瓜实蝇不同发育期及组织实时荧光定量PCR内参基因筛选
Selection of reference genes in Zeugodacus cucurbitae(Coquil-lett)different development periods and tissues by real-time fluorescence quantitative PCR
陈黔 1杨朗 2王凤英 2黄立飞 2曹雪梅 2张建民 3姜建军4
作者信息
- 1. 广西农业职业技术大学,广西南宁 530007;广西农业科学院植物保护研究所/农业农村部华南果蔬绿色防控重点实验室/广西作物病虫害生物学重点实验室,广西南宁 530007;长江大学农学院,湖北荆州 434025
- 2. 广西农业科学院植物保护研究所/农业农村部华南果蔬绿色防控重点实验室/广西作物病虫害生物学重点实验室,广西南宁 530007
- 3. 长江大学农学院,湖北荆州 434025
- 4. 广西农业职业技术大学,广西南宁 530007;广西农业科学院植物保护研究所/农业农村部华南果蔬绿色防控重点实验室/广西作物病虫害生物学重点实验室,广西南宁 530007
- 折叠
摘要
[目的]筛选出适用于瓜实蝇[Zeugodacus cucurbitae(Coquillett)]抗药性分子机制及生理分子机制研究的内参基因及最优组合数,为瓜实蝇抗药性机理研究打下基础.[方法]以不同发育期的敏感品系和抗阿维菌素品系瓜实蝇及其不同组织样本为试验材料,将8个候选内参基因通过实时荧光定量PCR进行表达,并利用geNorm、Norm-Finder、Bestkeeper和RefFinder软件对内参基因表达稳定性进行综合评估分析,筛选出最稳定表达的内参基因并明确最优组合数.[结果]geNorm分析结果显示,不同发育期处理内参基因表达稳定性排序为:RPS13=RpL32(0.269)>TUBE(0.623)>TBP(0.702)>β-tubulin(1.069)>G6PDH(1.366)>Actin3(1.596)>Actin2(1.923);不同组织处理内参基因表达稳定性排序为:RpL32=RPS13(0.132)>TBP(0.345)>TUBE(0.442)>β-tubulin(0.552)>G6PDH(0.780)>Actin2(1.101)>Actin3(1.417),最适内参基因个数为2.NormFinder分析结果显示,不同发育期处理内参基因表达稳定性排序为:RpL32(0.399)>RPS13(0.446)>TUBE(0.738)>G6PDH(0.912)>TBP(0.924)>β-tubulin(1.086)>Actin3(1.092)>Actin2(1.866);不同组织处理内参基因表达稳定性排序为:RpL32(0.090)>RPS13(0.117)>TBP(0.389)>TUBE(0.445)>β-tubulin(0.705)>G6PDH(0.877)>Actin2(1.009)>Actin3(1.567).BestKeeper分析结果显示,不同发育期处理内参基因表达稳定性排序为:TUBE>TBP>RPS13>RpL32>β-tubulin>G6PDH>Actin3>Actin2;不同组织处理内参基因表达稳定性排序为:β-tubulin>TBP>TUBE>RPS13>RpL32>G6PDH>Actin2>Actin3.RefFinder分析结果显示,不同发育期处理内参基因表达稳定性排序为:RpL32>RPS13>TUBE>TBP>G6PDH>β-tubulin>Actin3>Actin2;不同组织处理内参基因表达稳定性排序为RPS13>RpL32>TBP>β-tubulin>TUBE>G6PDH>Actin2>Actin3.[结论]RPS13和RpL32可组合作为瓜实蝇不同发育期、不同组织及阿维菌素抗性分子机制和生理分子机制研究的内参基因.
Abstract
[Objective]The purpose of the study was to screen the suitable reference gene and the optimal number of combinations for Zeugodacus cucurbitae(Coquillett)insecticides resistance and physiological molecular mechanism study,thus providing a foundation for the study of Z.cucurbitae insecticide resistance mechanism.[Method]Sensitive and abamectin resistant strains of Z.cucurbitae in different development periods and different tissue samples were used as test materials.The eight candidate reference genes were expressed by real-time fluorescence quantitative PCR.The expression stability of reference genes was analyzed by comprehensive assessment using geNorm,NormFinder,BestKeeper and Ref-Finder.The most stably expressed reference genes were selected and the optimal number of combinations was clarified.[Result]The results of geNorm analysis indicated that the expression stability rank of the reference genes treated in differe-nt development periods was RPS13=RpL32(0.269)>TUBE(0.623)>TBP(0.702)>β-tubulin(1.069)>G6PDH(1.366)>Actin3(1.596)>Actin2(1.923).The expression stability rank of the reference genes treated in different tissues was RpL32 = RPS13(0.132)>TBP(0.345)>TUBE(0.442)>β-tubulin(0.552)>G6PDH(0.780)>Actin2(1.101)>Actin3(1.417),and the optimal number of reference gene was 2.The results of NormFinder analysis indicated that the expres-sion stability rank of the reference genes treated in different development periods was RpL32(0.399)>RPS13(0.446)>TUBE(0.738)>G6PDH(0.912)>TBP(0.924)>β-tubulin(1.086)>Actin3(1.092)>Actin2(1.866).The expression sta-bility rank of the reference genes treated in different tissues was RpL32(0.090)>RPS13(0.117)>TBP(0.389)>TUBE(0.445)>β-tubulin(0.705)>G6PDH(0.877)>Actin2(1.009)>Actin3(1.567).The results of BestKeeper analysis indi-cated that the expression stability rank of the reference genes treated in different development periods was TUBE>TBP>RPS13>RpL32>β-tubulin>G6PDH>Actin3>Actin2.The expression stability rank of the reference genes treated in different tissues was β-tubulin>TBP>TUBE>RPS13>RpL32>G6PDH>Actin2>Actin3.The results of RefFinder analysis indicated that the expression stability rank of the reference genes treated in different development period was RpL32>RPS13>TUBE>TBP>G6PDH>β-tubulin>Actin3>Actin2.The expression stability rank of the reference genes treated in different tissue was RPS13>RpL32>TBP>β-tubulin>TUBE>G6PDH>Actin2>Actin3.[Conclusion]RPS13 and RpL32 can be combined as the reference gene for the study of different development periods,different tissues,abamectin resistance molecular mechanism and physiological molecular mechanism in Z.cucurbitae.
关键词
瓜实蝇/内参基因/组织/发育期/实时荧光定量PCRKey words
Zeugodacus cucurbitae(Coquillett)/reference gene/tissue/development period/real-time fluores-cence quantitative PCR引用本文复制引用
基金项目
国家自然科学地区基金项目(31960564)
广西科技重大专项(AA22036001)
广西农业科学院科技发展基金项目(2021JM75)
广西农业科学院科技发展基金项目(2021YT068)
出版年
2023
国家科技期刊平台
NSTL
万方数据