广西巴马小型猪BMP2基因克隆、生物信息学及组织表达分析

doi:10.16431/j.cnki.1671-7236.2021.07.004

摘要/Abstract

摘要： 本研究旨在对广西巴马小型猪骨形态发生蛋白2（bone morphogenetic proteins 2，BMP2）基因进行克隆及其结构和功能的预测，并检测其在广西巴马小型猪和长白猪中的表达差异。以广西巴马小型猪基因组DNA为模板，通过测序鉴定BMP2基因外显子区的单核苷酸多态性（SNPs），利用多种生物信息学软件对广西巴马小型猪BMP2基因启动子区、编码区（CDS）、3'UTR区进行了分析，预测分析广西巴马小型猪BMP2基因编码蛋白的基本理化性质、磷酸化位点、O-糖基化位点、跨膜区域、亲疏水性、蛋白质修饰结构、二级结构和三级结构等，并对miRNA结合位点及其相关通路进行分析，绘制了广西巴马小型猪和长白猪的组织表达谱。结果显示，试验成功克隆了广西巴马小型猪BMP2基因CDS区，总共1 188 bp，编码395个氨基酸；与猪参考基因组相比，广西巴马小型猪BMP2基因在G1663A、G1897C发生了同义突变，在C1896T（Pro→Leu）、T1971C（Gly→Ser）、G2000A（Leu→Ser）发生了3处错义突变；在3'UTR区域存在miR-142-5p、miR-129-5p等共13个miRNA结合区域；相似性比对结果显示，广西巴马小型猪与野猪、黄牛、水牛、山羊、绵羊、马、澳洲野犬、家犬、猕猴对应区段的相似性依次为99.92%、91.75%、91.84%、91.92%、91.92%、90.50%、89.43%、90.91%和90.91%；系统进化树结果表明，广西巴马小型猪与野猪的亲缘关系最近，与黄牛的亲缘关系相对较近，与猕猴亲缘关系较远；BMP2蛋白分子质量为44.55 ku，理论等电点为8.37，预测其有12个O-糖基化位点，42个磷酸化位点；预测分析发现该蛋白为亲水性蛋白，且是膜外蛋白。组织表达谱发现，广西巴马小型猪生长板软骨中BMP2基因表达量显著低于长白猪（P<0.05）。该研究为进一步探索广西巴马小型猪中BMP2基因的功能提供了理论基础，也为广西巴马小型猪品种资源的利用提供了重要参考。

关键词: 广西巴马小型猪; BMP2基因; 骨; 生物信息学分析

Abstract: This study was aimed to clone BMP2 gene of Guangxi Bama Mini pigs and conduct bioinformatics analysis such as structure and function prediction,and analyze the difference in BMP2 gene expression between Guangxi Bama Mini pigs and Landrace pigs.The genomic DNA of Guangxi Bama Mini pigs was used as template and single nucleotide polymorphism (SNPs) in the exon region of BMP2 gene were identified by sequencing.Then,using variety of bioinformatics software to carry out a series of analysis on the promoter region,coding region and 3'UTR region of BMP2 gene in Guangxi Bama Mini pigs.The basic physical and chemical properties,phosphorylation site,O-glycosylation site,transmembrane region,hydrophilicity and hydrophobicity,protein modification structure,and secondary and tertiary structure of BMP2 protein in Guangxi Bama Mini pigs were predicted and analyzed.The miRNA binding sites and related pathways were analyzed,and the tissue expression profiles of Guangxi Bama Mini pigs and Landrace pigs were drawn.The results showed that the CDS region of BMP2 gene in Guangxi Bama Mini pigs was 1 188 bp in length,encoding 395 amino acids.Compared with pig reference genome BMP2 gene of Guangxi Bama Mini pigs had synonymous mutations in G1663A and G1897C,three missense mutations in C1896T (Pro→Leu),T1971C (Gly→Ser) and G2000A (Leu→Ser),and 13 miRNA binding regions in 3'UTR region,including miR-142-5p and miR-129-5p.The homology of Guangxi Bama Mini pig with Sus scrofa,Bos taurus,Bubalus bubals,Capra hircus,Ovis aries,Equus cabalus,Canis lupus dingo,Canis lupus familiaris and Macaca mulatta was 99.92%,91.75%,91.84%,91.92%,91.92%,90.50%,89.43%,90.91% and 90.91%,respectively.The results of phylogenetic tree showed that Guangxi Bama Mini pigs had the highest similarity with Sus scrofa,had a relatively close genetic distance with Bos taurus,and had a relatively distant genetic relationship with Macaca mulatta.The molecular weight of BMP2 protein was 44.55 ku,and the theoretical isoelectric point was 8.37.It was predicted that there were 12 O-glycosylation sites and 42 phosphorylation sites.The prediction analysis showed that BMP2 protein was hydrophilic and was an extracellular protein.Tissue expression profile showed that the expression of BMP2 gene in growth plate cartilage of Guangxi Bama Mini pigs was significantly lower than Landrace pigs (P<0.05).This study provided a theoretical basis for further exploring the function of BMP2 gene in Guangxi Bama Mini pigs,and also provided an important reference for the utilization of Guangxi Bama Mini pigs breed resources.

Key words: Guangxi Bama Mini pigs; BMP2 gene; bone; bioinformatics analysis

中图分类号:

S828

奉玲丽, 司景磊, 莫家远, 刘笑笑, 田威龙, 程锋, 李月月, 郭亚芬, 梁晶, 兰干球. 广西巴马小型猪BMP2基因克隆、生物信息学及组织表达分析[J]. 中国畜牧兽医, 2021, 48(7): 2302-2312.

FENG Lingli, SI Jinglei, MO Jiayuan, LIU Xiaoxiao, TIAN Weilong, CHENG Feng, LI Yueyue, GUO Yafen, LIANG Jing, LAN Ganqiu. Cloning,Bioinformatics and Tissue Expression Analysis of BMP2 Gene in Guangxi Bama Mini Pig[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(7): 2302-2312.

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