
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)
许亚丽,董彬,丁国婵,桑迪,季荣,王晗
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DOI:10.7679/j.issn.2095-1353.2022.137
作者单位: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
中文关键词:意大利蝗;内参基因;表达稳定性;卵巢发育;实时荧光定量PCR
英文关键词:Calliptamus italicus; reference gene; expression stability; ovarian development; RT-qPCR
中文摘要:【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。
英文摘要:[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.