近零磁场下灰飞虱转录表达分析稳定性内参基因筛选  

Selection of Stable Internal Reference Genes for Transcript Expression Analyses in Laodelphax striatellus Under Near-Zero Magnetic Field

作　　者：刘凡奇 万贵钧 曾路影 李春绪 潘卫东[2] 陈法军[1] LIU FanQi;WAN GuiJun;ZENG LuYing;LI ChunXu;PAN WeiDong;CHEN FaJun(College of Plant Protection,Nanjing Agricultural University,Nanjing 210095;Beijing Key Laboratory of Bioelectromagetics,Institute of Electrical Engineering,Chinese Academy of Sciences,Beijing 100190)

机构地区：[1]南京农业大学植物保护学院,南京210095 [2]中国科学院电工研究所生物电磁学北京市重点实验室,北京100190

出　　处：《中国农业科学》2019年第19期3346-3356,共11页Scientia Agricultura Sinica

基　　金：国家自然科学基金(31470454,31701787);江苏省自然科学基金青年基金(BK20160717);中央高校基本科研业务费(KYZ201818,KJQN201820);国家重点研发计划(2017YFD0200400);江苏省“青蓝工程”优秀中青年学术带头人项目

摘　　要：【背景】地磁场(geomagnetic field,GMF)并不是稳定不变的,其随时间和空间时刻变化。目前,随着对动物磁生物学研究的日益深入,基于实时荧光定量PCR(qRT-PCR)技术开展的磁响应基因转录表达谱研究有力促进了磁响应通路的鉴定和磁感受机制的揭示。【目的】筛选近零磁场(near-zero magnetic field,NZMF)下短翅型灰飞虱(Laodelphax striatellus)稳定表达的内参基因,使对目的基因的定量分析更加准确。【方法】迁飞性昆虫灰飞虱采自江苏省农业科学院试验田并在室内使用TN1三叶期稻苗进行扩繁(温度:(25.0±1.0)℃,相对湿度:70%-90%,光周期:14L﹕10D)。采用亥姆霍兹线圈室内模拟近零磁场(NZMF;<500 nT)和地磁场(GMF;~50000 nT),人工模拟磁场强度有效处理空间为直径30 cm的球形空间,试验过程中严格控制除磁场强度外的环境因子(温度:(25.0±1.0)℃,相对湿度:75%,光周期:14L﹕10D)并利用磁通门计每日对人工模拟磁场进行校准和监测,灰飞虱连同TN1三叶期稻苗均置于试管中进行暴露处理,每隔两日与对照磁场中稻苗对调以避免稻苗潜在磁响应对灰飞虱的影响。利用Trizol法分别提取初羽化灰飞虱雌、雄成虫总RNA,检测各生物学重复RNA质量并调至含量一致,反转录为cDNA,利用qRT-PCR技术并结合常用内参筛选分析软件geNorm、NormFinder、BestKeeper以及在线综合分析系统RefFinder对在NZMF和GMF两种磁场强度下灰飞虱体内的内参基因稳定性进行评估筛选,其中,待评估的11个常用内参基因包括Actin1、Tubulin(α1TUB和α2TUB)、Elongation factor 1alpha(EF-1α)、Glyceraldehyde-3-phosphate dehydrogenase(GAPDH)、Ubiquitin(UBI)、Ribosomal protein S11(RPS11)、Ribosomal protein S15e(RPS15)、Ribosomal protein L8(RPL8)、Ribosomal protein L9(RPL9)和ADP ribosylation factor2(ARF2)。【结果】不同磁场环境(NZMF vs.GMF)下,灰飞虱短翅雌成虫EF-1α和RPL9表达稳定性在geNorm和NormFinder两种评估方�【Background】The geomagnetic field(GMF)is not constant,it can change with time and space.At present,with the development of research on animal magnetic biology,the study on transcriptional profiling of magnetic response genes with quantitative real-time PCR(qRT-PCR)has greatly promoted the identification of magnetic response pathway and the uncovering of magnetoreception mechanism.【Objective】The objective of this study is to screen the internal reference genes of the brachypterous small brown planthopper(Laodelphax striatellus)under near-zero magnetic field(NZMF),and to make the quantification of target genes more accurate.【Method】The population of L.striatellus,a migratory insect,was collected from the experimental fields of Jiangsu Academy of Agricultural Sciences and expanded indoors using TN1 three-leaf rice seedlings(temperature:(25.0±1.0)℃,relative humidity:70%-90%,photoperiod:14 L﹕10 D).Helmholtz coils system was used to simulate the near-zero magnetic field(NZMF;<500 nT)vs.geomagnetic field(GMF;~50000 nT),the artificial simulated magnetic field intensity was homogeneous within a spherical space with a diameter of 30 cm.During the experiment,environmental factors other than the magnetic field intensity were strictly controlled(temperature:(25.0±1.0)℃,relative humidity:75%,photoperiod:14 L﹕10 D)and the artificial simulated magnetic field was calibrated and monitored daily using a fluxgate magnetometer.The L.striatellus and TN1 three-leaf rice seedlings were placed in a test tube for exposure treatment.Every two days,the rice seedlings in control and treatment magnetic fields were swapped to avoid the influence triggered by the potential magnetic response of rice seedlings on L.striatellus.Trizol method was used to extract the total RNA of the female and male adults of L.striatellus,respectively.The quality of total RNA was inspected and adjusted to the same mass,and cDNA was then made by reverse transcription.Using qRT-PCR technique and combined with the common internal reference select

关 键 词：灰飞虱 近零磁场 磁场强度 实时荧光定量PCR 内参基因筛选 

分 类 号：S43[农业科学—农业昆虫与害虫防治]