Tissue Expression and Localization Anaysis of FGG in Female Reproductive Organs of Bos grunniens

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

Abstract

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

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.

Figures/Tables 10

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

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

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%

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

Fig. 4 FGG protein network interaction analysis

Fig. 5 Phylogenetic tree of FGG gene

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

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

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

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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