牛卵泡TEDDM1表达特点及其功能分析
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摘要
旨在研究牛卵泡跨膜附睾蛋白1(transmembrane epididymal protein 1, TEDDM1)的分子特征和立体结构,并结合TEDDM1在不同生理状态牛卵泡的表达特性分析其功能。本研究采集牛发情期第一卵泡波优势卵泡(dominant follicle, DF)和从属卵泡(subordinate follicle, SF),分别分离颗粒细胞(granulosa cells, GCs),提取总RNA后反转录,设计牛TEDDM1特异性引物进行PCR扩增、克隆、测序,获得全CDS区后运用生物信息学方法对序列结构进行分析;以牛RPLP0作为内参基因,在DF和SF中使用qRT-PCR对TEDDM1的表达量进行检测;兔抗TEDDM1一抗检测TEDDM1在牛卵泡中的表达和定位。结果表明,TEDDM1基因CDS区全长903 bp,编码300个氨基酸,有7次跨膜的α螺旋结构,属于典型的G蛋白偶联受体,该基因氨基酸序列与非洲野牛(Bison bison bison)序列相似性最高,为99.4%;功能域分析表明,TEDDM1分子中存在未知功能结构域,蛋白家族716(domains of unknown function protein families 716, DUF716)结构域。qRT-PCR分析表明,TEDDM1 mRNA在SF的表达量显著高于DF(P<0.05);免疫组化分析表明,TEDDM1在DF和SF的颗粒层和膜层细胞均有表达,特异性显色强度表明TEDDM1在SF颗粒层和膜层细胞表达量均高于DF。本研究为进一步探讨TEDDM1在牛卵泡发育过程中的调控作用及其在信号转导和激素调节中的功能提供了依据,为后期深入研究牛卵泡发育机理奠定基础。
The aim of this study was to identify molecular characteristic and three-dimensional structure of TEDDM1 in bovine follicle, its function was analyzed by combining expression characteristic of TEDDM1 in different physiology states of follicles. DF and SF in first follicular development wave during estrous cycle were collected, GCs were isolated, total RNA was extracted and the reverse transcription was performed by RT-PCR, the specific primers were designed to amplify TEDDM1 and the sequence was sequenced, bioinformatics method was used to analyze CDS region sequence structure; Using RPLP0 as reference gene, qRT-PCR was performed to detect the experssion of TEDDM1 in DF and SF; The rabbit anti-TEDDM1 antibody was used to dectet expression level and localization of TEDDM1 in bovine follicles. The results showed that full length of TEDDM1 CDS region was 903 bp, encoding 300 amino acids, contained 7 parallel alpha helical structures across the cell membrane, which was a typical G protein-coupled receptor, sequence of TEDDM1 had the highest similarity with Bison(99.4%); The functional domain analysis showed that DUF716 existed in TEDDM1. The results of qRT-PCR analysis showed that expression level of TEDDM1 mRNA in SF was significantly higher than that in DF(P<0.05); The immunohistochemical analysis showed that TEDDM1 was expressed in GCs and membrane cells layer of DF and SF, and specific color intensity result showed that expression of TEDDM1 in SF GCs and membrane cells was higher than that in DF. The study provides a basis for further study on regulatory effect of TEDDM1 in bovine follicular development, signal transduction and hormone regulation, and lays a foundation for further research on mechanism of bovine follicular development.
The aim of this study was to identify molecular characteristic and three-dimensional structure of TEDDM1 in bovine follicle, its function was analyzed by combining expression characteristic of TEDDM1 in different physiology states of follicles. DF and SF in first follicular development wave during estrous cycle were collected, GCs were isolated, total RNA was extracted and the reverse transcription was performed by RT-PCR, the specific primers were designed to amplify TEDDM1 and the sequence was sequenced, bioinformatics method was used to analyze CDS region sequence structure; Using RPLP0 as reference gene, qRT-PCR was performed to detect the experssion of TEDDM1 in DF and SF; The rabbit anti-TEDDM1 antibody was used to dectet expression level and localization of TEDDM1 in bovine follicles. The results showed that full length of TEDDM1 CDS region was 903 bp, encoding 300 amino acids, contained 7 parallel alpha helical structures across the cell membrane, which was a typical G protein-coupled receptor, sequence of TEDDM1 had the highest similarity with Bison(99.4%); The functional domain analysis showed that DUF716 existed in TEDDM1. The results of qRT-PCR analysis showed that expression level of TEDDM1 mRNA in SF was significantly higher than that in DF(P<0.05); The immunohistochemical analysis showed that TEDDM1 was expressed in GCs and membrane cells layer of DF and SF, and specific color intensity result showed that expression of TEDDM1 in SF GCs and membrane cells was higher than that in DF. The study provides a basis for further study on regulatory effect of TEDDM1 in bovine follicular development, signal transduction and hormone regulation, and lays a foundation for further research on mechanism of bovine follicular development.
引文
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[21] OKADA N,YAMAMOTO T,WATANABE M,et al.Identification of TMEM45B as a protein clearly showing thermal aggregation in SDS-PAGE gels and dissection of its amino acid sequence responsible for this aggregation[J].Protein Expr Purif,2011,77(1):118-123.
[22] SUN D,CHEN J,ZHOU Z S,et al.Ectopic expression of a proteinase inhibitor I4 (MtPiI4) gene from Medicago truncatula confers plant resistance to Pseudomonas syringae pv.Tomato DC3000[J].Plant Mol Biol Rep,2015,33(6):1686-1696.
[23] GERBER P A,HEVEZI P,BUHREN B A,et al.Systematic identification and characterization of novel human skin-associated genes encoding membrane and secreted proteins[J].PLoS One,2013,8(6):e63949.
[24] JUSTESEN J,HARTMANN R,KJELDGAARD N O.Gene structure and function of the 2′-5′-oligoadenylate synthetase family[J].Cell Mol Life Sci,2000,57(11):1593-1612.
[25] LEE S,STEWART S,NAGTEGAAL I,et al.Differentially expressed genes regulating the progression of ductal carcinoma in situ to invasive breast cancer[J].Cancer Res,2012,72(17):4574-4586.
[26] LAN Z J,LABUS J C,HINTON B T.Regulation of gamma-glutamyl transpeptidase catalytic activity and protein level in the initial segment of the rat epididymis by testicular factors:role of basic fibroblast growth factor[J].Biol Reprod,1998,58(1):197-206.
[27] FAISST S,MEYER S.Compilation of vertebrate-encoded transcription factors[J].Nucleic Acids Res,1992,20(1):3-26.
[28] AUDET M,BOUVIER M.Restructuring G-protein-coupled receptor activation[J].Cell,2012,151(1):14-23.
[29] ARITRO S,BETTEGOWDA A,JIMENEZ-KRASSEL F,et al.Cocaine-and amphetamine-regulated transcript regulation of follicle-stimulating hormone signal transduction in bovine granulosa cells[J].Endocrinology,2007,148(9):4400-4410.
[30] SEN A,LV L H,BELLO N,et al.Cocaine-and amphetamine-regulated transcript accelerates termination of follicle-stimulating hormone-induced extracellularly regulated kinase 1/2 and Akt activation by regulating the expression and degradation of specific mitogen-activated protein kinase phosphatases in bovine granulosa cells[J].Mol Endocrinol,2008,22(12):2655-2676.
[31] VITALE A M,GONZALEZ O M,PARBORELL F,et al.Inhibin a increases apoptosis in early ovarian antral follicles of diethylstilbestrol-treated ratsl[J].Biol Reprod,2002,67(6):1989-1995.
[2] GOODMAN A L,NIXON W E,JOHNSON D K,et al.Regulation of folliculogenesis in the cycling rhesus monkey:selection of the dominant follicle[J].Endocrinology,1977,100(1):155-161.
[3] IRELAND J J,ROCHE J F.Hypotheses regarding development of dominant follicles during a bovine estrous cycle[M]//ROCHE J F.Follicular growth and ovulation rate in farm animals.The Netherlands:Martinus Nijhoff Publishers,1987:1-18.
[4] SMITH G W,SEN A,FOLGER J K,et al.Putative role of cocaine- and amphetamine-regulated transcript (CARTPT) in dominant follicle selection in cattle[J].Soc Reprod Fertil,2010,67(S1):105-117.
[5] LV L H,JIMENEZ-KRASSEL F,SEN A,et al.Evidence supporting a role for cocaine- and amphetamine-regulated transcript(CARTPT) in control of granulosa cell estradiol production associated with dominant follicle selection in cattle[J].Biol Reprod,2009,81(3):580-586.
[6] SEN A,BETTEGOWDA A,JIMENEZ-KRASSEL F,et al.Cocaine- and amphetamine-regulated transcript regulation of follicle-stimulating hormone signal transduction in bovine granulosa cells[J].Endocrinology,2007,148(9):4400-4410.
[7] 李鹏飞.牛卵泡可卡因-苯丙胺调节转录肽(CART)受体的筛选[D].太谷:山西农业大学,2014.LI P F.Screening of cocaine-and amphetamine-regulated transcript peptide(CART) receptor of cattle follicle[D].Taigu:Shanxi Agricultural University,2014.(in Chinese)
[8] ZIMIN A V,DELCHER A L,FLOREA L,et al.A whole-genome assembly of the domestic cow,Bos Taurus[J].Genome Biol,2009,10:R42.
[9] YAMAZAKI K,ADACHI T,SATO K,et al.Identification and characterization of novel and unknown mouse epididymis-specific genes by complementary DNA microarray technology[J].Biol Reprod,2006,75(3):462-468.
[10] LI P F,MENG J Z,ZHU Z W,et al.Detection of genes associated with follicle development through tran-scriptome analysis of bovine ovarian follicles GCs[J].Curr Bioinform,2018,13(2):127-140.
[11] SHI L G,ZHAO H,REN Y S,et al.Effects of different levels of dietary selenium on the proliferation of spermatogonial stem cells and antioxidant status in testis of roosters[J].Anim Reprod Sci,2014,149(3-4):266-272.
[12] 李鹏飞,毕锡麟,王锴,等.CART在不同发育阶段牛卵泡颗粒细胞中的表达和定位[J].中国农业科学,2016,49(12):2389-2396.LI P F,BI X L,WANG K,et al.Research on the expression and localization of CART in bovine granulosa cells at different developmental stages[J].Scientia Agricultura Sinica,2016,49(12):2389-2396.(in Chinese)
[13] PUNTA M,COGGILL P C,EBERHARDT R Y,et al.The Pfam protein families database[J].Nucleic Acids Res,2012,40(D1):D290-D301.
[14] BATEMAN A,COGGILL P,FINN R D.DUFs:families in search of function[J].Acta Crystallogr Sect F Struct Biol Cryst Commun,2010,66(10):1148-1152.
[15] HOU C C,TIAN W,KLEIST T,et al.DUF221 proteins are a family of osmosensitive calcium-permeable cation channels conserved across eukaryotes[J].Cell Res,2014,24(5):632-635.
[16] PAVY N,BOYLE B,NELSON C,et al.Identification of conserved core xylem gene sets:conifer cDNA microarray development,transcript profiling and computational analyses[J].New Phytol,2008,180(4):766-786.
[17] SWARBRECK D,WILKS C,LAMESCH P,et al.The arabidopsis information resource (TAIR):gene structure and function annotation[J].Nucleic Acids Res,2008,36(D1):D1009-D1014.
[18] KO J H,BEERS E P,HAN K H.Global comparative transcriptome analysis identifies gene network regulating secondary xylem development in Arabidopsis thaliana[J].Mol Genet Genomics,2006,276(6):517-531.
[19] YOSHIDA A,SUZAKI T,TANAKA W,et al.The homeotic gene long sterile lemma (G1) specifies sterile lemma identity in the rice spikelet[J].Proc Natl Acad Sci U S A,2009,106(47):20103-20108.
[20] KROEGER T S,WATKINS K P,FRISO G,et al.A plant-specific RNA-binding domain revealed through analysis of chloroplast group II intron splicing[J].Proc Natl Acad Sci U S A,2009,106(11):4537-4542.
[21] OKADA N,YAMAMOTO T,WATANABE M,et al.Identification of TMEM45B as a protein clearly showing thermal aggregation in SDS-PAGE gels and dissection of its amino acid sequence responsible for this aggregation[J].Protein Expr Purif,2011,77(1):118-123.
[22] SUN D,CHEN J,ZHOU Z S,et al.Ectopic expression of a proteinase inhibitor I4 (MtPiI4) gene from Medicago truncatula confers plant resistance to Pseudomonas syringae pv.Tomato DC3000[J].Plant Mol Biol Rep,2015,33(6):1686-1696.
[23] GERBER P A,HEVEZI P,BUHREN B A,et al.Systematic identification and characterization of novel human skin-associated genes encoding membrane and secreted proteins[J].PLoS One,2013,8(6):e63949.
[24] JUSTESEN J,HARTMANN R,KJELDGAARD N O.Gene structure and function of the 2′-5′-oligoadenylate synthetase family[J].Cell Mol Life Sci,2000,57(11):1593-1612.
[25] LEE S,STEWART S,NAGTEGAAL I,et al.Differentially expressed genes regulating the progression of ductal carcinoma in situ to invasive breast cancer[J].Cancer Res,2012,72(17):4574-4586.
[26] LAN Z J,LABUS J C,HINTON B T.Regulation of gamma-glutamyl transpeptidase catalytic activity and protein level in the initial segment of the rat epididymis by testicular factors:role of basic fibroblast growth factor[J].Biol Reprod,1998,58(1):197-206.
[27] FAISST S,MEYER S.Compilation of vertebrate-encoded transcription factors[J].Nucleic Acids Res,1992,20(1):3-26.
[28] AUDET M,BOUVIER M.Restructuring G-protein-coupled receptor activation[J].Cell,2012,151(1):14-23.
[29] ARITRO S,BETTEGOWDA A,JIMENEZ-KRASSEL F,et al.Cocaine-and amphetamine-regulated transcript regulation of follicle-stimulating hormone signal transduction in bovine granulosa cells[J].Endocrinology,2007,148(9):4400-4410.
[30] SEN A,LV L H,BELLO N,et al.Cocaine-and amphetamine-regulated transcript accelerates termination of follicle-stimulating hormone-induced extracellularly regulated kinase 1/2 and Akt activation by regulating the expression and degradation of specific mitogen-activated protein kinase phosphatases in bovine granulosa cells[J].Mol Endocrinol,2008,22(12):2655-2676.
[31] VITALE A M,GONZALEZ O M,PARBORELL F,et al.Inhibin a increases apoptosis in early ovarian antral follicles of diethylstilbestrol-treated ratsl[J].Biol Reprod,2002,67(6):1989-1995.