抗药性灰飞虱稳定内参基因的筛选  

Identification of reference genes in resistant small brown plant hopper,Laodelphax striatellus

作　　者：王芮[1] 符蓉[1] 杨倩文[1] 慕莉莉[1] 吴敏[1] 

机构地区：[1]南京农业大学植物保护学院,江苏南京210095

出　　处：《南京农业大学学报》2016年第5期763-770,共8页Journal of Nanjing Agricultural University

基　　金：国家科技支撑计划项目(2012BAD19B00);南京农业大学实验教学示范中心项目(2015syzx014)

摘　　要：[目的]本文旨在筛选抗药性灰飞虱稳定的内参基因,使目标基因的定量更加准确。[方法]在利用抗性灰飞虱雌成虫转录组数据挖掘新候选内参基因[26S蛋白酶体非ATP酶的调节亚基3(PMSD3)、泛素结合酶E2D2类基因(UBE2D2)、蛋白磷酸酶1B类基因(PP1B-like)和真核翻译起始因子3亚基(e IF-3)]的基础上,结合传统持家基因[3-磷酸甘油醛脱氢酶(GAPDH)、延伸因子(EF-1)、肌动蛋白1(β-ACT1)、核糖基化因子(ARF)、60S核糖体蛋白L9基因(RPL9)、40S核糖体蛋白S15e(RPS15e)和精氨酸激酶(AK)],采用以ΔC_T值法、Best Keeper、Norm Finder、ge Norm软件法和在线工具Ref Finder分析上述候选内参基因在不同抗性灰飞虱雌成虫和若虫中表达量的稳定性。[结果]在3种不同抗性的灰飞虱雌成虫中EF-1和e IF-3基因表达最为稳定;在抗吡虫啉的灰飞虱若虫中EF-1和ARF基因表达稳定;在抗溴氰菊酯的灰飞虱若虫中EF-1和β-ACT1基因表达稳定;在抗毒死蜱的灰飞虱若虫中RPL9和EF-1基因表达稳定。[结论]研究明确了3种不同抗药性灰飞虱雌成虫和若虫中稳定表达的内参基因,为实时荧光定量PCR(qRT-PCR)技术提供了有力的技术支撑,对后续灰飞虱抗药性研究中关键目标基因的准确定量具有重要的意义。[Objectives]In order to acquire the accurate results of the expression levels of target genes in resistant Laodelphax striatellus,it is critical to perform the identification of reference genes for qRT-PCR normalization. [Methods]Four novel candidate reference genes[26S proteasome non-ATPase regulatory subunit（ PMSD3）,protein phosphatase 1B-like（ PP1B-like）,ubiquitinconjugating enzyme E2 D 2-like（ UBE2D2） and eukaryotic translation initiation factor 3 subunit（ e IF-3） ]were selected based on transcriptome data,and seven traditional housekeeping genes[glyceraldehyde phosphate dehydrogenase（ GAPDH）,elongation factor（ EF-1）,beta actin 1（ β-ACT1）,ribosylation factor（ ARF）,60 S ribosomal protein L9 gene（ RPL9）,40 S ribosomal protein S15e（ RPS15e） and arginine acid kinase（ AK） ]were analyzed in the expression stabilities by using delta CTvalues,Best Keeper,ge Norm,Norm Finder software and online tools of Ref Finder. [Results]The results showed that EF-1 and e IF-3 are stably expressed genes in female adults of three different resistant strains; EF-1 and ARF are stably expressed in imidacloprid resistant nymphs; EF-1 andβ-ACT1 are stably expressed in deltamethrin resistant nymphs; RPL9 and EF-1 are stably expressed in chlorpyrifos resistant nymphs.[Conclusions]This study has important practical value in accurate quantification of key target genes in resistant strains of L. striatellus.

关 键 词：灰飞虱 实时荧光定量PCR 内参基因 抗药性 

分 类 号：S435.112.3[农业科学—农业昆虫与害虫防治]