张家炜,郑哲,王庆恒,黄荣莲,杜晓东

• 1:广东海洋大学水产学院

摘要(Abstract)：

【目的】筛选光裸星虫体壁、肠道、肾管、吻、收吻肌以及体腔液细胞中稳定表达的内参基因。【方法】应用荧光定量PCR技术,对GAPDH、β-actin、TUB、TBP以及UBC等常用内参基因的表达情况进行分析,通过ge Norm、Norm Finder、Best Keeper以及在线内参筛选工具Ref Finder对8个内参基因表达稳定性进行评测。【结果】8个内参基因均可获得特异性扩增产物,但稳定性各异。通过累计积分法综合了4个软件给出的稳定性排序结果,候选内参基因表达稳定性综合排名为TBP(1)>TBP(2)>UBC>GAPDH>TUB(2)>TUB(1)>β-actin(2)>β-actin(1)。【结论】TBP(1)在光裸星虫全组织中的表达最稳定,可作为最适单内参基因。

关键词(KeyWords)： 荧光定量PCR;内参基因;光裸星虫

Abstract:

Keywords:

基金项目(Foundation): 广东省科技计划(2016A020209010);; 广东省自然科学基金(2017A030303075)

作者(Author): 张家炜,郑哲,王庆恒,黄荣莲,杜晓东

参考文献(References)：

• [1]CUTLER E B.The sipuncula:their systematics,biology,and evolution[M].New York:Cornell University Press,1994:36-38.
• [2]罗少杰,杨创业,王庆恒,等.光裸星虫4个野生群体的营养成分分析与品质评价[J].广东海洋大学学报,2016,36(1):25-30.
• [3]LIU Y,QIU C P.Calculated taste activity values and umami equivalences explain why dried Sha-Chong(Sipunculus nudus)is a valuable condiment[J].Journal of Aquatic Food Product Technology,2016,25(2):177-184.
• [4]ZHANG Q,DONG L F,TONG T,et al.Polysaccharides in Sipunculus nudus:Extraction condition optimization and antioxidant activities[J].Journal of Ocean University of China,2017,16(1):74-80.
• [5]LI J W,ZHU C B,GUO Y J,et al.Experimental study of bioturbation by Sipunculus nudus in a polyculture system[J].Aquaculture,2015,437:175-181.
• [6]李俊伟,朱长波,郭永坚,等.光裸方格星虫(Sipunculus nudus)生物扰动对混养系统沉积物及间隙水中营养物质的影响[J].渔业科学进展,2015,36(1):103-110.
• [7]王玉倩,薛秀花.实时荧光定量PCR技术研究进展及其应用[J].生物学通报,2016,51(2):1-6.
• [8]程海星,郭月英,任霆,等.实时荧光定量PCR技术原理及在食品检测中的应用[J].食品与发酵工业,2015,41(3):243-247.
• [9]陈旭,齐凤坤,康立功,等.实时荧光定量PCR技术研究进展及其应用[J].东北农业大学学报,2010,41(8):148-155.
• [10]ZHENG Z,LIANG J L,HUANG R L,et al.Identification of a novel mi R-146a from Pinctada martensii involved in the regulation of the inflammatory response[J].Fish&Shellfish Immunology,2016,54:40-45.
• [11]WANG Q H,YANG C Y,HAO R J,et al.Molecular characterization of CHST11 and its potential role in nacre formation in pearl oyster Pinctada fucata martensii[J].Electronic Journal of Biotechnology,2017,28:113-119.
• [12]PFAFFL M W.A new mathematical model for relative quantification in real-time RT–PCR[J].Nucleic Acids Research,2001,29(9):e45.
• [13]胡艳,宋迟,徐文娟,等.在发育性变化相关基因表达特性检测中内参基因的有效性和实时定量PCR解析方法的适用性[J].基因组学与应用生物学,2013,32(2):216-226.
• [14]VANDESOMPELE J,DE PRETER K D,PATTYN F,et al.Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[J].Genome Biology,2002,3(7):34.
• [15]ANDERSEN C L,JENSEN J L,?RNTOFT T F.Normalization of real-time quantitative reverse transcription-PCR data:a model-based variance estimation approach to identify genes suited for normalization,applied to bladder and colon cancer data sets[J].Cancer Research,2004,64(15):5245-5250.
• [16]PFAFFL M W,TICHOPAD A,PRGOMET C,et al.Determination of stable housekeeping genes,differentially regulated target genes and sample integrity:Best Keeper–Excel-based tool using pair-wise correlations[J].Biotechnology Letters,2004,26(6):509-515.
• [17]张翠彩,逄波,蒋秀高,等.霍乱弧菌基因表达分析中内参基因的选择[J].中国人兽共患病学报,2014,30(5):433-438.
• [18]张玉芳,赵丽娟,曾幼玲.基因表达研究中内参基因的选择与应用[J].植物生理学报,2014,50(8):1119-1125.
• [19]冯波,郭前爽,毛必鹏,等.松墨天牛化学感受组织荧光定量PCR内参基因的鉴定与筛选[J].昆虫学报,2016,59(4):427-437.
• [20]YANG H L,LIU J,HUANG S M,et al.Selection and evaluation of novel reference genes for quantitative reverse transcription PCR(q RT-PCR)based on genome and transcriptome data in Brassica napus L.[J].Gene,2014,538(1):113-122.
• [21]BAGéS S,ESTANY J,TOR M,et al.Investigating reference genes for quantitative real-time PCR analysis across four chicken tissues[J].Gene,2015,561(1):82-87.
• [22]杨丹,李清,王贵禧,等.平欧杂种榛实时荧光定量PCR内参基因的筛选与体系建立[J].中国农业科学,2017,50(12):2399-2410.
• [23]唐培安,张棋麟,薛昊,等.印度谷螟实时荧光定量PCR内参基因的选择[J].中国科学:生命科学,2016,46(10):1201-1209.
• [24]蒋婷婷,高燕会,童再康.石蒜属植物实时荧光定量PCR内参基因的选择[J].园艺学报,2015,42(6):1129-1138.
• [25]李瑞雪,余三淼,李夏,等.红苞凤梨实时荧光定量PCR分析中内参基因的筛选[J].热带亚热带植物学报,2017,25(3):250-256.
• [26]杨楠.1.TBP和TLP对h TERT的转录调节作用及机制2.DNA甲基化在宫颈癌诊断中的应用[D].北京:北京大学,2008.
• [27]KORNBERG R D.The molecular basis of eukaryotic transcription[J].Proceedings of the National Academy of Sciences of the United States of America,2007,104(32):12955-12961.
• [28]HARDIVILLE S,HAN G H,MA J F,et al.Nutrient regulation of TATA-Box binding protein by O-Glc NAcylation[J].The FASEB Journal,2017,31(S1):908.7.
• [29]RADONI?A,THULKE S,BAE H G,et al.Reference gene selection for quantitative real-time PCR analysis in virus infected cells:SARS corona virus,Yellow fever virus,Human Herpesvirus-6,Camelpox virus and Cytomegalovirus infections[J].Virology Journal,2005,2:7.
• [30]胡锦兴,彭德虎,梁小朋,等.低氧性肺动脉高压大鼠肺动脉平滑肌RT-PCR检测中内参基因的选择[J].广州医药,2015,46(4):1-6.
• [31]彭馥芝,冉茂良,翁波,等.猪睾丸组织定量PCR分析中内参基因的选择[J].中国农业科学,2017,50(15):3033-3041.
• [32]DOMINGUEZ R,HOLMES K C.Actin structure and function[J].Annual Review of Biophysics,2011,40:169-186.
• [33]陈明,李爱媛.肌动蛋白、肌动蛋白结合蛋白质和细胞运动的研究进展[J].生命科学,1997,9(1):1-5.
• [34]刘洪峰,高乐旋,胡永红.牡丹不同发育阶段种子和花瓣组织实时荧光定量PCR中内参基因的挖掘与筛选[J].农业生物技术学报,2015,23(12):1639-1648.
• [35]计红,王忠伟,郭景茹,等.产蛋前期和产蛋期籽鹅组织内参基因的稳定性[J].中国农业科学,2011,45(11):2260-2266.
• [36]张艳君,朱志峰,陆融,等.基因表达转录分析中内参基因的选择[J].生物化学与生物物理进展,2007,34(5):546-550.