单环刺螠soxb基因的克隆及其在幼虫发育中的表达特征
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摘要
SOXB作为重要的转录因子,参与了动物内胚层分化、消化道形成、神经细胞和感官细胞分化的调控。本研究利用RACE技术克隆了单环刺螠(Urechis unicinctus)2个soxb亚族基因soxb1(Uu-soxb1)和soxb2(Uu-soxb2)的cDNA全长序列,大小分别是1 871 bp和2 906 bp。在两个推导的氨基酸序列中均包含SOX家族的HMG-box结构和SOXB亚族蛋白特有的Group B homology序列,进化树分析它们分别与SOXB1和SOXB2聚类。原位杂交结果显示,Uu-soxb1和Uu-soxb2为母源表达基因;二者在担轮幼虫中的表达图式存在差异,其中早期担轮幼虫中Uu-soxb1 mRNA广泛分布于虫体中,Uu-soxb2 mRNA则集中于顶纤毛束基部和虫体后部(口后纤毛环之后),中期担轮幼虫中,Uu-soxb2阳性信号主要位于纤毛环及虫体的下半球;而Uu-soxb1 mRNA主要位于虫体下半球;之后的发育中二者的表达位点基本一致,即:晚期担轮幼虫中主要位于虫体后部,体节幼虫和蠕虫状幼虫中主要分布于消化道和体壁处。本研究结果表明,单环刺螠soxb在消化道发育过程中的表达模式与其他已报道动物类似,提示其在消化道发育中具有保守的功能。
SOXB, an important transcription factor, participates in the regulation of endoderm differentiation, digestive gut formation, regulation of neural and sensory cell differentiation in animals. In this study, two full-length cDNAs of soxb in Urechis unicinctus were cloned using RACE technique, which were 1 871 bp for soxb1(Uu-soxb1) and 2 906 bp for soxb2(Uu-soxb2). Both proteins contain the HMG-box of SOX family and the Group B homology domain of SOXB subfamily. Phylogenetic analysis suggested that they were clustered with members from SOXB1 and SOXB2 subfamilies, respectively. In situ hybridization showed that Uu-soxb1 and Uu-soxb2 were maternal expression genes. In early-trochophore, the Uu-soxb1 mRNA was visible broadly in whole larva, while Uu-soxb2 mRNA were located only in the base of apical tuft and the body part behind metatroch. In mid-trochophore,the Uu-soxb2 positive signals were mainly located around the peritroch and the lower hemispheres of the body, while the Uu-soxb1 mRNA was mainly located in the lower hemispheres of the body. After that, the expression patterns between Uu-soxb1 and Uu-soxb2 were basically consistent. The positive signals were restricted in the posterior part of body of late-trochophore.In somite larva and worm-like larva, their expressions were limited in the gut and body wall. Our data suggested that the expression pattern of soxb during the development of digestive gut is similar to that of other reported species, suggesting that it has a conservative function in the development of digestive gut.
SOXB, an important transcription factor, participates in the regulation of endoderm differentiation, digestive gut formation, regulation of neural and sensory cell differentiation in animals. In this study, two full-length cDNAs of soxb in Urechis unicinctus were cloned using RACE technique, which were 1 871 bp for soxb1(Uu-soxb1) and 2 906 bp for soxb2(Uu-soxb2). Both proteins contain the HMG-box of SOX family and the Group B homology domain of SOXB subfamily. Phylogenetic analysis suggested that they were clustered with members from SOXB1 and SOXB2 subfamilies, respectively. In situ hybridization showed that Uu-soxb1 and Uu-soxb2 were maternal expression genes. In early-trochophore, the Uu-soxb1 mRNA was visible broadly in whole larva, while Uu-soxb2 mRNA were located only in the base of apical tuft and the body part behind metatroch. In mid-trochophore,the Uu-soxb2 positive signals were mainly located around the peritroch and the lower hemispheres of the body, while the Uu-soxb1 mRNA was mainly located in the lower hemispheres of the body. After that, the expression patterns between Uu-soxb1 and Uu-soxb2 were basically consistent. The positive signals were restricted in the posterior part of body of late-trochophore.In somite larva and worm-like larva, their expressions were limited in the gut and body wall. Our data suggested that the expression pattern of soxb during the development of digestive gut is similar to that of other reported species, suggesting that it has a conservative function in the development of digestive gut.
引文
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[17] Aleksic J,Ferrero E,Fischer B,et al.The role of Dichaete in transcriptional regulation during Drosophila embryonic development[J].Bmc Genomics,2013,14(1):861-878.
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[22] 邵明瑜,张志峰,康庆浩,等.单环刺螠虫消化道组织学和细胞学[J].中国水产科学,2003,10(4):265-270.Shao M Y,Zhang Z F,Kang K h,et al.Histology and cytology of digestive tract in Urechis unicinctus VonDrasche[J].Journal of Fishery Sciences of China,2003,10(4):265-270.
[23] J萨姆布鲁克,D W 拉塞尔.分子克隆实验指南[M].北京:化学工业出版社,2008:707-721.Sambrook J,Russell D W.Molecular Cloning Experiment Guide[M].Beijing:Chemical Industry Press,2008:707-721.
[24] Fabioux C,Huvet A,Lelong C,et al.Oyster vasa-like gene as a marker of the germline cell development in Crassostrea gigas[J].Biochem Biophys Res Commun,2004,320(2):592-598.
[25] 冯政夫,邵明瑜,孙大鹏,等.栉孔扇贝Cf-dmrt4-like基因的克隆、序列特征及表达分析[J].中国水产科学,2010,17(5):930-940.FENG Z F,SHAO M Y,SUN D P.Cloning,characterization and expression analysis of Cf-dmrt4-like gene in Chlamys farreri[J].Journal of Fishery Sciences of China,2010,17(5):930-940.
[26] Zhong L,Wang D,Gan X,et al.Parallel expansions of sox transcription factor group B predating the diversifications of the arthropods and jawed vertebrates[J].Plos One,2011,6(1):e16570.
[27] Sur A,Magie C R,Seaver E C,et al.Spatiotemporal regulation of nervous system development in the annelid Capitella teleta[J].Evodevo,2017,8(1):13-36.
[28] Kenny A P,Kozlowski D,Oleksyn D W,et al.SpSoxB1,a maternally encoded transcription factor asymmetrically distributed among early sea urchin blastomeres[J].Development,1999,126(23):5473-5483.
[29] Schnitzler C E,Simmons D K,Pang K,et al.Expression of multiple Sox genes through embryonic development in the ctenophore Mnemiopsis leidyi,is spatially restricted to zones of cell proliferation[J].Evodevo,2014,5(1):15-32.
[30] Gouar M L,Guillou A,Vervoort M.Expression of a SoxB and a Wnt2/13 gene during the development of the mollusc Patella vulgata[J].Development Genes and Evolution,2004,214(5):250-256.
[31] He Y,Bao Z M,Guo H H,et al.Molecular cloning and characterization of SoxB2 gene from Zhikong scallop Chlamys farreri[J].Journal of Oceanology and Limnology,2013,31(6):1216-1225.
[32] Sánchezsoriano N,Russell S.Regulatory mutations of the Drosophila Sox gene Dichaete reveal new functions in embryonic brain and hindgut development.[J].Developmental Biology,2000,220(2):307-321.
[33] Daniel M,Marianne B F.The amphioxus soxB Family:Implications for the evolution of vertebrate placodes[J].International Journal of Biological Sciences,2007,3(6):356-364.
[2] Kamachi Y,Uchikawa M,Kondoh H.Pairing SOX off with partners in the regulation of embryonic development[J].Trends in Genetics Tig,2000,16(4):182-187.
[3] Schepers G E,Teasdale R D,Koopman P.Twenty pairs of sox:Extent,homology,and nomenclature of the mouse and human sox transcription factor gene families[J].Developmental Cell,2002,3(2):167-170.
[4] Bowles J,Schepers G,Koopman P.Phylogeny of the SOX family of developmental transcription factors based on sequence and structural indicators[J].Developmental Biology,2000,227(2):239-255.
[5] Uchikawa M,Kamachi Y,Kondoh H.Two distinct subgroups of Group B SOX genes for transcriptional activators and repressors:Their expression during embryonic organogenesis of the chicken[J].Mechanisms of Development,1999,84(1-2):103-120.
[6] Doerks T,Copley R R,Schultz J,et al.Systematic identification of novel protein domain families associated with nuclear functions[J].Genome Research,2002,12(1):47-56.
[7] Mizuseki K,Kishi M,Matsui M,et al.Xenopus Zic-related-1 and Sox-2,two factors induced by chordin,have distinct activities in the initiation of neural induction[J].Development,1998,125(4):579-586.
[8] Kamachi Y,Sockanathan S,Liu Q,et al.Involvement of SOX proteins in lens-specific activation of crystallin genes[J].Embo Journal,1995,14(14):3510-3519.
[9] Lowe C J,Wu M,Salic A,et al.Anteroposterior patterning in hemichordates and the origins of the chordate nervous system[J].Cell,2003,113(7):853-865.
[10] Taguchi S,Tagawa K,Humphreys T,et al.Group B sox genes that contribute to specification of the vertebrate brain are expressed in the apical organ and ciliary bands of Hemichordate larvae[J].Zoological Science,2002,19(1):57-66.
[11] Detlev A,Valérie L,Le G M,et al.atonal- and achaete-scute-related genes in the annelid Platynereis dumerilii:Insights into the evolution of neural basic-Helix-Loop-Helix genes[J].Bmc Evolutionary Biology,2008,8(1):170-183.
[12] Vidal B,Santella A,Serrano-Saiz E,et al.C.elegans SoxB genes are dispensable for embryonic neurogenesis but required for terminal differentiation of specific neuron types[J].Development,2015,142(14):2464-2477.
[13] Jager M,Quéinnec E,Chiori R,et al.Insights into the early evolution of SOX genes from expression analyses in a ctenophore[J].Journal of Experimental Zoology Part B Molecular and Developmental Evolution,2008,310B(8):650-667.
[14] Wei Z.Direct development of neurons within foregut endoderm of sea urchin embryos[J].Proceedings of the National Academy of Sciences of the United States of America,2011,108(22):9143-9147.
[15] Mashanov V S,Zueva O R,García-Arrarás J E.Expression of pluripotency factors in echinoderm regeneration[J].Cell and Tissue Research,2015,359(2):521-536.
[16] Shen S P,Aleksic J,Russell S.Identifying targets of the Sox domain protein Dichaete in the Drosophila,CNS via targeted expression of dominant negative proteins[J].Bmc Developmental Biology,2013,13(1):1-15.
[17] Aleksic J,Ferrero E,Fischer B,et al.The role of Dichaete in transcriptional regulation during Drosophila embryonic development[J].Bmc Genomics,2013,14(1):861-878.
[18] 方华华,王波.星斑川鲽仔稚鱼消化系统发育的组织学研究[J].中国农学通报,2011,27(14):50-54.Fang H H,Wang B.Histological studies on the development of digestive system in larval and juvenile starry flounder[J].Chinese Agricultural Science Bulletin,2011,27(14):50-54.
[19] Zambonino Infante J L,Cahu C L.Ontogeny of the gastrointestinal tract of marine fish larvae[J].Comparative Biochemistry and Physiology Part C,2001,130(4):477-487.
[20] 姜俊,冯琳,胡凯,等.谷氨酰胺与鱼类消化系统生长发育以及肠上皮细胞蛋白质合成和抗氧化能力的关系[J].动物营养学报,2011,23(5):735-739.Jang J,Feng L,Hu K,et al.Glutamine:The relationship with growth and development of digestive system,enterocyte protein synthesis and antioxidant ability in fish[J].Chinese Journal of Animal Nutrition,2011,23(5):735-739.
[21] 陈宗涛,张志峰,康庆浩,等.单环刺螠消化道的发生和分化[J].中国水产科学,2006,13(5):700-707.Chen Z T,Zhang Z F,Kang K H,et al.Ontogenesis and development of digestive canal in Urechis unicinctus[J].Journal of Fishery Sciences of China,2006,13(5):700-707.
[22] 邵明瑜,张志峰,康庆浩,等.单环刺螠虫消化道组织学和细胞学[J].中国水产科学,2003,10(4):265-270.Shao M Y,Zhang Z F,Kang K h,et al.Histology and cytology of digestive tract in Urechis unicinctus VonDrasche[J].Journal of Fishery Sciences of China,2003,10(4):265-270.
[23] J萨姆布鲁克,D W 拉塞尔.分子克隆实验指南[M].北京:化学工业出版社,2008:707-721.Sambrook J,Russell D W.Molecular Cloning Experiment Guide[M].Beijing:Chemical Industry Press,2008:707-721.
[24] Fabioux C,Huvet A,Lelong C,et al.Oyster vasa-like gene as a marker of the germline cell development in Crassostrea gigas[J].Biochem Biophys Res Commun,2004,320(2):592-598.
[25] 冯政夫,邵明瑜,孙大鹏,等.栉孔扇贝Cf-dmrt4-like基因的克隆、序列特征及表达分析[J].中国水产科学,2010,17(5):930-940.FENG Z F,SHAO M Y,SUN D P.Cloning,characterization and expression analysis of Cf-dmrt4-like gene in Chlamys farreri[J].Journal of Fishery Sciences of China,2010,17(5):930-940.
[26] Zhong L,Wang D,Gan X,et al.Parallel expansions of sox transcription factor group B predating the diversifications of the arthropods and jawed vertebrates[J].Plos One,2011,6(1):e16570.
[27] Sur A,Magie C R,Seaver E C,et al.Spatiotemporal regulation of nervous system development in the annelid Capitella teleta[J].Evodevo,2017,8(1):13-36.
[28] Kenny A P,Kozlowski D,Oleksyn D W,et al.SpSoxB1,a maternally encoded transcription factor asymmetrically distributed among early sea urchin blastomeres[J].Development,1999,126(23):5473-5483.
[29] Schnitzler C E,Simmons D K,Pang K,et al.Expression of multiple Sox genes through embryonic development in the ctenophore Mnemiopsis leidyi,is spatially restricted to zones of cell proliferation[J].Evodevo,2014,5(1):15-32.
[30] Gouar M L,Guillou A,Vervoort M.Expression of a SoxB and a Wnt2/13 gene during the development of the mollusc Patella vulgata[J].Development Genes and Evolution,2004,214(5):250-256.
[31] He Y,Bao Z M,Guo H H,et al.Molecular cloning and characterization of SoxB2 gene from Zhikong scallop Chlamys farreri[J].Journal of Oceanology and Limnology,2013,31(6):1216-1225.
[32] Sánchezsoriano N,Russell S.Regulatory mutations of the Drosophila Sox gene Dichaete reveal new functions in embryonic brain and hindgut development.[J].Developmental Biology,2000,220(2):307-321.
[33] Daniel M,Marianne B F.The amphioxus soxB Family:Implications for the evolution of vertebrate placodes[J].International Journal of Biological Sciences,2007,3(6):356-364.