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【目的】柑橘大实蝇Bactrocera minax(Enderlein)是一种危害严重的柑橘害虫。本研究旨在筛选柑橘大实蝇在特定条件下体内稳定表达的内参基因,以确保使用实时荧光定量PCR分析目标基因表达的可靠性。【方法】选择10种候选内参基因用于进行实时荧光定量PCR(qRT-PCR),利用5种软件对柑橘大实蝇在不同虫态下(低龄幼虫、3龄幼虫、1日龄蛹、80日龄蛹、160日龄蛹、雄成虫、雌成虫)以及成虫不同部位(成虫头、胸、腹、整体)中候选内参基因的Ct值进行分析,明确其表达的稳定性。【结果】在柑橘大实蝇不同虫态和成虫不同部位,10种候选内参基因的Ct值都处于15~30之间,各基因Ct值的不同表明各基因的表达量存在差异。综合分析各种软件对内参基因稳定性的排名,结合ge Norm软件对最佳内参基因数量的分析结果,推荐在不同虫态下采用UBQ,GAPDH和GST作为内参基因,在不同成虫部位中采用TUB,GAPDH和GST作为内参基因。【结论】为了获取可信的目标基因表达分析结果,建议根据不同条件选择使用不同的内参基因组合。本研究结果有利于进一步研究柑橘大实蝇在特定条件下的目标基因表达。
【Objective】 Citrus pest Bactrocera minax (Enderlein) is a kind of harmful citrus pest. The aim of this study was to screen for the reference genes that were stably expressed in vivo in specific conditions in the citrus fruit fly to ensure the reliability of target gene expression using real-time fluorescence quantitative PCR. 【Method】 Ten candidate reference genes were selected for real-time quantitative PCR (qRT-PCR). Five softwares were used to analyze the genetic diversity of Citrus tazeta in different insect states (young larvae, 3rd instar larvae, 1 day old pupae, Day pupa, 160 day old pupal, male adult and female adult) and Ct values of candidate internal reference genes in different parts of adults (adult head, chest, abdomen and whole body) were analyzed to confirm the stability of their expression. 【Result】 The results showed that the Ct values of 10 candidate reference genes were between 15 and 30 in different insect states and adult parts of Citrus testridae. The differences of Ct values showed that the expression of each gene was different. Comprehensive analysis of various software on the stability of internal reference gene sequencing, combined with ge Norm software analysis of the best number of internal reference genes recommended in different insect states using UBQ, GAPDH and GST as an internal reference gene in different adult parts of the use of TUB , GAPDH and GST as internal reference genes. 【Conclusion】 In order to obtain credible target gene expression analysis results, it is suggested to select different internal reference gene combinations according to different conditions. The results of this study are helpful to further study the target gene expression of the citrus fruit fly under certain conditions.