圆斑星鲽sox9基因的克隆与表达
|
摘要
本研究筛选到圆斑星鲽(Verasper variegatus)的性别相关基因sox9,并通过RACE技术获得了全长序列,基因全长为3287 bp,包括1431 bp的ORF,编码477个氨基酸,368 bp的5'UTR和1488 bp的3'UTR。在3'UTR中有多聚腺苷酸尾和加尾信号AATAAA。通过荧光定量PCR测定了sox9基因在圆斑星鲽成鱼不同组织中的表达水平,发现sox9基因在圆斑星鲽的脑、眼、鳃、心、肝、胆、肠、精巢、卵巢、肾和肌肉等各个组织中都有不同程度的表达。在鳃、脑和精巢组织中检测到较高水平的sox9转录,其中精巢中的转录水平显著高于其他组织,sox9基因在性腺中的表达显示出性别两相性差异。其在精巢中的表达水平要显著高于卵巢,说明sox9基因与雄性性腺发育相关。通过测定sox9基因在圆斑星鲽幼鱼不同发育时期(20、30、40、50、60、70和80日龄)的表达水平,发现其在20～50日龄表达量逐渐下降,在60日龄时表达量上升,推测表达量上升可能与幼鱼性腺分化相关。
Significant differences exist in the growth rate and body size of male and female spotted halibut(Verasper variegatus), with the females being significantly larger than the males. All-female cultivation can improve breeding efficiency. Studies investigating the mechanism of sex determination and features of sex differentiation may provide valuable information for all-female cultivation. In this study, we successfully identified the sox9 gene of spotted halibut. The total length is 3287 bp, including a 1431 bp ORF, encoding 477 amino acids; the 5' UTR is 368 bp and the 3' UTR is 1488 bp. Polyadenylic acid tails and AATAAA caudal signals were identified in the 3' UTR. The expression of the sox9 gene differed in the brain, eye, gills, heart, liver, gallbladder, intestine, testis, ovary, kidney, and muscle in spotted halibut. Transcript levels were high in the gill, brain, and testis, and were significantly higher in the testis than in the other tissues. A two-phase gender difference was observed in the gonads; sox9 expression was significantly higher in the testis than in the ovary, indicating that sox9 might be associated with male gonad development. The dynamic expression patterns of the sox9 gene in spotted halibut larvae gradually declined from 20 to 50 d after hatching, with a sudden rise observed after 60 d. This increased expression might be associated with differentiation of larval gonad.
Significant differences exist in the growth rate and body size of male and female spotted halibut(Verasper variegatus), with the females being significantly larger than the males. All-female cultivation can improve breeding efficiency. Studies investigating the mechanism of sex determination and features of sex differentiation may provide valuable information for all-female cultivation. In this study, we successfully identified the sox9 gene of spotted halibut. The total length is 3287 bp, including a 1431 bp ORF, encoding 477 amino acids; the 5' UTR is 368 bp and the 3' UTR is 1488 bp. Polyadenylic acid tails and AATAAA caudal signals were identified in the 3' UTR. The expression of the sox9 gene differed in the brain, eye, gills, heart, liver, gallbladder, intestine, testis, ovary, kidney, and muscle in spotted halibut. Transcript levels were high in the gill, brain, and testis, and were significantly higher in the testis than in the other tissues. A two-phase gender difference was observed in the gonads; sox9 expression was significantly higher in the testis than in the ovary, indicating that sox9 might be associated with male gonad development. The dynamic expression patterns of the sox9 gene in spotted halibut larvae gradually declined from 20 to 50 d after hatching, with a sudden rise observed after 60 d. This increased expression might be associated with differentiation of larval gonad.
引文
Adolfi MC,Carreira AC,Jesus LW,et al.Molecular cloning and expression analysis of dmrt1 and sox9 during gonad development and male reproductive cycle in the lambari fish,Astyanax altiparanae.Reproductive Biology and Endocrinology,2015,13:2
Berbejillo J,Martinez-Bengochea A,BedóG,et al.Expression of dmrt1 and sox9 during gonadal development in the Siberian sturgeon(Acipenser baerii).Fish Physiology and Biochemistry,2013,39(1):91-94
Dong T,Liu FJ,Hu HX,et al.Research progress on sex differentiation related genes on sturgeon.Biotechnology Bulletin,2015,31(10):62-70[东天,刘凤娇,胡红霞,等.鲟鱼性别分化相关基因研究进展.生物技术通报,2015,31(10):62-70]
Dong XL,Chen SL,JI XS,et al.Molecular cloning,charactcrization and expression analysis of Sox9a and Foxl2genes in half-smooth tongue sole(Cynoglossus semilaevis).Acta Oceanologica Sinica,2011,30(1):68-77
Fernandino JL,Guilgur LG,Strobl-Mazzulla PH,et al.Molecular cloning of SOX9,DMRT1 and SF1 cDNA partial sequences in the pejerrey fish Odontesthes bonariensis(Atheriniformes).Fish Physiology and Biochemistry,2003,28(1-4):145-146
Foster JW,Dominguez-Steglich MA,Guioli S,et al.Campomelic dysplasia and autosomal sex reversal caused by mutations in an SRY-related gene.Nature,1994,372(6506):525-530
Ge J,Chen S,Liu C,et al.Characterization of the global transcriptome and microsatellite marker information for spotted halibut Verasper variegatus.Genes and Genomics,2017,39(3):307-316
Hou L,Cui YX,Yao B.Progress in sexual determination related SOX9.Chinese Journal of Birth Health and Heredity,2007,15(8):1-2,9[侯林,崔英霞,姚兵.性别决定相关基因SOX9研究进展.中国优生与遗传杂志,2007,15(8):1-2,9]
Kobayashi T,Kajirua-Kobayashi H,Guan G,et al.Sexual dimorphic expression of DMRT1 and Sox9a during gonadal differentiation and hormone-induced sex reversal in the teleost fish Nile tilapia(Oreochromis niloticus).Developmental Dynamics,2008,237(1):297-306
Li YJ,Wu LM,Li XJ.A review on the genes related with sex determination and differentiation in teleosts.Journal of Henan Normal University(Natural Science Edition),2017,45(4):72-78[李永婧,吴利敏,李学军.硬骨鱼类性别决定与分化相关基因研究进展.河南师范大学学报(自然科学版),2017,45(4):72-78]
Li ZH.Gynogenesis induced and histological observation on gonadal sex differentiation in barfin flounder(Verasper moseri).Master’s Thesis of Northeast Agricultural University,2009,28-34[李忠红.条斑星鲽Verasper moseri雌核发育诱导及性别分化过程的初步研究.东北农业大学硕士研究生学位论文,2009,28-34]
Luo YS,Hu W,Liu XC,et al.Molecular cloning and m RNAexpression pattern of Sox9 during sex reversal in orange-spotted grouper(Epinephelus coioides).Aquaculture,2010,306(1-4):322-328
Ma LM.Study on sex related genes Sox3,Sox9 and Dmrt1 in black rockfish,Sebastes Schlegeli.Doctoral Dissertation of Ocean University of China,2014,79-91[马丽曼.许氏平鲉性别相关基因Sox3、Sox9和Dmrt1的研究.中国海洋大学博士研究生学位论文,2014,79-91]
Nakamura S,Watakabe I,Nishimura T,et al.Analysis of medaka sox9 orthologue reveals a conserved role in germ cell maintenance.PLoS One,2012,7(1):e29982
Rodríguez-MaríA,Yan YL,Bre Miller RA,et al.Characterization and expression pattern of zebrafish anti-Müllerian hormone(amh)relative to sox9a,sox9b,and cypl9ala,during gonad development.Gene Expression Patterns,2005,5:655-667
Smith EK,Guzmán JM,Luckenbach JA.Molecular cloning,characterization,and sexually dimorphic expression of five major sex differentiation-related genes in a Scorpaeniform fish,sablefish(Anoplopoma fimbria).Comparative Biochemistry and Physiology Part B:Biochemistry and Molecular Biology,2013,165(2):125-137
Volff JN.Genome evolution and biodiversity in teleost fish.Heredity,2005,94(3):280-294
Wen AY,You F,Sun P,et al.Study on cloning and expression of gene sox9 in olive flounder(Paralichthys olivaceus).Advances in Marine Science,2011,29(1):97-104[文爱韵,尤锋,孙鹏,等.牙鲆sox9基因的克隆与表达研究.海洋科学进展,2011,29(1):97-104]
Xia W,Zhou L,Yao B,et al.Differential and spermatogenic cell-specific expression of DMRT1 during sex reversal in protogynous hermaphroditic groupers.Molecular and Cellular Endocrinology,2007,263(1-2):156-172
Ye JS,Wang XQ,Ma S,et al.Studies on biological and advances of Verasper variegates.Fisheries Economy Research,2006(6):5-7[叶建生,王兴强,马甡,等.圆斑星鲽的生物学特性及其研究进展.渔业经济研究,2006(6):5-7]
Yu JH,Li JL,Cao LP,et al.Isolation and analysis of Sox9derived from yellow catfish(Pelteobagrus fulvidraco).Journal of Agricultural Biotechnology,2005,13(5):620-623[俞菊华,李建林,曹丽萍,等.黄颡鱼Sox9基因的分离及分析.农业生物技术学报,2005,13(5):620-623]
Zhou R,Liu L,Guo Y,et al.Similar gene structure of two Sox9a genes and their expression patterns during gonadal differentiation in a teleost fish,rice field eel(Monopterus albus).Molecular Reproduction and Development,2003,66(3):211-217
Berbejillo J,Martinez-Bengochea A,BedóG,et al.Expression of dmrt1 and sox9 during gonadal development in the Siberian sturgeon(Acipenser baerii).Fish Physiology and Biochemistry,2013,39(1):91-94
Dong T,Liu FJ,Hu HX,et al.Research progress on sex differentiation related genes on sturgeon.Biotechnology Bulletin,2015,31(10):62-70[东天,刘凤娇,胡红霞,等.鲟鱼性别分化相关基因研究进展.生物技术通报,2015,31(10):62-70]
Dong XL,Chen SL,JI XS,et al.Molecular cloning,charactcrization and expression analysis of Sox9a and Foxl2genes in half-smooth tongue sole(Cynoglossus semilaevis).Acta Oceanologica Sinica,2011,30(1):68-77
Fernandino JL,Guilgur LG,Strobl-Mazzulla PH,et al.Molecular cloning of SOX9,DMRT1 and SF1 cDNA partial sequences in the pejerrey fish Odontesthes bonariensis(Atheriniformes).Fish Physiology and Biochemistry,2003,28(1-4):145-146
Foster JW,Dominguez-Steglich MA,Guioli S,et al.Campomelic dysplasia and autosomal sex reversal caused by mutations in an SRY-related gene.Nature,1994,372(6506):525-530
Ge J,Chen S,Liu C,et al.Characterization of the global transcriptome and microsatellite marker information for spotted halibut Verasper variegatus.Genes and Genomics,2017,39(3):307-316
Hou L,Cui YX,Yao B.Progress in sexual determination related SOX9.Chinese Journal of Birth Health and Heredity,2007,15(8):1-2,9[侯林,崔英霞,姚兵.性别决定相关基因SOX9研究进展.中国优生与遗传杂志,2007,15(8):1-2,9]
Kobayashi T,Kajirua-Kobayashi H,Guan G,et al.Sexual dimorphic expression of DMRT1 and Sox9a during gonadal differentiation and hormone-induced sex reversal in the teleost fish Nile tilapia(Oreochromis niloticus).Developmental Dynamics,2008,237(1):297-306
Li YJ,Wu LM,Li XJ.A review on the genes related with sex determination and differentiation in teleosts.Journal of Henan Normal University(Natural Science Edition),2017,45(4):72-78[李永婧,吴利敏,李学军.硬骨鱼类性别决定与分化相关基因研究进展.河南师范大学学报(自然科学版),2017,45(4):72-78]
Li ZH.Gynogenesis induced and histological observation on gonadal sex differentiation in barfin flounder(Verasper moseri).Master’s Thesis of Northeast Agricultural University,2009,28-34[李忠红.条斑星鲽Verasper moseri雌核发育诱导及性别分化过程的初步研究.东北农业大学硕士研究生学位论文,2009,28-34]
Luo YS,Hu W,Liu XC,et al.Molecular cloning and m RNAexpression pattern of Sox9 during sex reversal in orange-spotted grouper(Epinephelus coioides).Aquaculture,2010,306(1-4):322-328
Ma LM.Study on sex related genes Sox3,Sox9 and Dmrt1 in black rockfish,Sebastes Schlegeli.Doctoral Dissertation of Ocean University of China,2014,79-91[马丽曼.许氏平鲉性别相关基因Sox3、Sox9和Dmrt1的研究.中国海洋大学博士研究生学位论文,2014,79-91]
Nakamura S,Watakabe I,Nishimura T,et al.Analysis of medaka sox9 orthologue reveals a conserved role in germ cell maintenance.PLoS One,2012,7(1):e29982
Rodríguez-MaríA,Yan YL,Bre Miller RA,et al.Characterization and expression pattern of zebrafish anti-Müllerian hormone(amh)relative to sox9a,sox9b,and cypl9ala,during gonad development.Gene Expression Patterns,2005,5:655-667
Smith EK,Guzmán JM,Luckenbach JA.Molecular cloning,characterization,and sexually dimorphic expression of five major sex differentiation-related genes in a Scorpaeniform fish,sablefish(Anoplopoma fimbria).Comparative Biochemistry and Physiology Part B:Biochemistry and Molecular Biology,2013,165(2):125-137
Volff JN.Genome evolution and biodiversity in teleost fish.Heredity,2005,94(3):280-294
Wen AY,You F,Sun P,et al.Study on cloning and expression of gene sox9 in olive flounder(Paralichthys olivaceus).Advances in Marine Science,2011,29(1):97-104[文爱韵,尤锋,孙鹏,等.牙鲆sox9基因的克隆与表达研究.海洋科学进展,2011,29(1):97-104]
Xia W,Zhou L,Yao B,et al.Differential and spermatogenic cell-specific expression of DMRT1 during sex reversal in protogynous hermaphroditic groupers.Molecular and Cellular Endocrinology,2007,263(1-2):156-172
Ye JS,Wang XQ,Ma S,et al.Studies on biological and advances of Verasper variegates.Fisheries Economy Research,2006(6):5-7[叶建生,王兴强,马甡,等.圆斑星鲽的生物学特性及其研究进展.渔业经济研究,2006(6):5-7]
Yu JH,Li JL,Cao LP,et al.Isolation and analysis of Sox9derived from yellow catfish(Pelteobagrus fulvidraco).Journal of Agricultural Biotechnology,2005,13(5):620-623[俞菊华,李建林,曹丽萍,等.黄颡鱼Sox9基因的分离及分析.农业生物技术学报,2005,13(5):620-623]
Zhou R,Liu L,Guo Y,et al.Similar gene structure of two Sox9a genes and their expression patterns during gonadal differentiation in a teleost fish,rice field eel(Monopterus albus).Molecular Reproduction and Development,2003,66(3):211-217