畜牧兽医学报 ›› 2017, Vol. 48 ›› Issue (9): 1579-1590.doi: 10.11843/j.issn.0366-6964.2017.09.003

• 遗传育种 • 上一篇    下一篇

ADAM10基因编码区及其剪接体克隆、序列分析与组织表达研究

陈闻达, 裴悦, 周晓龙, 赵阿勇, 杨松柏*   

  1. 浙江农林大学动物科技学院, 临安 311300
  • 收稿日期:2017-03-09 出版日期:2017-09-23 发布日期:2017-09-14
  • 通讯作者: 杨松柏,讲师,博士,主要从事猪功能基因组与分子育种研究,E-mail:sbyang@zafu.edu.cn
  • 作者简介:陈闻达(1995-),男,浙江宁波人,本科生,主要从事猪分子育种研究,E-mail:cwd619619@163.com
  • 基金资助:

    国家自然科学基金(31501921);浙江省自然科学基金(LQ15C170001);浙江省农业(畜禽)新品种选育重大科技专项(2016C02054-3)

Coding Sequence Cloning, Alternative Splicing, Sequence Analysis and Tissue Expression of Porcine ADAM10 Gene

CHEN Wen-da, PEI Yue, ZHOU Xiao-long, ZHAO A-yong, YANG Song-bai*   

  1. College of Animal Science and Technology, Zhejiang A & F University, Lin'an 311300, China
  • Received:2017-03-09 Online:2017-09-23 Published:2017-09-14

摘要:

旨在克隆猪ADAM10基因及其剪接体的编码区(Coding sequence,CDS),利用生物信息学方法分析其序列结构和生物学功能,并揭示ADAM10基因及其剪接体mRNA在猪组织中的表达特征。根据GenBank中公布的猪ADAM10基因的序列信息,设计特异性引物,运用反转录PCR(Reverse transcription-PCR,RT-PCR)方法结合T/A克隆技术进行ADAM10基因及其剪接体编码区的克隆,利用生物信息学方法分析ADAM10及其剪接体蛋白的结构,同时用半定量PCR(Semi-quantitative PCR)分析猪ADAM10基因完整型及其剪接体mRNA在猪不同组织中的表达谱。结果显示,克隆和测序获得两种ADAM10 CDS序列,其中完整型CDS长2 247 bp,编码748个氨基酸;剪接型CDS缺失外显子2~7和部分外显子8,其长度为1 344 bp,编码447个氨基酸。和完整型蛋白相比,猪ADAM10剪接体编码的蛋白不存在前引导序列,但存在完整的信号肽序列和跨膜结构域,并含有金属蛋白酶域和解聚素结构域,其三级结构也和完整型一致。序列比对和进化树分析表明,猪ADAM10完整型蛋白在物种间具有较高的保守性。半定量PCR分析表明,ADAM10基因完整型mRNA在脾中表达量较高,在肾、乳腺、腿肌、输卵管、卵巢、子宫、小肠等组织中低水平表达;ADAM10基因剪接体mRNA在肺中表达量较高,在脾和胃中也有低水平表达。本研究为进一步探究猪ADAM10基因的结构和生物学功能提供了基础资料。

关键词: 猪, ADAM10基因, 克隆, 剪接体, 组织表达

Abstract:

The objective of this study was to clone the coding sequence (CDS) of porcine ADAM10 gene and its splicing variant, analyze the gene structure and functions using bioinformatics methods, and investigate the mRNA levels of ADAM10 gene and its splicing variant in different tissues in porcine. According to the sequence of porcine ADAM10 gene in GenBank, the specific primers were designed for cloning the CDS of ADAM10 gene and its splicing variant using reverse transcription-PCR (RT-PCR) and T/A cloning methods. The protein structure of ADAM10 and its splicing variant were analyzed through bioinformatics methods. The mRNA expression patterns of porcine ADAM10 gene and its splicing variant in different tissues in porcine were analyzed by semi-quantitative PCR. The results showed that the complete CDS of ADAM10 gene was 2 247 bp, encoding 748 amino acids. The CDS of splicing variant, missing the sequence of exon 2-7 and partial exon 8, had a length of 1 344 bp which encoded 447 amino acids. Compared with the intact protein, the splicing variant protein missed the leading peptide sequence. However, the splicing variant protein contained signal peptide, transmembrane domain and metalloproteinase and depolymerized domains. Futhermore, the tertiary structures of the 2 proteins were identical. Sequence alignment and phylogenetic analysis showed that the intact ADAM10 protein was highly conserved among species. Semi-quantitative PCR analysis showed that the intact mRNA of ADAM10 gene was highly expressed in spleen and lowly in kidney, mammary gland, leg muscle, fallopian tube, ovary, uterus and small intestine; the mRNA of splicing variant was highly expressed in lung and lowly in spleen and stomach. Our study provides the basic materials for further studying on the structure and biological function of porcine ADAM10 gene.

Key words: pig, ADAM 10 gene, cloning, splicing variant, tissue expression

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