多浪羊BMP7基因克隆、生物信息学分析及其在不同初情期阶段组织表达分析

doi:10.16431/j.cnki.1671-7236.2023.07.004

摘要/Abstract

摘要： 【目的】对多浪羊骨形态发生蛋白-7（bone morphogenetic protein-7，BMP7）基因序列进行克隆和生物信息学分析，并检测多浪羊在初情期3个阶段5个组织中的表达规律，为探究BMP7基因在多浪羊初情期中的生物学功能提供参考。【方法】采用PCR扩增多浪羊BMP7基因序列并克隆测序，利用Mega 7.0软件构建系统发育树，利用生物信息学软件分析BMP7蛋白的理化性质和结构功能；采用实时荧光定量PCR检测BMP7基因在初情期前、初情期、初情期后多浪羊下丘脑、垂体、卵巢、输卵管、子宫5个组织中的表达量。【结果】试验成功获得多浪羊BMP7基因序列长1 334 bp，其中编码区长1 296 bp，编码431个氨基酸，与绵羊和牛的亲缘关系最近，其次是人、猴、马，与小鼠和犬的亲缘关系最远。生物信息学分析显示，BMP7蛋白分子式为C₂₂₀₀H₃₃₇₄N₆₃₂O₆₂₈S₁₇，理论等电点（pI）为8.17，原子总数为6 851个，不稳定指数为53.18，脂溶性系数76.96，总平均亲水性为―0.419，为不稳定亲水碱性蛋白；二级结构主要以α-螺旋及无规则卷曲为主，三级结构预测结果与二级结构一致。实时荧光定量PCR检测显示，BMP7基因在多浪羊初情期前、初情期、初情期后均有表达，其中在下丘脑中表达量显著高于其他组织（P<0.05）；在初情期前到初情期的下丘脑和子宫中BMP7基因表达量显著上升（P<0.05），初情期到初情期后BMP7基因表达量显著下降（P<0.05）。【结论】试验成功克隆了多浪羊BMP7基因序列，该基因在多浪羊初情期前后3个不同阶段的5个组织中均有表达，结果为探究BMP7基因在多浪羊初情期中的作用提供了参考。

关键词: 多浪羊; BMP7基因; 克隆; 初情期

Abstract: 【Objective】 The sequence of bone morphogenetic protein-7 (BMP7) gene in Dolang sheep was cloned and bioinformatics analyzed, and the expression patterns of five tissues in three stages of puberty were detected, which provided reference for exploring the biological function of BMP7 gene in Dolang sheep during the puberty.【Method】 The sequence of BMP7 gene in Dolang sheep was amplified by PCR, and cloned and sequenced, the phylogenetic tree was constructed using Mega 7.0 software.The physicochemical properties and structural function of BMP7 protein was analyzed by bioinformatics software.The expression of BMP7 gene in hypothalamus, pituitary gland, ovary, oviduct and uterus of Dolang sheep at prepuberty, puberty and postpuberty were detected by Real-time quantitative PCR.【Result】 The BMP7 gene sequence of Dolang sheep was successfully cloned, which was 1 334 bp long, and the CDS sequence was 1 296 bp which encoded 431 amino acids.The BMP7 gene sequence of Dolang sheep had the closer genetic relationship with Ovis aries and Bos taurus, followed by Homo sapiens, Macaca mulatta and Equus caballus, and had the furthest genetic relationship with Mus musculus and Canis lupus familiaris.Bioinformatics analysis results showed that the molecular formula of BMP7 protein was C₂₂₀₀H₃₃₇₄N₆₃₂O₆₂₈S₁₇, the theoretical isoelectric point (pI) was 8.17, the total number of atoms was 6 851, the instability index was 53.18, the lipid solubility coefficient was 76.96, and the total average hydrophilicity was-0.419.BMP7 protein was an unstable hydrophilic basic protein.The secondary structure was mainly consisted of alpha helix and random coil, and the prediction result of tertiary structure was consistent with the secondary structure.Real-time quantitative PCR results showed that BMP7 gene was expressed in prepuberty, puberty and postpuberty of Dolang sheep, and the expression in hypothalamus was significantly higher than that in other tissues (P<0.05).The expression of BMP7 gene was significantly increased in hypothalamus and uterus from prepuberty to puberty (P<0.05), while decreased significantly from puberty to postpuberty (P<0.05).【Conclusion】 The BMP7 gene sequence of Dolang sheep was cloned successfully, and this gene was expressed in five tissues at three different stages of puberty.The results provided a reference for exploring the role of BMP7 gene in puberty of Dolang sheep.

Key words: Dolang sheep; BMP7 gene; cloning; puberty

中图分类号:

王晨光, 隋志远, 张永杰, 张志帅, 李孝君, 邢凤. 多浪羊BMP7基因克隆、生物信息学分析及其在不同初情期阶段组织表达分析[J]. 中国畜牧兽医, 2023, 50(7): 2629-2638.

WANG Chenguang, SUI Zhiyuan, ZHANG Yongjie, ZHANG Zhishuai, LI Xiaojun, XING Feng. Cloning and Bioinformatics Analysis of BMP7 Gene and Its Tissue Expression at Different Stages of Puberty in Dolang Sheep[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(7): 2629-2638.

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