桉蝙蛾幼虫实时荧光定量PCR内参基因的筛选  

Screening of reference genes for RT-qPCR analysis in Endoclita signifier Walker larvae

作　　者：陈晓 邱峙嵩 苏小燕 许原 曹丽沙 杨振德[1] 胡平 CHEN Xiao;QIU Zhi-Song;SU Xiao-Yan;XU Yuan;CAO Li-Sha;YANG Zhen-De;HU Ping(Forestry College of Guangxi University,Nanning 530004,China)

机构地区：[1]广西大学林学院,南宁530004

出　　处：《环境昆虫学报》2023年第4期1016-1026,共11页Journal of Environmental Entomology

基　　金：广西壮族自治区科技基地和人才专项(2020AC19057);广西壮族自治区自然科学基金面上项目(2020JJA130068);国家自然科学基金青年科学基金(32001321)。

摘　　要：本研究通过筛选桉蝙蛾Endoclita signifer Walker幼虫不同龄期与不同体节中稳定表达的内参基因,为桉蝙蛾基因表达研究提供参考。通过实时荧光定量PCR技术测定5个候选内参基因(ACTIN、GAPDH、TUB、RIB、EF)在桉蝙蛾幼虫不同龄期(3龄、5龄、9龄、12龄)与不同体节(头部、胸部、腹部)及全样品(由所有样品组成)处理中的表达量,后利用GeNorm、NormFinder、BestKeeper对5个候选基因的稳定性进行评估,最后由RefFinder综合分析结果,选出最佳的内参基因。基于4种分析方法的评估可知,桉蝙蛾5龄和9龄幼虫的不同体节、不同龄期幼虫的头部和胸部中内参基因的最佳数目为2,而3龄和12龄幼虫的不同体节、不同龄期幼虫的腹部及全样本中内参基因的最佳数目为3。3龄、5龄、9龄和12龄幼虫的不同体节中可分别选择ACTIN+RIB+GAPDH、EF+RIB、GAPDH+EF和ACTIN+RIB+GAPDH作为最稳定的内参基因组合;EF+RIB、RIB+GAPDH和EF+GAPDH+ACTIN可分别作为不同龄期幼虫头部、胸部和腹部的最佳内参基因组合;综合考虑桉蝙蛾幼虫不同龄期与不同体节的影响时,可选择RIB、ACTIN和GAPDH作为内参基因。Screening the reference genes stably expressed in different instars and different body segments of Endoclita signifer Walker larvae,which provided a reference for gene expression study of E.signifer.Expression levels of five candidate reference genes(ACTIN,GAPDH,TUB,RIB and EF)in different instars(3 rd,5 th,9 th and 12 th instars),different body segments(head,thorax and abdomen)and the all samples were determined by real-time quantitative PCR.GeNorm,NormFinder and BestKeeper were used to evaluate the stability of 5 candidate genes.Finally,RefFinder comprehensively analyzed the results and selected the best reference gene.Optimal number of reference genes was 2 in different body segments of 5 th and 9 th instar E.signifer larvae,head and thorax of different instar larvae,and 3 in different body segments of 3 rd and 12 th instar larvae,abdomen of different instar larvae,and the all samples.ACTIN+RIB+GAPDH,EF+RIB,GAPDH+EF and ACTIN+RIB+GAPDH could be selected as the most stable reference gene combinations in different segments of 3 rd,5 th,9 th and 12 th instar larvae,respectively.EF+RIB,RIB+GAPDH and EF+GAPDH+ACTIN could be used as the most stable reference gene combinations in the head,thorax and abdomen of larvae at different instars,respectively.RIB,ACTIN and GAPDH could be selected as reference genes when the effects of different instars and different body segments of E.signifer larvae were considered.

关 键 词：桉蝙蛾 幼虫 龄期 体节 内参基因 RT-QPCR 

分 类 号：Q963[生物学—昆虫学] S433[农业科学—农业昆虫与害虫防治]