosensory tissue, using RT-q PCR.