半滑舌鳎gata5基因的克隆及表达分析
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摘要
【目的】研究半滑舌鳎(Cynoglossussemilaevis)gata5基因(csgata5)的序列特征及该基因在半滑舌鳎性腺分化、成熟过程中的表达模式。【方法】通过克隆获得csgata5编码区序列,利用荧光定量PCR(RT-qPCR)方法检测csgata5 mRNA在不同类型性腺、胚胎和性腺发育中的表达模式,借助原位杂交技术定位csgata5 mRNA表达的细胞类型。【结果】克隆获得csgata5 cDNA序列1 777 bp,包含1 167 bp的编码区(Coding sequence,CDS),262 bp的5′UTR和348 bp的3′UTR区,预测csgata5编码388个氨基酸残基(aa),分子质量为41.9 ku,等电点为8.90,在C端含有两个典型的GATA锌指结构域;基因组分析显示csgata5位于半滑舌鳎10号染色体上,包含7个外显子和6个内含子区。RT-qPCR分析显示csgata5 mRNA在2龄雄鱼和伪雄鱼精巢中表达高于卵巢;csgata5m RNA主要定位在卵母细胞、精原细胞和精子中。胚胎发育过程中,csgata5m RNA表达在1细胞期至囊胚期呈下降趋势,之后上升,至原肠胚期达到峰值,随后降低并维持较低水平至孵化出膜。在精巢发育过程中,csgata5m RNA表达水平呈升高趋势,在1龄达到检测的峰值;在卵巢发育过程中,csgata5m RNA表达趋势类似精巢,表达水平低于同期精巢。【结论】csgata5参与半滑舌鳎原肠胚期的组织器官分化,具有促进原始生殖细胞向雄性生殖细胞分化及雄性生殖细胞发育的作用。
【Objective】In order to elucidate the gene function of gata5 of Chinese tongue sole(Cynoglossus semilaevis)(csgata5), the sequence characteristics and the expression patterns of this gene in Chinese tongue sole were studied.【Methods】The coding region of csgata5 was obtained by cloning and the expression pattern of csgata5 m RNA in embryonic development and gonadal development was detected by real-time PCR(RT-qPCR). In situ hybridization was used to localize the expression of csgata5 mRNA in the gonads.【Results】The csgata5 cDNA sequence is 1 777 bp in length and contains the entire coding region of 1 167 bp, the 262 bp 5′UTR and 348 bp 3′UTR. The location of csgata5 is on chromose 10, and it includes seven exons and six introns. The predicted protein of csgata5 contains 388 amino acid residues(aa), the molecular weight is 41.9 ku, the isoelectric point is 8.90, and it has two typical GATA zinc finger binding domains at the C-terminus. Phylogenetic analysis shows that the same GATA proteins are clustered together, and the clustering pattern is independent of species.The expression levels of csgata5 mRNA are higher in the testis of two-years old male and pseudomale than in the ovary of two-years old female and it is mainly localized in the oocytes, spermatogonia and sperm. During embryonic development, the expression levels of csgata5 m RNA decrease from the cleavage stage to the blastocyst stage, peak at the gastrula stage, and then decrease and maintain at a lower level until hatching. During the development of testis, the expression levels of csgata5 m RNA tend to increase, reaching the peak of detection at the age of one-year old. In the ovary, the expression profile of csgata5 mRNA is similar to that of the testis, and the expression level is lower than that of the testis.【Conclusion】These results show that csgata5 may be involved in the differentiation of organs in the gastrula stage of Chinese tongue sole; it has the effect of promoting the differentiation of primordial germ cells into male germ cells and the development of male germ cells.
【Objective】In order to elucidate the gene function of gata5 of Chinese tongue sole(Cynoglossus semilaevis)(csgata5), the sequence characteristics and the expression patterns of this gene in Chinese tongue sole were studied.【Methods】The coding region of csgata5 was obtained by cloning and the expression pattern of csgata5 m RNA in embryonic development and gonadal development was detected by real-time PCR(RT-qPCR). In situ hybridization was used to localize the expression of csgata5 mRNA in the gonads.【Results】The csgata5 cDNA sequence is 1 777 bp in length and contains the entire coding region of 1 167 bp, the 262 bp 5′UTR and 348 bp 3′UTR. The location of csgata5 is on chromose 10, and it includes seven exons and six introns. The predicted protein of csgata5 contains 388 amino acid residues(aa), the molecular weight is 41.9 ku, the isoelectric point is 8.90, and it has two typical GATA zinc finger binding domains at the C-terminus. Phylogenetic analysis shows that the same GATA proteins are clustered together, and the clustering pattern is independent of species.The expression levels of csgata5 mRNA are higher in the testis of two-years old male and pseudomale than in the ovary of two-years old female and it is mainly localized in the oocytes, spermatogonia and sperm. During embryonic development, the expression levels of csgata5 m RNA decrease from the cleavage stage to the blastocyst stage, peak at the gastrula stage, and then decrease and maintain at a lower level until hatching. During the development of testis, the expression levels of csgata5 m RNA tend to increase, reaching the peak of detection at the age of one-year old. In the ovary, the expression profile of csgata5 mRNA is similar to that of the testis, and the expression level is lower than that of the testis.【Conclusion】These results show that csgata5 may be involved in the differentiation of organs in the gastrula stage of Chinese tongue sole; it has the effect of promoting the differentiation of primordial germ cells into male germ cells and the development of male germ cells.
引文
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[10]邓思平,陈松林,刘本伟,等.半滑舌鳎脑芳香化酶基因cDNA克隆及表达分析[J].动物学研究,2008,29(1):17-24.
[11]DONG X L,CHEN S L,JI X S,et al.,Molecular cloning,characterization and expression analysis of Sox9a and Foxl2 genes in half-smooth tongue sole(Cynoglossus semilaevis)[J].Acta Oceanologica Sinica,2011,30(1):68-77.
[12]WANG L N,ZHU Y,XU W T,et al.,Molecular characterization of Pod1 during sex development in Chinese tongue sole(Cynoglossus semilaevis)[J].Biochemical and Biophysical Research Communications,2017,494(3/4):714-718.
[13]CUI Z,LIU Y,WANG W W,et al,Genome editing reveals dmrt1 as an essential male sex-determining gene in Chinese tongue sole(Cynoglossus semilaevis)[J].Scientific Reports,2017,7:42213.
[14]DONG Z D,ZHANG N,LIU Y,et al.Expression analysis and characterization of zglp1 in the Chinese tongue sole(Cynoglossus semilaevis)[J].Gene,2019,683:72-79.
[15]PEVNY L,SIMON M C,ROBERTSON E,et al.Erythroid differentiation in chimaeric mice blocked by a targeted mutation in the gene for transcription factor GATA-1[J].Nature,1991,349(6306):257-260.
[16]MERIKA M,ORKIN S H.DNA-binding specificity of GATA family transcription factors[J].Molecular&Cellular Biology,1993,13(7):3999-4010.
[17]KATSUMURA K R,BRESNICK E H.The GATA factor revolution in hematology[J].Blood,2017,129(15):2092-2102.
[18]LIN H Y,ZENG D,LIANG Y K,et al.GATA3 and TRPS1 are distinct biomarkers and prognostic factors in breast cancer:database mining for GATA family members in malignancies[J].Oncotarget,2017,8(21):34750-34761.
[19]LOWRY J A,ATCHLEY W R.Molecular evolution of the GATA family of transcription factors:conservation within the DNA-binding domain[J].Journal of Molecular Evolution,2000,50(2):103-115.
[20]PATIENT R K,MCGHEE J D.The GATA family(vertebrates and invertebrates)[J].Current Opinion in Genetics&Development,2002,12(4):416-422.
[21]WEN B,YUAN H,LIU X H,et al.GATA5 Sumoylation is indispensable for zebrafish cardiac development[J].Biochimica et Biophysica Acta(BBA)-General Subjects,2017,1861(7):S0304416517300880.
[22]HEMPEL M,CASAR T T,DIEHL T,et al.Compound heterozygous GATA5 mutations in a girl with hydrops fetalis,congenital heart defects and genital anomalies[J].Human Genetics,2017,136(3):339-346.
[23]LIU J X,ZHANG W,SUN Y,et al.Molecular characterization and expression profiles of GATA6 in tongue sole(Cynoglossus semilaevis)[J].Comp Biochem Physiol B Biochem Mol Biol,2016,198:19-26.
[24]LIU J X,ZHANG W,DU X X,et al.Molecular characterization and functional analysis of the GATA4 in tongue sole(Cynoglossus semilaevis)[J].Comparative Biochemistry&Physiology Part B,2016,193(2):1-8.
[25]BIELINSKA M,SEEHRA A,TOPPARI J,et al.GATA-4is required for sex steroidogenic cell development in the fetal mouse[J].Developmental Dynamics,2010,236(1):203-213.
[26]KETOLA I,PENTIK?INEN V,VASKIVUO T,et al.Expression of transcription factor GATA-4 during human testicular development and disease[J].Journal of Clinical Endocrinology&Metabolism,2000,85(10):3925-3931.
[27]WATANABE K,CLARKE T R,LANE A H,et al.Endogenous expression of Müllerian inhibiting substance in early postnatal rat Sertoli cells requires multiple steroidogenic factor-1 and GATA-4-binding sites[J].Proceedings of the National Academy of Sciences of the United States of America,2000,97(4):1624-1629.
[28]ROBERT N M,TREMBLAY J J,VIGER R S.Friend of GATA(FOG)-1 and FOG-2 differentially repress the GATA-dependent activity of multiple gonadal promoters[J].Endocrinology,2002,143(10):3963-3973.
[29]BOUMA G J,WASHBURN L L,ALBRECHT K H,et al.Correct dosage of Fog2 and Gata4 transcription factors is critical for fetal testis development in mice[J].Proceedings of the National Academy of Sciences of the United States of America,2007,104(38):14994-14999.
[30]LI J Z,CHEN W J,WANG D S,et al.GATA4 is involved in the gonadal development and maturation of the teleost fish tilapia,Oreochromis niloticus[J].Journal of Reproduction&Development,2012,58(2):237-242.
[31]WEBER H.SYMES C E,WALMSLEY M E,et al.Arole for GATA5 in Xenopus endoderm specification[J].Development,2000,127(20):4345-4360.
[32]MOLKENTIN J D,TYMITZ K M,RICHARDSON J A,et al.Abnormalities of the genitourinary tract in female mice lacking GATA5[J].Mol cell biol,2000,20(14):5256-5260.
[33]ZHANG L Y,LIU W J,SHAO C W,et al.Cloning,expression and methylation analysis of piwil2 in half-smooth tongue sole(Cynoglossus semilaevis)[J].Marine Genomics,2014,18:45-54.
[34]叶凯.尼罗罗非鱼GATA因子表达及其功能的初步研究[D].重庆:西南大学,2012.
[35]张未丽,刘智皓,吴风瑞,等.转录因子GATA-2在脊椎动物发育过程中的作用[J].四川动物,2009,28(6):945-948.
[36]LIU X M,LI Z,WANG B,et al.GATA4 is a transcriptional regulator of R-spondin1 in Japanese flounder(Paralichthys olivaceus)[J].Gene,2018,648:68-75
[37]LI Z,LIU X,SUN Y,et al.Roles of GATA6 during gonadal development in Japanese flounder:gonadogenesis,regulation of gender-related genes,estrogen formation and gonadal function maintenance[J].International Journal of Molecular Sciences,2017,18(1):160.
[38]DE SOUSA P A,CAVENEY A,WESTHUSIN M E,et al.Temporal patterns of embryonic gene expression and their dependence on oogenetic factors[J].Theriogenology,1998,49(1):115-128.
[39]马馨,张胜,杨树宝,等.母源效应蛋白在基因组印记维持中的作用[J].遗传,2014,36(10):959-964.
[40]柳学周,徐永江,刘乃真,等.半滑舌鳎卵巢发育的组织学和形态数量特征研究[J].渔业科学进展,2009,30(6):25-35.
[41]刘长琳.半滑舌鳎麻醉及性腺分化的研究[D].武汉:华中农业大学,2007.
[42]吴莹莹.半滑舌鳎精子发生及其受精过程[D].青岛:中国海洋大学,2008.
[2]ZHUANG Z M,WU D,ZHANG S C,et al.G-banding patterns of the chromosomes of tonguefish Cynoglossus semilaevis Gunther,1873[J].Journal of Applied Ichthyology,2010,22(5):437-440.
[3]季相山,陈松林,马洪雨,等.半滑舌鳎养殖群体中自然性逆转伪雄鱼的发现[J].水产学报,2010,34(2):322-327.
[4]SHAO C W,LI Q Y,CHEN S L,et al.Epigenetic modification and inheritance in sexual reversal of fish[J].Genome Research,2014,24(4):604-615.
[5]CHEN S L,LI J,DENG S P,et al.Isolation of female-Specific AFLP markers and molecular identification of genetic sex in half-smooth tongue sole(Cynoglossus semilaevis)[J].Marine Biotechnology(New York),2007,9(2):273-280.
[6]刘洋,陈松林,高峰涛,等.半滑舌鳎性别特异微卫星标记的SCAR转化及其应用[J].农业生物技术学报,2014,22(6):787-792.
[7]董忠典,张宁,徐文腾,等.一种快速鉴定半滑舌鳎遗传性别的方法[J].农业生物技术学报,2016,24(5):764-769.
[8]LI H L,XU W T,ZHANG N,et al.Two figla homologues have disparate functions during sex differentiation in half-smooth tongue sole(Cynoglossus semilaevis)[J].Scientific Reports,2016,6(1):28219.
[9]LI H L,XU WT,ZHU Y,et al.Characterization and expression pattern of r-spondin1 in Cynoglossus semilaevis[J].Journal of Experimental Zoology Part BMolecular and Developmental Evolution.2017,328(8):772-780.
[10]邓思平,陈松林,刘本伟,等.半滑舌鳎脑芳香化酶基因cDNA克隆及表达分析[J].动物学研究,2008,29(1):17-24.
[11]DONG X L,CHEN S L,JI X S,et al.,Molecular cloning,characterization and expression analysis of Sox9a and Foxl2 genes in half-smooth tongue sole(Cynoglossus semilaevis)[J].Acta Oceanologica Sinica,2011,30(1):68-77.
[12]WANG L N,ZHU Y,XU W T,et al.,Molecular characterization of Pod1 during sex development in Chinese tongue sole(Cynoglossus semilaevis)[J].Biochemical and Biophysical Research Communications,2017,494(3/4):714-718.
[13]CUI Z,LIU Y,WANG W W,et al,Genome editing reveals dmrt1 as an essential male sex-determining gene in Chinese tongue sole(Cynoglossus semilaevis)[J].Scientific Reports,2017,7:42213.
[14]DONG Z D,ZHANG N,LIU Y,et al.Expression analysis and characterization of zglp1 in the Chinese tongue sole(Cynoglossus semilaevis)[J].Gene,2019,683:72-79.
[15]PEVNY L,SIMON M C,ROBERTSON E,et al.Erythroid differentiation in chimaeric mice blocked by a targeted mutation in the gene for transcription factor GATA-1[J].Nature,1991,349(6306):257-260.
[16]MERIKA M,ORKIN S H.DNA-binding specificity of GATA family transcription factors[J].Molecular&Cellular Biology,1993,13(7):3999-4010.
[17]KATSUMURA K R,BRESNICK E H.The GATA factor revolution in hematology[J].Blood,2017,129(15):2092-2102.
[18]LIN H Y,ZENG D,LIANG Y K,et al.GATA3 and TRPS1 are distinct biomarkers and prognostic factors in breast cancer:database mining for GATA family members in malignancies[J].Oncotarget,2017,8(21):34750-34761.
[19]LOWRY J A,ATCHLEY W R.Molecular evolution of the GATA family of transcription factors:conservation within the DNA-binding domain[J].Journal of Molecular Evolution,2000,50(2):103-115.
[20]PATIENT R K,MCGHEE J D.The GATA family(vertebrates and invertebrates)[J].Current Opinion in Genetics&Development,2002,12(4):416-422.
[21]WEN B,YUAN H,LIU X H,et al.GATA5 Sumoylation is indispensable for zebrafish cardiac development[J].Biochimica et Biophysica Acta(BBA)-General Subjects,2017,1861(7):S0304416517300880.
[22]HEMPEL M,CASAR T T,DIEHL T,et al.Compound heterozygous GATA5 mutations in a girl with hydrops fetalis,congenital heart defects and genital anomalies[J].Human Genetics,2017,136(3):339-346.
[23]LIU J X,ZHANG W,SUN Y,et al.Molecular characterization and expression profiles of GATA6 in tongue sole(Cynoglossus semilaevis)[J].Comp Biochem Physiol B Biochem Mol Biol,2016,198:19-26.
[24]LIU J X,ZHANG W,DU X X,et al.Molecular characterization and functional analysis of the GATA4 in tongue sole(Cynoglossus semilaevis)[J].Comparative Biochemistry&Physiology Part B,2016,193(2):1-8.
[25]BIELINSKA M,SEEHRA A,TOPPARI J,et al.GATA-4is required for sex steroidogenic cell development in the fetal mouse[J].Developmental Dynamics,2010,236(1):203-213.
[26]KETOLA I,PENTIK?INEN V,VASKIVUO T,et al.Expression of transcription factor GATA-4 during human testicular development and disease[J].Journal of Clinical Endocrinology&Metabolism,2000,85(10):3925-3931.
[27]WATANABE K,CLARKE T R,LANE A H,et al.Endogenous expression of Müllerian inhibiting substance in early postnatal rat Sertoli cells requires multiple steroidogenic factor-1 and GATA-4-binding sites[J].Proceedings of the National Academy of Sciences of the United States of America,2000,97(4):1624-1629.
[28]ROBERT N M,TREMBLAY J J,VIGER R S.Friend of GATA(FOG)-1 and FOG-2 differentially repress the GATA-dependent activity of multiple gonadal promoters[J].Endocrinology,2002,143(10):3963-3973.
[29]BOUMA G J,WASHBURN L L,ALBRECHT K H,et al.Correct dosage of Fog2 and Gata4 transcription factors is critical for fetal testis development in mice[J].Proceedings of the National Academy of Sciences of the United States of America,2007,104(38):14994-14999.
[30]LI J Z,CHEN W J,WANG D S,et al.GATA4 is involved in the gonadal development and maturation of the teleost fish tilapia,Oreochromis niloticus[J].Journal of Reproduction&Development,2012,58(2):237-242.
[31]WEBER H.SYMES C E,WALMSLEY M E,et al.Arole for GATA5 in Xenopus endoderm specification[J].Development,2000,127(20):4345-4360.
[32]MOLKENTIN J D,TYMITZ K M,RICHARDSON J A,et al.Abnormalities of the genitourinary tract in female mice lacking GATA5[J].Mol cell biol,2000,20(14):5256-5260.
[33]ZHANG L Y,LIU W J,SHAO C W,et al.Cloning,expression and methylation analysis of piwil2 in half-smooth tongue sole(Cynoglossus semilaevis)[J].Marine Genomics,2014,18:45-54.
[34]叶凯.尼罗罗非鱼GATA因子表达及其功能的初步研究[D].重庆:西南大学,2012.
[35]张未丽,刘智皓,吴风瑞,等.转录因子GATA-2在脊椎动物发育过程中的作用[J].四川动物,2009,28(6):945-948.
[36]LIU X M,LI Z,WANG B,et al.GATA4 is a transcriptional regulator of R-spondin1 in Japanese flounder(Paralichthys olivaceus)[J].Gene,2018,648:68-75
[37]LI Z,LIU X,SUN Y,et al.Roles of GATA6 during gonadal development in Japanese flounder:gonadogenesis,regulation of gender-related genes,estrogen formation and gonadal function maintenance[J].International Journal of Molecular Sciences,2017,18(1):160.
[38]DE SOUSA P A,CAVENEY A,WESTHUSIN M E,et al.Temporal patterns of embryonic gene expression and their dependence on oogenetic factors[J].Theriogenology,1998,49(1):115-128.
[39]马馨,张胜,杨树宝,等.母源效应蛋白在基因组印记维持中的作用[J].遗传,2014,36(10):959-964.
[40]柳学周,徐永江,刘乃真,等.半滑舌鳎卵巢发育的组织学和形态数量特征研究[J].渔业科学进展,2009,30(6):25-35.
[41]刘长琳.半滑舌鳎麻醉及性腺分化的研究[D].武汉:华中农业大学,2007.
[42]吴莹莹.半滑舌鳎精子发生及其受精过程[D].青岛:中国海洋大学,2008.