摘要
本研究旨在克隆绵羊基质Gla蛋白(MGP)基因的蛋白质编码序列(CDS),制备特异抗体,并检测该基因在绵羊卵巢组织中的表达分布。以卵泡总cDNA为模板,扩增获得绵羊MGP基因CDS区全长序列;合成绵羊MGP蛋白C末端15个氨基酸长度的多肽,免疫小鼠,制备抗血清;利用实时荧光定量PCR(qPCR)和免疫印迹技术分析其在不同组织和不同大小的卵泡中的表达水平;通过免疫组织化学方法分析其在卵巢组织中的表达分布特征。结果表明,成功获得绵羊MGP基因的CDS区全长片段(467 bp),编码103个氨基酸,理论相对分子量为12230.96,等电点为9.27;N末端19个氨基酸序列被预测为信号肽序列,成熟肽有84个氨基酸,为外泌性蛋白质,无胞内区;MGP总蛋白质氨基酸序列与山羊、牛的同源蛋白质相似性分别为100.0%、99.0%,与人、小鼠的相似性均为85.4%。MGP mRNA和蛋白质在绵羊心、肝、脾、肺、肾等组织中的相对表达量较低,在卵巢、输卵管、子宫等生殖系统或组织中的相对表达量较高,在黄体中的相对表达量也较高;MGP在卵巢组织颗粒细胞、卵丘细胞中均有表达,但在闭锁卵泡中不表达。本研究结果可为进一步研究MGP在卵巢组织中的生物学功能提供参考。
The aim of this study was to clone the protein coding sequence(CDS)of sheep matrix Gla protein(MGP)gene to prepare specific antibody and to detect the expression distribution of the gene in sheep ovarian tissues.Total sheep follicle cDNA was used as the template to amplify and obtain the full-length sequence of the CDS region of sheep MGP gene.Peptide with a length of 15 amino acids from the C-terminal end of the sheep MGP protein was synthesized and was used to immunize mouse and perpare antiserum.Expression levels of MGP in different tissues and follicles of varying sizes were analyzed by real-time fluorescence quantitative PCR(qPCR)and immunoblotting technology.Immunohistochemistry method was used to analyze the expression and distribution characteristics of MGP in ovarian tissue.The results revealed that the overall fragment(467 bp)of sheep MGP gene CDS region was obtained successfully,and the fragment encoded 103 amino acids,with a theoretical relative molecular weight of 12230.96 and an isoelectric point of 9.27.Sequence composed of 19 amino acids from the N-terminal was predicted as the signal peptide sequence,and the mature peptide contained 84 amino acids,which was extra-membranous domain protein without intracellular region.The total protein amino acid sequences of MGP shared similarities with those of the homologous proteins from goats and cows of 100.0%and 99.0%respectively,and the similarity with human and mouse was 85.4%.The relative expression levels of MGP mRNA and protein were low in sheep tissues such as heart,liver,spleen,lung and kidney,but were high in reproductive systems or tissues,such as ovary,fallopian tube and uterus,and the relative expression level was also high in corpus luteum.The expression levels of MGP granulosa cells and cumulus cells were high,whereas it didn’t expressed in atresia follicles.The study result can provide a basis for further investigation of the biological role of MGP in ovarian tissues.
作者
张欣如
古丽米热·阿不都热依木
陈莹
马秀玲
林嘉鹏
汪立芹
黄俊成
吴阳升
ZHANG Xinru;Gulimire·Abudureyimu;CHEN Ying;MA Xiuling;LIN Jiapeng;WANG Liqin;HUANG Juncheng;WU Yangsheng(Key Laboratory of Genetics Breeding and Reproduction of Grass Feeding Livestock,Ministry of Agriculture and Rural Affairs/Key Laboratory of Animal Biotechnology of Xinjiang/Institute of Biotechnology,Xinjiang Academy of Animal Science,Urumqi 830011,China;College of Animal Science,Xinjiang Agriculture University,Urumqi 830052,China)
出处
《江苏农业学报》
CSCD
北大核心
2024年第7期1276-1284,共9页
Jiangsu Journal of Agricultural Sciences
基金
新疆维吾尔自治区自然科学基金项目(2021D01A59)。
