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目的:筛选适用于褐飞虱Nilaparvata lugens成虫期目的基因表达水平检测的内参基因。方法:根据褐飞虱转录组数据初步筛选候选内参基因，采用RT-qPCR技术检测候选内参基因RPS11、β-actin和18S(18S rRNA)在褐飞虱不同发育阶段的表达水平，分别按照若虫阶段、成虫阶段、若虫-成虫阶段(包含若虫期和成虫期)3种情况分析内参基因在不同发育阶段的表达稳定性，用geNorm、NormFinder、BestKeeper和ΔCt 4种方法对数据进行处理，并用RefFinder对以上4种方法进行内参基因稳定性综合分析。结果:在褐飞虱若虫阶段，geNorm、NormFinder、BestKeeper和ΔCt 4种方法均表明RPS11基因的稳定性最好；在褐飞虱成虫阶段和若虫-成虫阶段，ΔCt和NormFinder方法分析表明，18S作为内参基因更稳定，而BestKeeper分析显示RPS11基因比18S稳定，geNorm分析表明RPS11与18S基因稳定性相同，4种方法均表明β-actin基因的稳定性最差。RefFinder综合以上4种分析方法，结果显示3个内参基因稳定性由强到弱依次为：18S、RPS11和β-actin。分别以RPS11、β-actin和18S为内参基因，检测NlATG3基因在褐飞虱成虫阶段羽化后不同天数的相对表达量，发现NlATG3基因在褐飞虱成虫羽化后不同天数的表达规律因内参基因的不同而出现差异。结论:内参基因RPS11在褐飞虱若虫时期表达稳定，但18S更适用于检测褐飞虱成虫阶段羽化后不同天数目的基因的表达水平。
Abstract：Aims: This paper aims to screen the internal reference genes suitable for detecting the expression level of target genes at the adult stage of brown planthopper Nilaparvata lugens. Methods: The candidate internal reference genes were screened according to the transcriptome data of brown planthopper; and the expression levels of the candidate internal reference genes RPS11, β-actin and 18S(18S rRNA) were detected by RT-qPCR at different developmental stages. The expression stability of internal reference genes at different developmental stages was analyzed for three situations: the nymph stage, the adult stage and the nymph-adult stage(including the nymph stage and the adult stage). Reffinder was used to comprehensively analyze the stability of internal reference genes based on the analysis of the four methods of geNorm, NormFinder, BestKeeper and ΔCt. Results: At the nymph stage of brown planthopper, the four methods of georm, NormFinder, BestKeeper and ΔCt showed that the stability of RPS11 gene was the best. At the adult stage and the nymph-adult stage of brown planthopper, ΔCt and NormFinder analysis showed that 18S was more stable as an internal reference gene, while BestKeeper analysis showed that RPS11 gene was more stable than 18S. GeNorm analysis showed that RPS11 and 18S were the same in stability. But all four methods showed that β-actin gene had the worst stability. Reffinder was used to integrate the results from the above four analysis methods; and it showed that the stability of the three internal reference genes from strong to weak was: 18S, RPS11 and β-actin. The relative expression of the NlATG3 gene in brown planthopper adults at different posteclosional stages was detected using RPS11, β-actin and 18S as internal reference genes; and it showed that the expression pattern of NlATG3 gene was different due to different internal reference genes. Conclusions: The expression of the internal reference gene RPS11 is stable during the nymph stage of brown planthopper; but 18S is more suitable for detecting the expression level of the target in brown planthopper adults at different posteclosional stages.
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基本信息:
DOI：
中图分类号:S435.112.3
引用信息:
[1]矫启启,叶成龙,冯娅琳等.褐飞虱若虫、成虫期荧光定量PCR内参基因的筛选[J].中国计量大学学报,2022,33(03):443-452.
基金信息:
国家自然科学基金项目(No.31672026,U21A20223,32011530117); 浙江省科技计划项目(No.LY20C140005,2019C02015,2022C02047); 中国计量大学科研标志性发展专项项目(No.2020YW27)