大灰象甲实时定量PCR内参基因的筛选  

Screening of reference genes for quantitative real-time PCR in Sympiezomias velatus (Coleoptera: Curculionidae)

作　　者：李晓[1] 李建文[2] 成波[1] 李伟[2] 孙文秀[2] 高华援[3] 鞠倩[1] 姜晓静[1] 杜龙[1] 曲春娟 曲明静[1] LI Xiao;LI Jian-Wen;CHENG Bo;LI Wei;SUN Wen-Xiu;GAO Hua-Yuan;JU Qian;JIANG Xiao-Jing;DU Long;QU Chun-Juan;QU Ming-Jing(Shandong Peanut Research Institute,Qingdao,Shandong 266100,China;College of Life Science,Yangtze University,Jingzhou,Hubei 434025,China;Peanut Research Institute,Jilin Academy of Agricultural Sciences,Gongzhuling,Jilin 136100,China)

机构地区：[1]山东省花生研究所,山东青岛266100 [2]长江大学生命科学学院,湖北荆州434025 [3]吉林省农业科学院花生研究所,吉林公主岭136100

出　　处：《昆虫学报》2018年第11期1284-1294,共11页Acta Entomologica Sinica

基　　金：山东省农业科学院青年科研基金项目(2016YQN15);国家花生产业技术体系(CARS-14);国家自然科学基金项目(31401741)

摘　　要：【目的】筛选出适合分析大灰象甲Sympiezomias velatus成虫不同组织中基因表达水平的内参基因。【方法】利用转录组测序技术获得大灰象甲管家基因序列作为候选内参基因,采用实时荧光定量PCR(qRT-PCR)技术分析候选基因在大灰象甲雌雄成虫触角、头、胸、腹和足中的表达量;并利用geNorm, NormFinder和BestKeeper软件及在线工具RefFinder评价候选基因的表达稳定性。以大灰象甲气味结合蛋白1(odorant bindng protein 1, OBP1)基因为目标基因验证候选基因在大灰象甲成虫不同组织中的表达稳定性。【结果】基于大灰象甲转录组数据首次鉴定得到β-肌动蛋白基因(ACT)、3-磷酸甘油醛脱氢酶基因(GAPDH)、18S核糖体RNA基因(18S rRNA)、60S核糖体蛋白L12基因(RPL12)、60S核糖体蛋白L32基因(RPL32)、40S核糖体蛋白S20基因(RPS20)、延伸因子2基因(EF2)、α-微管蛋白基因(TUA)和β-微管蛋白基因(TUB)共9个管家基因序列。geNorm分析结果显示,RPL12和RPS20是最稳定表达的内参基因,而BestKeeper和NormFinder分析结果显示最稳定表达的内参基因分别是TUA和TUB。综合各分析方法得出9个候选基因中TUB,TUA,RPS20和RPL12是最稳定表达的内参基因,而18S rRNA,ACT和GAPDH这3个广泛应用的内参基因则表现出最低的表达稳定性。最后以OBP1为目标基因对稳定性不同的4个候选基因进行稳定性验证,发现以TUB和RPL12为内参基因,OBP1在成虫不同组织之间的表达模式基本一致;而以RPL32为内参基因,表达模式与应用TUB作为内参基因时稍有不同,使用18S rRNA作为内参基因得到的OBP1表达模式则与应用TUB作为内参基因时的完全不一致。【结论】TUB,TUA,RPS20和RPL12可以作为分析大灰象甲成虫不同组织中基因表达水平的内参基因,为后续基因表达研究奠定了基础。【Aim】 This study aims to screen out the suitable reference genes for gene expression analysis in different tissues of adult Sympiezomias velatus . 【Methods】 The house-keeping gene sequences of S.velatus were acquired and adopted as the candidate reference genes through transcriptome sequencing technique. Their mRNA expression levels in different tissues （antenna, head, thorax, abdomen and leg） of male and female adults of S. velatus were investigated by qRT-PCR. The expression stabilities of these candidate genes were evaluated by using three software-based （geNorm, NormFinder and BestKeeper） and one web-based （RefFinder） comprehensive analysis tools. And the expression stabilities of the candidate reference genes in different adult tissues of S. velatus were further validated by using the odorant binding protein 1 gene （ OBP 1） as the target gene. 【Results】 Nine house-keeping genes, including beta-actin gene （ ACT ）, glyceraldehyde-3-phosphate gene （ GAPDH ）, 18S ribosomal RNA （18S rRNA）, 60S ribosomal protein L12 gene （ RPL 12）, 60S ribosomal protein L32 gene （ RPL 32）, 40S ribosomal protein S20 gene （ RPS 20）, elongation factor 2 gene （ EF 2）, α-tubulin gene （ TUA ）, and β-tubulin gene （ TUB ）, were identified for the first time based on the S. velatus transcriptome data. It was revealed that RPL 12 and RPS 20 were the most stable reference genes based on the geNorm analysis, while TUA and TUB were the most stable reference genes based on the BestKeeper analysis and NormFinder analysis, respectively. Comprehensive analysis showed that TUB, TUA, RPS 20 and RPL 12 were the most stable reference genes, while three of the most widely used reference genes including 18S rRNA, ACT and GAPDH , were the least stable. Finally, the validation result of expression stability of four candidate reference genes with OBP 1 as the target gene showed that the expression patterns of OBP 1 in different tissues of adult S. velatus were basically coincident when TUB a

关 键 词：大灰象甲 实时定量PCR 内参基因 基因表达分析 表达稳定性 

分 类 号：Q966[生物学—昆虫学]