南方农业学报2023，Vol.54Issue(11)：3256-3264,9.DOI:10.3969/j.issn.2095-1191.2023.11.013
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
摘要
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分类
农业科技引用本文复制引用
陈黔,杨朗,王凤英,黄立飞,曹雪梅,张建民,姜建军..瓜实蝇不同发育期及组织实时荧光定量PCR内参基因筛选[J].南方农业学报,2023,54(11):3256-3264,9.基金项目
国家自然科学地区基金项目(31960564) （31960564）
广西科技重大专项(AA22036001) （AA22036001）
广西农业科学院科技发展基金项目(2021JM75,2021YT068) Regional Project of National Natural Science Foundation of China(31960564) （2021JM75,2021YT068）
Guangxi Science and Technology Key Project(AA22036001) （AA22036001）
Science and Technology Development Project of Guangxi Academy of Agri-cultural Sciences(2021JM75,2021YT068) （2021JM75,2021YT068）
