印楝素胁迫下草地贪夜蛾幼虫实时荧光定量PCR内参基因表达稳定性评价  

Expression Stability of Reference Genes in Larvae of Spodoptera frugipferda Under Azadirachtin Stress by Real-Time Quantitative PCR Analysis

作　　者：舒本水 黄玉婷 余萱悦 刘翠婷 谢心怡 沈皓 林进添 SHU Benshui;HUANG Yuting;YU Xuanyue;LIU Cuiting;XIE Xinyi;SHEN Hao;LIN Jintian(College of Agriculture&Biology,Zhongkai University of Agriculture and Engineering/Guangzhou Key Laboratory of Subtropical Fruit Tree Pest and Disease Outbreak Control,Guangzhou 510225,China)

机构地区：[1]仲恺农业工程学院农业与生物学院/广州市亚热带果树重大疫情控制重点实验室,广东广州510225

出　　处：《广东农业科学》2024年第8期21-30,共10页Guangdong Agricultural Sciences

基　　金：国家自然科学基金(32102221);广东省重点领域研发计划项目(2020B020223004);广东省大学生创新创业训练计划项目(S202311347039)。

摘　　要：【目的】选择合适的内参基因对实时荧光定量PCR技术结果的准确性具有重要意义。通过评估印楝素胁迫下草地贪夜蛾(Spodoptera frugiperda J.E Smith)幼虫候选内参基因在不同样品中的表达稳定性,确定最适内参基因,为后续靶标基因功能分析和印楝素分子毒理机制解析提供基础。【方法】选取草地贪夜蛾α-TUB、β-1-TUB、Actin、EF1α、EF2、GAPDH、RPL3和RPL138个候选内参基因,通过ΔCt、GeNorm、NormFinder、BestKeeper和RefFinder软件对印楝素胁迫下各候选内参基因在草地贪夜蛾幼虫整虫、表皮、中肠、脂肪体和马氏管样品中转录水平表达量的稳定性进行评估,采用GeNorm软件确定最适内参基因数目,最终确定印楝素胁迫下不同样品中最适内参基因组合。【结果】印楝素胁迫下草地贪夜蛾幼虫样品中,ΔCt和Normfinder分析发现α-TUB和β-1-TUB的表达稳定性最高,GeNorm分析发现RPL3和RPL13的表达稳定性最高,BestKeeper分析则是α-TUB和RPL13表达稳定性最高。表皮样品中,β-1-TUB和α-TUB在ΔCt、GeNorm和Normfinder中表达稳定性最高,Actin和β-1-TUB在BestKeeper评估中最稳定。在中肠组织中,ΔCt分析发现EF2和β-1-TUB表达最稳定,GeNorm发现EF1α和RPL3表达最稳定,Normfinder分析发现EF2和β-1-TUB表达最稳定,BestKeeper分析发现RPL13和RPL3表达最稳定。在脂肪体组织中,ΔCt、GeNorm和Normfinder分析下RPL3和EF1α表达稳定性最高,BestKeeper分析则认为RPL13和RPL3稳定性最高。在马氏管组织中,ΔCt分析发现RPL3和EF2表达最稳定,GeNorm评估EF1α和EF2表达最稳定,Normfinder分析发现β-1-TUB和EF2表达最稳定,BestKeeper分析发现RPL13和β-1-TUB表达最稳定。此外,GeNorm软件分析发现各样品中最适内参基因数目均为2个。RefFinder综合分析结果为,α-TUB和β-1-TUB在幼虫和表皮组织样品中表达稳定性最高;EF2和RPL3在幼虫中肠和马氏管组织样品中表达稳定性最高;RPL3和EF1α�【Objective】Real-time quantitative PCR is one of the most conventional and efficient techniques for studying the mRNA expression level of target genes.The proper selection of reference genes is of great significance for the accuracy of the results.The study aimed to identify the most suitable reference genes by evaluating the expression stability of candidate reference genes in different samples of Spodoptera frugiperda larvae under azadirachtin stress.The results provided a basis for functional analysis of target genes and molecular toxicological mechanism analysis of azadirachtin.【Method】Eight candidate reference genes,includingα-TUB,β-1-TUB,Actin,EF1α,EF2,GAPDH,RPL3 and RPL3,of S.frugiperda were selected.The expression stability of these genes in different samples of larvae,cuticle,midgut,fat body and malpighian tube under azadirachtin stress was evaluated by theΔCt,GeNorm,NormFinder,BestKeeper and RefFinder software.And the optimal number of reference genes were determined by GeNorm software.Finally,the best reference gene combinations in different samples were comfirmed.【Result】When exposed to azadirachtin stress,in larvae samples,the expression stability ofα-TUB andβ-1-TUB was the highest byΔCt and Normfinder analyses;the expression stability of RPL3 and RPL13 was the highest by GeNorm analysis;and the expression stability ofα-TUB and RPL13 was the highest by BestKeeper analysis.In cuticle samples,β-1-TUB andα-TUB were rated as the most stable genes inΔCt,GeNorm and Normfinder,while Actin andβ-1-TUB were the most stable in BestKeeper.In the midgut tissue,ΔCt analysis found that EF2 andβ-1-TUB expression was the most stable;GeNorm assessment showed that EF1αand RPL3 expression was the most stable;Normfinder considered that EF2 andβ-1-TUB expression was the most stable,and BestKeeper analysis found that RPL13 and RPL3 expression was the most stable.ΔCt,GeNorm and Normfinder analyses showed that RPL3 and EF1αwere the most stable in fat body tissue,while BestKeeper analysis showed t

关 键 词：实时荧光定量PCR 草地贪夜蛾 印楝素 内参基因 表达稳定性 

分 类 号：S433[农业科学—农业昆虫与害虫防治]