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2018, 23(2): 91-98.

杂色鲍幼虫变态及TR干扰下内参基因的筛选

1. 集美大学水产学院, 福建 厦门 361021;

2. 农业部东海海水健康养殖重点实验室, 福建 厦门 361021

收稿日期:2017-09-07
修回日期:2017-10-26

基金项目:   国家自然科学基金项目(41006105,41176152,3170130721);福建省自然科学基金项目(2015J01142,2016J01163);福建省高校新世纪优秀人才支持计划项目(B15138);青岛海洋科学与技术国家实验室开放基金项目(OF2015NO11) 

关键词: 杂色鲍 , 内参基因 , 稳定性

The Stability Comparison of Reference Genes in Metamorphosis and TR RNAi of Haliotis diversicolor Larvae

1. Fisheries College, Jimei University, Xiamen 361021, China;

2. Key Laboratory of Healthy Mariculture for the East China Sea, Ministry of Agriculture, Xiamen 361021, China

Received Date:2017-09-07
Accepted Date:2017-10-26

Keywords: Haliotis diversicolor , reference gene , stability

内参基因的稳定性对实时定量PCR的结果影响巨大。采用GeNorm、normFinder和RefFinder三款软件比较了鲍变态和干扰幼虫甲状腺激素受体(TR)两种情况下内参基因的稳定性。软件分析了EIF5AOAZ1YB1RPL3RPS9ACT等6个候选内参基因的Ct值,据此获得各基因的稳定值。在杂色鲍幼虫的变态过程中,EIF5A稳定性最高,排名平均数为1,表明该基因可以作为这一阶段实时定量PCR的内参基因,但是在干扰TR情况下,EIF5A的稳定性下降,排名平均数为3.91。RPS9则与之相反,在变态过程该基因表达稳定性最差,而在干扰TR时表达稳定性最好。ACT在两种情况下稳定性较好,排名平均数均位列第二位,可以作为这两种情况下的通用内参基因。其他基因的稳定性在这两种情况下也发生剧烈变换。这表明内参基因的稳定与样品具体处理情况关系密切。为了保证定量PCR结果的准确性,需要针对具体处理情况相应地进行内参基因的稳定性评估。

The stability of reference gene strongly impacts the results of real time quantitative PCR(RT-qPCR).The stability of reference gene was analyzed by GeNorm,normFinder and RefFinder in larvae during metamorphosis or under TR RNAi.EIF5A,OAZ1,YB1,RPL3,RPS9 and ACT were choosed as candidate reference genes.The Ct of candidate reference gene was calculated to evaluate its stability by the three softwares.The stability of EIF5A was the highest and ranked the 1st during metamorphosis,which means EIF5A is a good reference gene during metamorphosis,However,its stability decreased in larvae under TR RNAi.On the contrary,RPS9 had a worst stability of the six candidate reference genes during metamorphosis,and had a best stability in larvae under TR RNAi.Only ACT had a good stability and its stability ranked 2nd in metamorphosis and TR RNAi.This mean that ACT is a universal reference gene.The stabilities of other genes also had dramatic changes between metamorphosis and TR RNAi.These results meanthat a gene's stability has a strong tie with the treatment of samples.In order to ensure accurate results of RT-qPCR,each reference gene should be analyzed in a specific experiment.

参考文献

[1] 董晓丽,王加启,卜登攀,等.内参基因在实时定量PCR中应用的研究进展[J].中国畜牧兽医,2009(9):83-85.
[2] 朱芷葳,董常生.持家基因作为相对定量内标物的稳定性比较[J].生物技术通讯,2006,17(5):807-809.
[3] CHEN J.Stable expression of Y-box protein 1 gene in early development of the abalone Haliotis diversicolor[J].International Journal of Developmental Biology,2012,56(5):369-375.
[4] 王国栋,张丽莉,王艺磊.鲍幼虫变态分子机制的研究进展[J].集美大学学报(自然科学版),2012,17(2):101-108.
[5] VANDESOMPELE J,DE PRETER K,PATTYNF,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):research0034.
[6] ANDERSEN C L,JENSEN J L,ØRNTOFT 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.
[7] ZHU X,LI X,CHEN W,et al.Evaluation of new reference genes in papaya for accurate transcript normalization under different experimental conditions[J].PLoS ONE,2012,7(8):e44405.
[8] SILVER N,BEST S,JIANG J,et al.Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR[J].BMC Molecular Biology,2006,7(1):33.
[9] 陈烨,李凯,周宇荀,等.小鼠基因转录表达分析中内参基因的优选[J].中国实验动物学报,2011,19(3):197-202.
[10] 冯焱,李建芳,张芬鹊,等.LPS诱导的免疫应激对肉鸡组织内参基因稳定性的影响[J].中国家禽,2015,37(2):31-36.
[11] ZHENG W,SUN L.Evaluation of housekeeping genes as references for quantitative real time RT-PCR analysis of gene expression in Japanese flounder(Paralichthys olivaceus)[J].Fish & Shellfish Immunology,2011,30(2):638-645.
[12] 吴玉,翟渊粉,黄明霞,等.家蚕常用内参基因稳定性分析及丝蛋白相关基因表达调控研究[J].中国细胞生物学学报,2013(4):423-431.
[13] 鲍相渤,刘卫东,姜冰,等.内参基因在虾夷扇贝定量PCR中表达稳定性的比较[J].水产科学,2011,30(10):603-608.
[14] LI A L,LI H Y,JIN B F,et al.Anovel eIF5A complex functions as a regulator of p53 and p53-dependent apoptosis[J].J Biol Chem,2004,279(47):49251-49258.
[15] KANG H A,HERSHEY J W.Effect of initiation factor eIF-5A depletion on protein synthesis and proliferation of Saccharomyces cerevisiae[J].J Biol Chem,1994,269:3934-3940.
[16] ZUK D,JACOBSON A.A single amino acid substitution in yeast eIF-5A results in mRNA stabilization[J].EMBO J,1998,17:2914-2925.
[17] LIU Z,DUGUAY J,MA F,et al.Modulation of eIF5A1 expression alters xylem abundance in Arabidopsis thaliana[J].J Exp Bot,2008,59:939-950.
[18] 肖云峰,刘辉,吕国栋,等.细粒棘球绦虫核糖体蛋白S9基因克隆鉴定及其在不同发育阶段表达分析[J].中国病原生物学杂志,2014(2):150-154.
[19] LI N.Insulin-like growth factor binding protein 7,a member of insulin-like growth factor signal pathway,involved in immune response of small abalone Haliotis diversicolor[J].Fish & Shellfish Immunology,2012,33(2):229-242.
[20] KOHNO K,IZUMI H,UCHIUMI T,et al.The pleiotropic functions of the Y-box-binding protein,YB-1[J].Bioessays,2003,25(7):691-8.
[21] HAYASHI S,MURAKAMI Y,MATSUFUJI S.Ornithine decarboxylase antizyme:a novel type of regulatory protein[J].Trends in Biochemical Sciences,1996,21(1):27-30.

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杂色鲍幼虫变态及TR干扰下内参基因的筛选