骨桥蛋白基因在黔北麻羊性腺轴中的表达及其对颗粒细胞中繁殖相关基因的影响  

Expression of Osteopontin Gene in the Gonad Axis of Qianbei Ma Goat and Its Effect on Reproductive Related Genes in Granulosa Cells

作　　者：赵佳福[1,2] 周明帅 杨秀远 徐畅[1,2] ZHAO Jiafu;ZHOU Mingshuai;YANG Xiuyuan;XU Chang(Key Laboratory of Animal Genetics,Breeding and Reproduction in the Plateau Mountainous Region,Ministry of Education,Guizhou University,Guiyang,550025;College of Animal Science,Guizhou University,Guiyang,550025)

机构地区：[1]贵州大学高原山地动物遗传育种与繁殖教育部重点实验室,贵阳550025 [2]贵州大学动物科学学院,贵阳550025

出　　处：《基因组学与应用生物学》2023年第3期285-294,共10页Genomics and Applied Biology

基　　金：贵州省科技支撑计划(黔科合支撑[2022]一般089);高原山地动物遗传育种与繁殖教育部重点实验室(贵州大学)开放课题基金资助项目(黔教合KY字[2020]244);贵州大学引进人才科研项目[贵大人基合字(2020)074号];黔南州科技计划项目[黔南科合(2021)27号]共同资助。

摘　　要：本研究的目的是探究骨桥蛋白(osteopontin,OPN)基因与黔北麻羊(Capra hircus)繁殖性状间的关系。本研究通过ProtParam和NetPhos等生物在线软件对黔北麻羊OPN的理化性质和结构功能进行分析,克隆OPN基因,构建OPN基因的真核表达载体。运用实时定量PCR法检测OPN基因在单羔、多羔黔北麻羊的垂体、下丘脑、子宫、卵巢和输卵管中的表达情况。随后,将OPN基因的真核表达载体转染至卵巢颗粒细胞中,检测过表达OPN基因对繁殖相关基因骨形态发生蛋白15(bone morphogenetic protein 15,BMP15)、骨形态发生蛋白受体1B(bone morphogenetic protein receptor 1B,BMPR-1B)和促卵泡激素β亚基(follicle stimulating hormoneβ-subunit,FSHβ)表达量的影响。生物信息学分析结果表明,OPN是一种具有磷酸化位点的不稳定亲水性蛋白质,其蛋白质分子的质量为31115.11 Da,理论等电点为4.39,化学式为C_(1309)H_(2046)N_(368)O_(501)S_(5),共编码279个氨基酸,其中携带的正电荷残基总数(Arg+Lys)为28个,负电荷残基总数(Asp+Glu)为66个。与其他动物相比,黔北麻羊OPN基因与反刍动物绵羊和牛的同源性较高,遗传距离较近。实时定量PCR结果表明,OPN基因在多羔组黔北麻羊的垂体、下丘脑、卵巢中的表达量极显著高于单羔组的(P<0.01),且过表达OPN基因后,极显著上调了繁殖相关基因BMP15的表达(P<0.01)。以上结果说明,OPN基因的表达量与黔北麻羊繁殖率呈正相关关系,可作为影响黔北麻羊繁殖力的多羔候选基因进行深入研究。The aim of this study was to investigate the relationship between the osteopontin(OPN)gene and reproductive traits of Qianbei Ma Coat(Capra hircus).In this study,the physical and chemical properties and structural functions of OPN were analyzed by biological online software such as ProtParam and NetPhos,and the OPN gene was cloned and the eukaryotic expression vector of OPN gene was constructed.Real time quantitative PCR was used to analyze and detect the expression of OPN gene in the pituitary gland,hypothalamus,uterus,ovary and fallopian tube of Qianbei Ma goat in monotocous and polytocous lambs.Subsequently,the eukaryotic expression vector of OPN gene was transfected into ovarian granulosa cells to detect the effects of overexpression of OPN gene on the expression levels of reproductive-related genes bone morphogenetic protein 15(BMP15),bone morphogenetic protein receptor 1B(BMPR-1B)and follicle stimulating hormoneβ-subunit(FSHβ).Bioinformatics analysis results showed that OPN was an unstable hydrophilic protein with phosphorylation site.The mass of protein molecules was 31115.11 Da,theoretical isoelectric point was 4.39,chemical formula was C_(1309)H_(2046)N_(368)O_(501)S_(5),encoding 279 amino acids.The total number of positively charged residues(Arg+Lys)and negatively charged residues(Asp+Glu)was 28 and 66 respectively.Therefore,OPN was a negatively charged amino acid sequence.Compared with other animals,the OPN gene of Qianbei Ma goat had higher homology and closer genetic distance with sheep and cattle.The real time quantitative PCR results showed that the expression levels of OPN gene in the pituitary gland,hypothalamus and ovary of the multi-lambing group were significantly higher than those of the single lambing group(P<0.01),and the overexpression of OPN gene could significantly up-regulate the expression of reproductive related gene BMP15(P<0.01).These results indicate that the expression of OPN gene is positively correlated with the reproductive rate of goats,and it can be used as a candidate gene

关 键 词：黔北麻羊 骨桥蛋白基因 生物信息学分析 卵巢颗粒细胞 繁殖相关基因 

分 类 号：S827[农业科学—畜牧学]