牦牛FGG组织表达与雌性生殖器官中定位分析

doi:10.13560/j.cnki.biotech.bull.1985.2022-0216

摘要/Abstract

摘要：

旨在克隆获得牦牛纤维蛋白原γ链（FGG）基因，明确其在组织中的表达特性，探讨FGG对母牦牛繁殖的影响。采集母牦牛的心、肝、脾、肺、卵巢、输卵管和子宫等组织样以及颗粒细胞，利用RT-PCR克隆FGG基因并利用RT-qPCR和免疫组化检测其组织表达。获得到牦牛FGG基因cDNA序列，编码区全长1 332 bp，编码443个氨基酸，FGG蛋白属于酸性亲水稳定蛋白。FGG基因核苷酸序列进化树显示牦牛先与黄牛聚为一类。RT-qPCR显示FGG基因在检测的7个组织均有表达，肝脏表达量最高，显著高于其他组织（P<0.05）；在卵泡不同发育阶段的颗粒细胞中均有表达，且大卵泡颗粒细胞表达量最高，显著高于其他发育阶段（P<0.05）；妊娠期卵巢和子宫中表达量显著高于空怀期（P<0.05）。IHC显示FGG蛋白主要在卵巢颗粒细胞、卵泡腔、输卵管黏膜和子宫内膜中表达。牦牛FGG基因在物种间具有较高的保守性，组织表达广泛，可能在牦牛繁殖调控中发挥着重要作用。

关键词: 牦牛, 纤维蛋白原γ链, 表达特性, 荧光定量PCR, 免疫组化

Abstract:

The aim of this research is to clone the fibrinogen gamma chain（FGG）gene in yak（Bos grunniens）, clarify its expression characteristics in tissues, and explore the effects of FGG on female yak reproduction. Tissue samples and granulosa cells were collected from the heart, liver, spleen, lung, ovary, oviduct and uterus of adult female yaks, RT-PCR was used to clone Yak FGG gene, and RT-qPCR and immunohistochemistry（IHC）were to detect the tissue expression of yak FGG mRNA. The cDNA sequence of yak FGG gene was obtained with coding region of 1 332 bp encoding 443 amino acids. FGG protein was an acid hydrophilic stable protein. The phylogenetic tree based on nucleotide sequence of FGG gene showed that yaks were first grouped with cattle（Bos taurus）. RT-qPCR demonstrated that FGG gene expressed in the detected 7 genes, the expression in the liver was the highest and was significantly higher than that in other tissues（P<0.05）. There was FGG expression in the granulosa cells at follicular development, the highest in large ones, significantly higher than that in other developmental stages（P<0.05）. Its expression in the ovary and uterus during pregnancy was significantly higher than that of non-pregnant yaks（P<0.05）. IHC results revealed that FGG protein was mainly expressed in the granulosa cells, follicular cavity, oviduct mucosal epithelium and endometrium of yaks. FGG gene in yak is highly conservative among species, widely expressed in different tissues, and may play an important role in the regulation of yak reproduction.

Key words: Bos grunniens, fibrinogen gamma chain, expression characteristics, RT-qPCR, immunocytochemistry

蒋旭东, 刘宇, 邬建飞, 胡双阁, 卢建远, 字向东. 牦牛FGG组织表达与雌性生殖器官中定位分析[J]. 生物技术通报, 2022, 38(11): 286-294.

JIANG Xu-dong, LIU Yu, WU Jian-fei, HU Shuang-ge, LU Jian-yuan, ZI Xiang-dong. Tissue Expression and Localization Anaysis of FGG in Female Reproductive Organs of Bos grunniens[J]. Biotechnology Bulletin, 2022, 38(11): 286-294.

图/表 10

表1 PCR和荧光定量PCR引物序列

Table 1 Primer sequences for PCR and real-time quantitative PCR

基因Gene	引物序列Primer sequence（5'-3'）	扩增产物长度Amplified product size/bp	用途Usage	退火温度Annealing temperature /℃
FGG	F：ACACCATAGTTGGTCCTCG R：GAATGAACAGCTCAGGGCAAA	1 363	克隆	59
FGG	F：CAGGTAGTATCGGTTGGTGG R：CCATGTGGCCCAAATAATGCC	132	RT-qPCR	60
GAPDH	F：TGTTGGGATCTGACCTGCC R：AAGTCGCAGGAGACAACCTG	135	RT-qPCR	60

图1 牦牛FGG基因的PCR扩增结果 M：DL2000 DNA maker；1和2：FGG引物PCR扩增产物

Fig. 1 PCR amplification result of yak FGG gene M：DL2000 DNA maker；1 and 2：PCR amplification products of FGG primers

图2 牦牛和其他物种间FGG氨基酸序列比对黑色：同源性=100%；灰色：同源性>50%；白色：同源性<50%

Fig. 2 Alignment of amino acid sequence of FGG between yak（B. grunniens）and other species Black：Homology=100%. Gray：Homology>50%. White：Homology<50%

图3 牦牛FGG蛋白二级结构预测 h：α-螺旋；e：延伸链；t：β-转角；c：无规卷曲

Fig. 3 Secondary structure prediction of yak FGG protein h：α-helix. e：Extended chain. t：β-turn. c：Random coil

图4 FGG蛋白网络互作析图

Fig. 4 FGG protein network interaction analysis

图5 FGG基因系统进化树

Fig. 5 Phylogenetic tree of FGG gene

图6 空怀母牦牛FGG基因在不同组织的相对表达水平不同小写字母表示差异显著（P<0.05），字母相同表示差异不显著。下同

Fig. 6 Expressions of FGG gene in different tissues of non-pregnant female yak The different lowercase letters indicate that the difference is significant（P<0.05）.The same below

图7 空怀母牦牛FGG基因在不同发育阶段卵泡颗粒细胞的表达水平

Fig. 7 Expressions of FGG gene in follicular granulosa cells of non-pregnant female yak at different developmen-tal stages

图8 母牦牛FGG基因在生殖器官的相对表达水平 A：卵巢；B：输卵管；C：子宫

Fig. 8 Relative expressions of FGG gene in the reproductive organs of female yak A：Ovary. B：Oviduct. C：Uterus

图9 FGG蛋白在母牦牛生殖器官中的定位 A、B、D、F：FGG组；C、E、G：阴性对照；A、B、C：卵巢；D、E：输卵管；F、G：子宫；a：卵母细；b：颗粒细胞；c：基膜；e：初级卵泡；f：卵泡腔；g：黏膜上皮；h：子宫内膜；k：腺上皮；j：基质细胞

Fig. 9 Location of FGG protein in the reproductive organs of female yak A，B，D，F：FGG group；C，E，G：negative control. A，B，C：Ovary；D，E：oviduct；F，G：Uterus. a：Oocytes；b：granulosa cells；c：basement membrane；e：primary follicles；f：follicular cavity；g：mucosal epithelium；h：endometrium；k：glandular epithelium；j：stromal cells

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