陆川猪Occludin基因克隆及生物信息学分析

doi:10.16431/j.cnki.1671-7236.2019.11.002

摘要/Abstract

摘要： 本试验旨在获得陆川猪Occludin基因的编码区（CDS）序列，并对其进行生物信息学分析。根据GenBank中公布的猪Occludin基因序列（登录号：NM_001163647.2）设计1对特异性引物，采集健康陆川猪的回肠组织提取总RNA并反转录成cDNA，以cDNA作为模板进行RT-PCR扩增并获得目的基因片段，将其插入pMD18-T载体，筛选阳性克隆菌测序正确后并利用生物信息学软件对所获序列进行分析。结果表明，试验成功克隆获得陆川猪Occludin基因CDS，长度为1 569 bp，共编码522个氨基酸。序列比对结果显示，陆川猪Occludin基因与参考序列的同源性为99.7%，存在3个差异位点，其中第16 bp处C→T为错义突变，引起第6位的亮氨酸变成苯丙氨酸；第1 059 bp处A→G和第1 218 bp处C→T均为同义突变，该错义突变位点可能是陆川猪肠道屏障功能与其他猪种不同的原因。同源性比对结果显示，陆川猪Occludin基因与小鼠、牛、人、果蝇、猕猴和犬的同源性分别为83.8%、89.5%、88.1%、84.1%、88.3%和86.6%。系统进化树分析发现，陆川猪与牛的遗传距离最近，与犬的遗传距离最远。Occludin蛋白分子质量为59.13 ku，氨基酸组成中丝氨酸含量较高（9.2%），在肽链N端为Met，Occludin蛋白在水溶液中280 nm消光系数为96 415，不稳定指数为62.85，属于不稳定蛋白。疏水性分析结果表明，Occludin蛋白是亲水性蛋白。陆川猪Occludin蛋白存在5个跨膜螺旋区，不存在信号肽，包含2个超家族结构域：MARVEL超家族结构域和Occludin-ELL超家族结构域。二级结构预测结果显示，陆川猪Occludin蛋白中α-螺旋、延伸链及无规则卷曲分别占41.00%、5.36%和53.64%，三级结构与二级结构相一致。本试验成功克隆了陆川猪Occludin基因CDS并进行了生物信息学分析，为深入探讨Occludin基因对地方猪种肠道屏障功能的影响提供参考。

关键词: 陆川猪; Occludin基因; 克隆; 生物信息学分析

Abstract: The aim of this study was to obtain the coding sequence (CDS) of Occludin gene in Luchuan pig and carry out bioinformatics analysis of the sequence.A pair of specific primers were designed according to porcine Occludin gene sequence (accession No.:NM_001163647.2) published on GenBank,the jejunum tissue samples of Luchuan pig were collected,and RNA was extracted and then was reverse transcribed to synthesize cDNA.The target fragment was obtained by RT-PCR amplification,PCR products then were inserted into the pMD18-T vector,and then screened positive clones was sequenced.The obtained sequence was analyzed by bioinformatics software after sequencing.The results showed that the size of Occludin gene CDS in Luchuan pig was 1 569 bp,encoding 522 amino acids.The results of sequence alignment showed that the homology was 99.7% with reference sequence,and there were 3 different sites,the C→T was a missense mutation in the 16th position,which caused the leucine turns to phenylalanine at the 6th position,and the A→G at 1 059 bp position and the C→T at 1 218 bp position were synonymous mutations.This missense mutation site might be the reason of the intestinal barrier function in Luchuan pig was different from other pig breeds.The homology of Occludin gene in Luchuan pig were 83.8%,89.5%,88.1%,84.1%,88.3% and 86.6% with Mus musculus,Bos taurus,Homo sapiens,Mesocricetus auratus,Macaca nemestrina and Canis lupus,respectively.Phylogenetic tree analysis showed that the genetic distance between Luchuan pig and Bos taurus was the closest,and the genetic distance was the furthest with Canis lupus.The molecular weight of Occludin protein was 59.13 ku,the amino acid composition of the serine had a high content (9.2%).At the N-terminus of the peptide chain (Met),Occludin protein had an extinction coefficient of 96 415 in an aqueous solution and an instability index of 62.85,which was an unstable protein.Hydrophobic analysis results indicated that Occludin protein was a hydrophilic protein.There were five transmembrane helix regions of Occludin protein and two superfamily domains (MARVEL superfamily domain and Occludin-ELL superfamily domain) in Luchuan pig,and it had no signal peptide.The secondary structure prediction results showed that the alpha helix,extended chain and random coil of Occludin protein in Luchuan pig accounted for 41.00%,5.36% and 53.64%,respectively,and the tertiary structure was consistent with the secondary structure.This study successfully cloned and analyzed the sequence of Occludin gene CDS in Luchuan pig,which provided a reference for further study of the effect of Occludin gene on the intestinal barrier function of local pig breeds.

Key words: Luchuan pig; Occludin gene; cloning; bioinformatics analysis

中图分类号:

瞿秋红, 谢红月, 夏琴, 潘鹏, 蒋钦杨, 黄艳娜. 陆川猪Occludin基因克隆及生物信息学分析[J]. 中国畜牧兽医, 2019, 46(11): 3144-3153.

QU Qiuhong, XIE Hongyue, XIA Qin, PAN Peng, JIANG Qinyang, HUANG Yanna. Cloning and Bioinformatics Analysis of Occludin Gene in Luchuan Pig[J]. China Animal Husbandry and Veterinary Medicine, 2019, 46(11): 3144-3153.

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