牦牛FAF1基因的分子特征及其在不同阶段卵巢、输卵管、子宫中的表达

doi:10.13560/j.cnki.biotech.bull.1985.2019-0958

摘要/Abstract

摘要： FAF1（Fas-associated factor-1）在多种细胞中可与Fas蛋白结合,介导细胞凋亡的启动,其基因突变可导致分裂期胚胎死亡。旨在探索牦牛FAF1基因的分子特征及其在不同阶段的生物学作用。试验选取雌性牦牛3个不同时期（卵泡期、黄体期和妊娠期第3个月,以下将妊娠期第3个月简称为妊娠期）的卵巢、输卵管和子宫,克隆牦牛FAF1基因,采用实时荧光定量PCR（qRT-PCR）、免疫组织化学和Western blot（WB）方法对其基因和蛋白的表达水平进行检测和定位。成功克隆出牦牛FAF1基因的编码区（CDS）,长度为1 953 bp（GenBank登录号：MK416195）,编码650个氨基酸,基因特征分析显示该基因具有高度保守性,其编码的蛋白为含5个蛋白结合位点的非跨膜、可溶性蛋白,主要分布于细胞核（52.2%）、线粒体（26.1%）、细胞质（13.0%）、高尔基体（4.3%）、细胞骨架（4.3%）。qRT-PCR结果显示：卵巢中FAF1基因在卵泡期表达最高,妊娠期次之,黄体期最低;输卵管中黄体期最高,子宫中卵泡期最高,妊娠期次之,黄体期最低;蛋白水平显示：妊娠期输卵管和子宫FAF1蛋白相对表达量显著高于卵泡期和黄体期,卵巢在黄体期的表达量最高,卵泡期次之,妊娠期最低。免疫组织化学（Immunohistochemistry,IHC）结果显示不同阶段FAF1蛋白在同一组织中表达部位并无明显的差异,在卵巢中主要表达部位为卵巢生殖上皮、颗粒细胞、卵泡膜细胞和黄体细胞（黄体期）;在输卵管中主要表达部位为黏膜上皮细胞;在子宫中主要表达部位为子宫内膜细胞、子宫腺。FAF1基因和蛋白的表达存在显著差异,揭示其对牦牛的生殖生理调控具有重要意义。

关键词: FAF1, 生殖, 生物信息学分析, 表达

Abstract: FAF1（fas-associated factor-1）binds to Fas protein in a variety of cells,mediating the initiation of apoptosis,and its gene mutation maylead to the death of embryos in the mitotic phase. The purpose of this study is to explore the molecular characteristics of Bosgrunniens FAF1 gene and its biological role in reproductive cycle. The FAF1 gene was cloned from ovaries,fallopian tubes and uterus of female B.grunniensat 3 different stages（luteal,follicular and the third month of pregnancy, the third month of pregnancy is hereinafter referred to as pregnancy）. The expression levels of genes and proteins were detected and located by real-time fluorescence quantitative PCR（qRT-PCR）,immunohistochemistry and Western blot. As results,the coding region（CDS）of B. grunniens FAF1 gene was successfully cloned in this study,its length was 1 953 bp（GenBank：MK416195）,encoding 650 amino acids. Genetic traits analysis showed that the gene was highly conservative,its encoded protein was non-transmembrane and soluble protein containing 5 protein binding sites,mainly distributed in the nucleus（52.2%）,mitochondria（26.1%）,the cytoplasm（13.0%）,golgi body（4.3%）,and cytoskeleton（4.3%）. The results of qRT-PCR demonstrated that the expression of FAF1 gene in the ovary was the highestat the follicular stage,followed by the pregnancy stage and the lowest attheluteal stage.The expressionin the oviductwas the highestat theluteal stage. The expression in the ovarywas the highest atthe follicular stage,the second atthe pregnancy stage,and the lowestat the luteal stage. The protein level showed that the relative expression of FAF1 protein in the fallopian tube and uterus during pregnancy was significantly higher than that atthe follicular and lutealstages. The expression of FAF1 protein in the ovary was the highest in the lutealstage,followed by the follicular stage,and the lowest in the pregnancystage. Immunohistochemistry（IHC）results revealed that there was no significant difference in the expression sites of FAF1 protein in the same tissue at different stages. The main expression sites in ovary were ovarian reproductive epithelium,granulosa cells,follicular membrane cells and corpus luteum cells（follicular phase）. The main expression sites in the fallopian tube were mucosal epithelial cells. The main expression sites in the uterus were endometrial cells and uterine glands. In conclusion,the expressions of FAF1 gene and protein in different reproductive cycles of the same reproductive organ were significantly different,which indicated that it was of great significance to the physiological regulation of B.grunniens reproduction.

Key words: FAF1, reproduction, bioinformatics analysis, express

王静瑜, 王萌, 潘阳阳, 王靖雷, 张瑞, 马睿, 胡学权, 仇晓飞, 崔燕, 余四九, 徐庚全. 牦牛FAF1基因的分子特征及其在不同阶段卵巢、输卵管、子宫中的表达[J]. 生物技术通报, 2020, 36(7): 80-89.

WANG Jing-yu, WANG Meng, PAN Yang-yang, WANG Jing-lei, ZHANG Rui, MA Rui, HU Xue-quan, QIU Xiao-fei, CUI Yan, YU Si-jiu, XU Geng-quan. Molecular Characteristics of Bosgrunniens FAF1 Gene and Its Expression in Ovaries,Fallopian Tubes and Uterus at Different Stages[J]. Biotechnology Bulletin, 2020, 36(7): 80-89.

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