意大利蝗Calliptamus italicus卵巢基因表达RT-qPCR定量内参基因的筛选  

Selection of reference genes for RT-qPCR of ovarian gene expression in Calliptamus italicus(Orthopera:Acrididae)

作　　者：许亚丽 董彬 丁国婵 桑迪 季荣 王晗 XU Ya-Li;DONG Bin;DING Guo-Chan;SANG Di;JI Rong;WANG Han(International Research Center for the Collaborative Containment of Cross-Border Pests in Central Asia,Xinjiang Key Laboratory of Special Species Conservation and Regulatory Biology,College of Life Sciences,Xinjiang Normal University,Urumqi 830054,China;Manasi Station of Forecast and Control on Locusts and Rodents,Manasi 832200,China)

机构地区：[1]新疆师范大学生命科学学院,中亚区域跨境有害生物联合控制国际研究中心,新疆特殊物种保护与调控生物学实验室,乌鲁木齐830054 [2]新疆玛纳斯县蝗虫鼠害预测预报防治站,玛纳斯832200

出　　处：《应用昆虫学报》2022年第6期1362-1371,共10页Chinese Journal of Applied Entomology

基　　金：新疆维吾尔自治区自然科学基金项目(2021D01A124);新疆维吾尔自治区创新环境(人才、基地)建设专项(2022D04002)。

摘　　要：【目的】意大利蝗Calliptamus italicus是新疆草原重要害虫,研究其繁殖相关基因的生物学功能,对于害虫防控靶标筛选具有重要意义。为研究意大利蝗雌虫卵巢发育过程中功能基因的表达动态及调控功能,首先要选择适合的内参基因,以确保RT-qPCR定量结果的准确性。【方法】选择β-actin、EF1-a、β-tubulin、α-tubulin、GAPDH、SDHA、GST、RPL32和TBP为候选内参基因,采用RT-qPCR技术检测其在意大利蝗卵巢发育过程中的表达水平,通过GeNorm、NormFinder、BestKeeper和RefFinder评估其表达稳定性。以Hex2、VgR、Sox9a和Cyp6bg10为目标基因,验证候选基因在不同发育阶段卵巢组织中的表达稳定性。【结果】GeNorm、NormFinder和BestKeepe分析结果表明,β-tubulin、RPL32、GAPDH、EF1-α基因均表现出良好的稳定性。通过GeNorm判断配对变异值确定的内参基因最佳数目,结合RefFinder软件综合分析,表明意大利蝗卵巢发育过程中最优内参组合为RPL32和EF1-α。【结论】本研究获得意大利蝗卵巢发育过程中表达最稳定的内参基因组合,可用于后续的基因表达定量研究。[Objectives]To identify stable reference genes for RT-qPCR investigation of the expression and regulatory functions of genes related to ovarian development of Calliptamus italicus(L.), an important pest in Xinjiang’s grasslands.[Methods]Nine genes including β-actin, EF1-a, β-tubulin, α-tubulin, GAPDH, SDHA, GST, RPL32 and TBP, were selected as candidate reference genes. The expression levels of these genes were detected with quantitative real-time PCR and their stability of expression evaluated with GeNorm, NormFinder, BestKeeper, and RefFinder. Their stability in different tissues and developmental stages was verified using Hex2, VgR, Sox9a and Cyp6bg10 as the target gene. [Results]β-tubulin, RPL32,GAPDH and EF1-α were the most stable genes. Based on comprehensive analysis using GeNorm and RefFinder, the best combination of reference genes for the oocytes of different developmental stages was RPL32 and EF1-α. [Conclusion]The most stable genes identified in this study could be suitable reference genes for analyzing gene expression in C. italicus.

关 键 词：意大利蝗 内参基因 表达稳定性 卵巢发育 实时荧光定量PCR 

分 类 号：S433.2[农业科学—农业昆虫与害虫防治]