牦牛TGF-β1基因克隆及在雌性生殖系统主要器官中的表达定位

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

摘要/Abstract

摘要：

转化生长因子β1（transforming growth factor beta1,TGF-β1）是一种多功能的生长与分化因子,广泛调控机体的多个生理病理过程,具有重要的生物学功能和广阔的应用前景。试验采集牦牛发情期和妊娠期卵巢、输卵管和子宫组织,克隆牦牛TGF-β1基因,利用实时荧光定量PCR（qRT-PCR）、免疫组织化学（immunohistochemistry,IHC）、蛋白质免疫印迹（Western blot,WB）等方法,对牦牛TGF-β1在基因和蛋白水平上进行定量分析。结果显示,牦牛TGF-β1基因（GenBank No.MZ004937）高度保守,牦牛TGF-β1核苷酸序列与普通牛（Bos taurus）序列相比,存在差异,所编码氨基酸由丙氨酸突变为甘氨酸。与普通牛（Bos taurus）、瘤牛×普通牛（Bos indicus×Bos taurus）亲缘关系最近,与犬（Canine）亲缘关系最远,其编码的蛋白为稳定的亲水性膜蛋白。牦牛TGF-β1在卵泡期、黄体期、妊娠期的卵巢、输卵管和子宫组织中均有表达,在卵巢中,妊娠期的表达量显著高于卵泡期和黄体期（P<0.05）;在输卵管中,TGF-β1基因妊娠期表达量极显著高于卵泡期和黄体期（P<0.01）;在子宫中,卵泡期和妊娠期表达显著高于黄体期（P<0.05）。IHC结果显示,牦牛TGF-β1主要在卵巢生殖上皮、卵泡膜、卵泡颗粒层、黄体细胞、输卵管黏膜上皮细胞、子宫腺（UG）和子宫内膜细胞中表达。研究结果为进一步探索TGF-β1参与牦牛生殖生理的分子机制提供基础数据,以期为探讨高原哺乳动物对高寒环境的适应性提供理论依据。

关键词: 牦牛, TGF-β1, 基因克隆, 生物信息学分析, 表达

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

王楠, 张瑞, 潘阳阳, 何翃宏, 王靖雷, 崔燕, 余四九. 牦牛TGF-β1基因克隆及在雌性生殖系统主要器官中的表达定位[J]. 生物技术通报, 2022, 38(6): 279-290.

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.

图/表 10

表1 引物序列及长度

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

图1 D-TGF-β1、GAPDH基因 PCR扩增电泳 M：DL2000 DNA相对分子质量标准;1-3：黄体期输卵管、子宫、卵巢;4-6：妊娠期输卵管、子宫、卵巢;7-9：卵泡期输卵管、子宫、卵巢

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

图2 TGF-β1 PCR扩增电泳 M：DL2000 DNA 相对分子质量标准;1：黄体期输卵管

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

图3 不同物种间TGF-β1基因序列比对黑色箭头处为第414位、1 168位核苷酸

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

图4 TGF-β1基因的系统进化树 “★”为本实验所得

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

图5 牦牛TGF-β1蛋白生物信息学分析结果 A：牦牛TGF-β1蛋白的二级结构预测,长竖线区：α螺旋（h）,中竖线区：延伸链（e）,短竖线区：无规则卷曲（c）;B：牦牛TGF-β1蛋白的三级结构预测;C：TGF-β1基因编码蛋白的亲疏水性;D：牦牛TGF-β1蛋白跨膜区域分析;E：TGF-β1磷酸化位点分析

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

图6 TGF-β1 mRNA在不同组织中的相对表达量 A：卵巢;B：输卵管;C：子宫;内参基因：GAPDH;n=4。不同字母代表差异显著（P<0.05）,下同

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

图7 TGF-β1和GAPDH蛋白在不同组织中的检测结果 A：卵巢;B：输卵管;C：子宫 1：卵泡期;2：黄体期;3：妊娠期

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

图8 TGF-β1蛋白在不同组织中的相对表达量 A：卵巢;B：输卵管;C：子宫;内参蛋白：GAPDH;n=4

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

图9 TGF-β1蛋白在在不同组织中的分布 A-D：阳性表达;A、A1、A2、B、B1、B2：卵巢;C、C1、C2：输卵管;D、D1、D2：子宫;E-H：阴性对照;SG：卵泡颗粒层 EM：黏膜上皮;GE：生殖上皮;TF：卵泡膜;CL：黄体细胞;EP：子宫内膜;UG：子宫腺;sg：浆液腺

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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