猪ADAM10基因编码区及其剪接体克

发布时间：2018-02-13 11:04

  本文关键词： 猪 ADAM基因 克隆 剪接体 组织表达　出处：《畜牧兽医学报》2017年09期 　论文类型：期刊论文

【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ADAM10CDS序列,其中完整型CDS长2 247bp,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 344bp,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。
[Abstract]:To clone the coding region of porcine ADAM10 gene and its splicing sequence sequence, the sequence structure and biological function of porcine ADAM10 gene were analyzed by bioinformatics. According to the sequence information of porcine ADAM10 gene published in GenBank, specific primers were designed. Reverse transcription PCR(Reverse transcription-PCRR-RT-PCR method and T / A cloning technique were used to clone the coding region of ADAM10 gene and splice. Bioinformatics was used to analyze the structure of ADAM10 and its splice protein. At the same time, semi-quantitative PCR(Semi-quantitative PCR was used to analyze the expression profiles of porcine ADAM10 gene intact type and splice mRNA in different tissues. The results showed that two kinds of ADAM10CDS sequences were obtained by cloning and sequencing, among which the complete CDS was 2247bplong, encoding 748 amino acids. Splicing type CDS deletion exon 2, exon 7 and partial exon 8, with a length of 1 344 BP, encode 447 amino acids. Compared with the complete protein, porcine ADAM10 splice encoding protein does not contain a preleading sequence, but has a complete signal peptide sequence and a transmembrane domain. It also contains metalloproteinase domain and depolymerization domain, and its tertiary structure is consistent with the complete type. Sequence alignment and evolutionary tree analysis show that, The semi-quantitative PCR analysis showed that the intact type mRNA of ADAM10 gene was highly expressed in spleen, including kidney, mammary gland, leg muscle, fallopian tube, ovary, uterus, oviduct, ovary, uterus, oviduct, ovaries, uterus, kidney, mammary gland, leg muscle, oviduct, ovary and uterus. The expression of ADAM10 gene splicing mRNA in small intestine and other tissues was higher in lung and lower in spleen and stomach. This study provided basic data for further study on the structure and biological function of porcine ADAM10 gene.
【作者单位】： 浙江农林大学动物科技学院;
【基金】：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)
【分类号】：Q78;S828

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