猪硒蛋白P基因克隆、鉴定及组织mRNA相对表达量分析

汤加勇, 赵华, 何爱华, 周继昌, 贾刚, 刘光芒, 陈小玲, 蔡景义

doi:10.3969/j.issn.1006-267x.2016.03.027

动物营养学报 >

2016 , Vol. 28 >Issue 3: 858 - 863

DOI: https://doi.org/10.3969/j.issn.1006-267x.2016.03.027

研究简报 Short Communications

猪硒蛋白P基因克隆、鉴定及组织mRNA相对表达量分析

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1. 四川农业大学动物营养研究所, 成都 611130;
2. 深圳市慢性病防治中心分子生物学实验室, 深圳 518020

汤加勇(1981-),男,四川乐山人,实验师,硕士,研究领域为营养与分子生物学。E-mail:410699653@qq.com

收稿日期: 2015-09-20

网络出版日期: 2016-03-14

基金资助

国家自然科学基金面上项目(30871844,31272468);四川隆达畜牧科技有限公司项目(2015SCLD001)

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Cloning, Identification and Tissue mRNA Relative Expression Level Analysis of Porcine Selenoprotein P Gene

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1. Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, China;
2. Molecular Biology Lab, Shenzhen Center for chronic Disease Control, Shenzhen 518020, China

Received date: 2015-09-20

Online published: 2016-03-14

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摘要

本试验旨在克隆、鉴定猪硒蛋白P基因(Sepp1),并探明其在猪不同组织中的mRNA相对表达量,为以猪为模型研究硒蛋白P(SelP)的功能奠定基础。根据表达序列标签(EST)序列设计引物,利用cDNA末端快速克隆(3'-RACE)技术从猪肝脏总RNA中扩增出含开放阅读框(ORF)至polyA片段,然后与EST序列进行拼接;采用荧光定量PCR技术考察Sepp1在猪9个组织中的mRNA相对表达量。结果显示:1)扩增出共1 707 bp的片段,测序后与EST拼接获得了2 109 bp的猪Sepp1序列,并提交至NCBI GenBank数据库,序列号为EF113596.2;该基因1 170 bp的ORF编码区和对应的氨基酸残基与人相应序列分别有83.72%和75.64%序列同源性,其编码390个氨基酸,含有14个硒代半胱氨酸(Sec)残基,分别位于第59、267、286、309、311、327、339、352、354、361、376、378、385和387位。2)Sepp1 mRNA在猪组织中广泛分布,在肝脏中具有最高分布,依次为甲状腺 >肾脏 >睾丸 >下丘脑 >脾脏 >垂体 >心脏 >肌肉。本试验成功克隆、鉴定了猪Sepp1,检测了其在猪不同组织中表达分布情况,为其进一步以猪为模型探讨其功能奠定了基础。

关键词： 猪; Sepp1; 克隆; 荧光定量PCR

本文引用格式

汤加勇, 赵华, 何爱华, 周继昌, 贾刚, 刘光芒, 陈小玲, 蔡景义 . 猪硒蛋白P基因克隆、鉴定及组织mRNA相对表达量分析[J]. 动物营养学报, 2016 , 28(3) : 858 -863 . DOI: 10.3969/j.issn.1006-267x.2016.03.027

Abstract

The objective of this experiment was to clone and identify porcine selenoprotein P gene (Sepp1), and investigate its mRNA relative expression level in porcine tissues for further study of its roles using pig models. Total RNA was extracted from pig liver for 3'-RACE with primer designed according to a Sepp1-like EST sequence, and assembled with the EST sequence. We further investigated the mRNA relative expression level in 9 tissues of pigs by the qPCR technology. The results showed as follows: 1) 1 707 bp cDNA fragment of the Sepp1 containing the open reading frame (ORF) till to its poly (A) tail was isolated, and a 2 109 bp full length cDNA was acquired. The sequence of porcine Sepp1 was submitted to NCBI GenBank with accession number of EF113596.2. The 1 170 bp ORF share an 83.72% identity to that of human, while their amino acid sequences had 75.64% identity. The ORF of porcine Sepp1 encode 390 amino acids, which has 14 selenocysteine (Sec) encoded by TGA codon, the Sec residues located at positions of 59, 267, 286, 309, 311, 327, 339, 352, 354, 361, 376, 378, 385 and 387 from N-to C-terminal of the mature SelP. 2) The Sepp1 mRNA was wildly expressed in various tissues and exhibited the highest mRNA distribution in liver, followed by thyroid, kidney, testis, hypothalamus, spleen, pituitary, heart and muscle. In conclusion, the porcine Sepp1 is cloned and identified; also its mRNA distribution in porcine tissues is investigated in present study, which offers an effective alternative to further study its biology roles using pig models.

Key words： pig; Sepp1; clone; RT-qPCR

参考文献

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