六点始叶螨内参基因筛选及其在超氧化物歧化酶基因EsSOD表达分析中的应用  

Selection of Reference Genes for Transcription Analysis of Eotetranychus sexmaculatus Superoxide Dismutase Gene EsSOD

作　　者：梁晓 陈青[1,2] 伍春玲[1,2] 方永军 LIANG Xiao;CHEN Qing;WU Chunling;FANG Yongjun(Rubber Research Institute,Chinese Academy of Tropical Agricultural Sciences,Haikou,Hainan 571101,China;Environment and Plant Protection Institute,Chinese Academy of Tropical Agricultural Sciences/Key Laboratory of Pests Comprehensive Governance for Tropical Crops,Ministry of Agriculture and Rural Affairs/Hainan Engineering Research Center for Biological Control of Tropical Crops Diseases and Insect Pests/Hainan Key Laboratory for Monitoring and Control of Tropical Agricultural Pests,Haikou,Hainan 571101,China)

机构地区：[1]中国热带农业科学院橡胶研究所,海南海口571101 [2]中国热带农业科学院环境与植物保护研究所/农业农村部热带作物有害生物综合治理重点实验室/海南省热带作物病虫害生物防治工程技术研究中心/海南省热带农业有害生物监测与控制重点实验室,海南海口571101

出　　处：《热带作物学报》2021年第1期175-181,共7页Chinese Journal of Tropical Crops

基　　金：国家自然科学基金青年基金项目(No.31601649);农业农村部橡胶树生物学与遗传资源利用重点实验室开放课题项目(No.RRI-KLOF201602);海南省重点研发计划项目(No.ZDYF2018037)。

摘　　要：为了筛选稳定内参基因分析六点始叶螨超氧化物歧化酶基因EsSOD的表达量,本研究采用geNorm、Bestkeeper、Normfinder和RefFinder软件分析6个候选内参基因actin、gapdh、rpl13、α-tub、β-tub和18sRNA在六点始叶螨幼螨、前若螨、后若螨和雌成螨中的表达稳定性。结果表明,根据geNorm软件分析得出6个候选内参引物的稳定性从大到小的排序为:actin>β-tub>rpl13>α-tub>gapdh>18sRNA;根据NormFinder软件分析得出的稳定性从大到小的排序为:rpl13>β-tub>actin>α-tub>18sRNA>gapdh;根据BestKeeper软件分析得出的稳定性从大到小的排序为:β-tub>rpl13>actin>gapdh>α-tub>18sRNA;最终根据RefFinder软件的综合分析结果,actin和β-tub是稳定性最佳的2个内参引物。分别以actin和β-tub为内参进行EsSOD基因表达量的RT-qPCR分析,结果表明,与取食感螨橡胶树种质‘IAN2904’后EsSOD表达量相比,不同龄期六点始叶螨取食抗螨橡胶树种质‘IRCI12’后EsSOD表达量均降低。本研究获得了可用于六点始叶螨EsSOD表达量分析的稳定内参引物,为橡胶树种质抗螨性分子机理研究奠定了理论基础。In order to screen stable reference genes in analyzing the expression level of Eotetranychus sexmaculatus superoxide dismutase gene EsSOD,six candidate genes,actin,gapdh,rpl13,α-tub,β-tub and 18SRNA,were analyzed for their expression stability in different life stages(i.e.,larva,protonymph,deutonymph and female adults)of E.sexmaculatus by using geNorm,Bestkeeper,Normfinder and RefFinder analysis methods.The results showed that according to the geNorm analysis,the stability ranking of the six candidate genes was listed as:actin>β-ub>rpl13>α-tub>gapdh>18sRNA,according to the NormFinder analysis,the stability ranking of the six candidate genes was listed as:rpl13>β-tub>actin>α-tub>18sRNA>gapdh,according to the BestKeeper analysis,the stability ranking of the six candidate genes was listed as:rpl13>β-tub>actin>α-tub>18sRNA>gapdh,the order of the stability of six candidate primers was analyzed by BestKeeper software.The qualitative order from large to small was:β-tub>rpl13>actin>gapdh>α-tub>18sRNA.Finally,according to the comprehensive analysis results of Reffinder software,actin andβ-tub were the best two reference genes,and were subjected to the subsequent RT-qPCR analysis of EsSOD transcription.The results showed that compared with those feeding on mite-susceptible clutivars IAN2904,the expression of EsSOD in mite feeding on mite-resistant clutivars IRCI12 was decreased.Stable reference genes in analyzing the transcription of EsSOD might provide theoretical basis for the study of molecular mechanism of mite resistance in rubber tree.

关 键 词：六点始叶螨 内参基因 超氧化物歧化酶基因EsSOD 基因表达 

分 类 号：S433.5[农业科学—农业昆虫与害虫防治]