Cloning of Bos grunniens TGF-β1 Gene and Its Expression in Major Organs of Female Reproductive System

doi:10.13560/j.cnki.biotech.bull.1985.2021-1152

Abstract

Abstract:

Transforming growth factor β1（TGF-β1）is a multifunctional growth and differentiation factor,which regulates many physiological and pathological processes of the body,and has important biological functions and broad application prospects. In this study,we collected ovarian,oviductal and uterine tissues from yaks during estrus and pregnancy,cloned the yak TGF-β1 gene,and performed the quantitative analysis of yak TGF-β1 at the gene and protein levels using quantitative real-time fluorescence PCR（qRT-PCR）,immunohistochemistry（IHC）,and protein immunoblotting（Western blot）. The results showed that TGF-β1 gene（GenBank No.MZ004937）was highly conserved,and the nucleotide sequence differed from the TGF-β1 nucleotide sequence of Bos taurus,and the encoded amino acid was mutated from alanine to glycine. It was most closely related to Bos taurus,Bos indicus×Bos taurus,and most distantly related to Canine. The encoded protein was a stably hydrophilic transmembrane protein. TGF-β1 was expressed in ovary,oviduct and uterus tissues of yak during follicular phase,luteal phase and gestation phase. In ovary,the expression of TGF-β1 during gestation phase was significantly higher than that during follicular phase and luteal phase（P<0.05）. In oviduct,the expression of TGF-β1 gene in gestation phase was significantly higher than that in follicular and luteal phases（P<0.01）. In utero,the expression in follicular phase and gestation phase was significantly higher than that in luteal phase（P<0.05）. Immunohistochemistry（IHC）results indicated that TGF-β1 was mainly expressed in ovarian reproductive epithelium,follicular membrane,granular layer of follicle,luteal cells,tubal mucosal epithelial cells,uterine gland（UG）and endometrium cells. The results will provide basic data for further exploring the molecular mechanism of TGF-β1 in reproductive physiology of yaks,and provide theoretical basis for exploring the adaptability of plateau mammals to alpine environment.

Key words: yak, TGF-β1, gene cloning, bioinformatics analysis, expression

WANG Nan, ZHANG Rui, PAN Yang-yang, HE Hong-hong, WANG Jing-lei, CUI Yan, YU Si-jiu. Cloning of Bos grunniens TGF-β1 Gene and Its Expression in Major Organs of Female Reproductive System[J]. Biotechnology Bulletin, 2022, 38(6): 279-290.

Figures/Tables 10

Table 1 Primer sequences and length

引物Primer	引物序列Primer sequence（5'-3'）	产物长度Product size/bp	退火温度Annealing temperature Tm/℃	GenBank登录号GenBank No.
D-TGF-β1	F：ACTACTACGCCAAGGAGGTCAC	153	60	MZ004937
	R：TTCCGAGAATCTTAGCTGCA	153	60	MZ004937
TGF-β1	F：GCAAACAGACCCTCCTACCTTT	1 173	60	MZ004937
	R：TCCTTAAATACAGTCCTGGTGAG	1 173	60	MZ004937
GAPDH	F：GGGTCATCATCTCTGCACCT	178	60	EU195062.1
	R：TGGTCATAAGTCCCTCCACG	178	60	EU195062.1

Fig. 1 D-TGF-β1 and GAPDH PCR amplification electrop-horesis M：DL2000 DNA marker. 1-3：Fallopian tubes,uterus and ovaries in luteal phase. 4-6：Fallopian tubes,uterus and ovaries in pregnancy phase. 7-8：Fallopian tubes,uterus and ovaries in follicular phase

Fig. 2 TGF-β1 PCR amplification electrophoresis M：DL2000 DNA marker. 1：Fallopian tubes

Fig. 3 Sequence alignment of TGF-β1 gene among different species Black arrow points to nucleotide 414 and 1 168

Fig.4 Phylogenetic tree of TGF-β1gene “★”indicates that the data is obtained from this experiment

Fig. 5 Results of yak TGF-β1 protein via bioinformatics analysis A：The secondary structure prediction of TGF-β1 protein in Bos grunniens. The long vertical zone：Alpha helix（h）. The middle vertical zone：Extended stand（e）. The short vertical zone：Random coil（c）. B：The tertiary structure prediction of TGF-β1 protein in Bos grunniens. C：Hydrophilicity and hydrophobicity of the protein encoded by TGF-β1 gene. D：The protein transmembrane regional analysis of TGF-β1 protein in Bos grunniens. E：TGF-β1 phosphorylation site analyses

Fig. 6 Relative expressions of TGF-β1 mRNA in different tissues A：Ovary. B：Fallopian tubes. C：Uterus. Reference gene：GAPDH;n=4. Different letters indicate significant difference（P<0.05）,the same below

Fig. 7 Detection results of TGF-β1 and GAPDH protein in different tissues A：Ovary. B：Fallopian tubes. C：Uterus. 1：Follicular phase. 2：Luteal phase. 3：Pregnancy

Fig. 8 Relative expressions of TGF-β1 protein in different tissues A：Ovary. B：Fallopian tubes. C：Uterus. Reference protein：GAPDH;n=4

Fig. 9 Distribution of TGF-β1 proteins in different tissues A-D：Positive expression. A,A1,A2,A3,B,B1,and B2：Ovary. C,C1,and C2：Fallopian tube. D,D1,D2：Uterus. E-H：Negative control.SG：Follicle granular layer. EM：Mucosalepithelium. GE：Germinal epitnelium. TF：Theca follicle. CL：Corpus luteum verum. EP：Epitheliummucosae.UG：Uterineglands. sg：Serous gland

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