长牡蛎Calbin和Tyrosinase基因的克隆及其在幼虫生长发育中的表达分析
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摘要
贝类养殖是我国海水养殖业中重要的一部分。贝类人工大规模养殖中关键环节是早期幼虫能否正常发育，这对幼虫变态和变态后生长率、存活率有很大影响，直接影响到生产效益，因此了解幼虫发育机制对于生产实践具有重要意义。
长牡蛎(Crassostrea gigas)在我国沿海均有分布，以广东、福建较多，为南方沿海主要养殖品种之一，是重要的经济贝类。本文以长牡蛎幼虫为研究对象，分别对长牡蛎幼虫发育过程中神经发育相关的钙结合蛋白(CgCalbin)和初生壳形成相关的酪氨酸酶(CgTyrosinase)两个基因进行了克隆，分析了基因在牡蛎幼虫四个发育时期的时空表达特征，解析了可能的功能。
从长牡蛎幼虫cDNA文库中获得CgCalbin序列，进行了序列分析比对并构建了进化树。通过Real time PCR发现该基因mRNA表达量在幼虫的担轮期及成体肝胰脏中最高。整装原位杂交结果显示CgCalbin的mRNA主要存在于担轮期幼虫的神经节部位，推断该基因与长牡蛎幼虫神经发育相关。
根据D形幼虫与稚贝的消减文库信息，利用染色体步移法以及3' RACE法在幼虫中克隆到CgTyrosinase全长cDNA序列。通过Real time PCR发现该基因在稚贝中表达最高，提示该基因可能与变态过程、早期贝壳形成有关。
通过对上述两种基因的研究，分别探讨了长牡蛎幼虫神经发育相关、初生壳形成相关的基因及在幼虫中的表达分析，为研究贝类生长发育调控的分子机理提供了新的依据。
Bivalve culture is one of most widely aquaculture in China. Early larvae have greatinfluence on larval metamorphosis and growth rate after metamorphosis. If the early larvaehave normal growth, it can bring great production efficiency. Larval development is a keyphase in large scale bivalve culture industry. So investigation of molecular mechanism inlarval development is very important for production practice.
As an important economic bivalve, Crassostrea gigas distributes in the coastal areasand it is the main breeding species for the southern coast. In this paper, we studied CgCalbingene which is a EF-hand Calcium-binding protein and CgTyrosinase gene which is relatedto the formation of prodissconchⅠ. We obtained sequences of the two genes, and analyzedtemporal and spatial expression in C. gigas larvae and possible functions of CgCalbin generespectively.
CgCalbin sequence was obtained from larvae cDNA library of oyster. Sequence wasthen analyzed and phylogenetic tree was constructed. The real time PCR analysis revealedthat CgCalbin has the highest mRNA expression level in trochophore, and then theD-shaped larvae, the spat, and has the lowest level in veliger. The different tissue analysisshowed that CgCalbin has a very high mRNA expression level in liver, gonad and gill, andhas a very low level in mantle and muscle. The result of whole mount in situ hybridizationshowed that CgCalbin was located at the position of ganglion in trochophore stage,indicating CgCalbin may plays a role in neuromodulation.
EST sequence was selected from the subtranctive library of D-shape larvae and the spat.The full-length of CgTyrosinase was cloned by genome walking and RACE method. Thesequence of CgTyrosinase was analysed, and the real time PCR revealed that the mRNAlevel is highest in the spat. This indicates that the gene plays an important role in themetamorphosis and formation of shell in larvae.
In conclusion, the two genes (CgCalbin, CgTyrosinase) associated withneuromodulation and prodissconchⅠformation, have been characterized and analyzed respectively. The investigation will be helpful for studying the molecular mechanism ofgrowth and development in bivalve larvae.
长牡蛎(Crassostrea gigas)在我国沿海均有分布，以广东、福建较多，为南方沿海主要养殖品种之一，是重要的经济贝类。本文以长牡蛎幼虫为研究对象，分别对长牡蛎幼虫发育过程中神经发育相关的钙结合蛋白(CgCalbin)和初生壳形成相关的酪氨酸酶(CgTyrosinase)两个基因进行了克隆，分析了基因在牡蛎幼虫四个发育时期的时空表达特征，解析了可能的功能。
从长牡蛎幼虫cDNA文库中获得CgCalbin序列，进行了序列分析比对并构建了进化树。通过Real time PCR发现该基因mRNA表达量在幼虫的担轮期及成体肝胰脏中最高。整装原位杂交结果显示CgCalbin的mRNA主要存在于担轮期幼虫的神经节部位，推断该基因与长牡蛎幼虫神经发育相关。
根据D形幼虫与稚贝的消减文库信息，利用染色体步移法以及3' RACE法在幼虫中克隆到CgTyrosinase全长cDNA序列。通过Real time PCR发现该基因在稚贝中表达最高，提示该基因可能与变态过程、早期贝壳形成有关。
通过对上述两种基因的研究，分别探讨了长牡蛎幼虫神经发育相关、初生壳形成相关的基因及在幼虫中的表达分析，为研究贝类生长发育调控的分子机理提供了新的依据。
Bivalve culture is one of most widely aquaculture in China. Early larvae have greatinfluence on larval metamorphosis and growth rate after metamorphosis. If the early larvaehave normal growth, it can bring great production efficiency. Larval development is a keyphase in large scale bivalve culture industry. So investigation of molecular mechanism inlarval development is very important for production practice.
As an important economic bivalve, Crassostrea gigas distributes in the coastal areasand it is the main breeding species for the southern coast. In this paper, we studied CgCalbingene which is a EF-hand Calcium-binding protein and CgTyrosinase gene which is relatedto the formation of prodissconchⅠ. We obtained sequences of the two genes, and analyzedtemporal and spatial expression in C. gigas larvae and possible functions of CgCalbin generespectively.
CgCalbin sequence was obtained from larvae cDNA library of oyster. Sequence wasthen analyzed and phylogenetic tree was constructed. The real time PCR analysis revealedthat CgCalbin has the highest mRNA expression level in trochophore, and then theD-shaped larvae, the spat, and has the lowest level in veliger. The different tissue analysisshowed that CgCalbin has a very high mRNA expression level in liver, gonad and gill, andhas a very low level in mantle and muscle. The result of whole mount in situ hybridizationshowed that CgCalbin was located at the position of ganglion in trochophore stage,indicating CgCalbin may plays a role in neuromodulation.
EST sequence was selected from the subtranctive library of D-shape larvae and the spat.The full-length of CgTyrosinase was cloned by genome walking and RACE method. Thesequence of CgTyrosinase was analysed, and the real time PCR revealed that the mRNAlevel is highest in the spat. This indicates that the gene plays an important role in themetamorphosis and formation of shell in larvae.
In conclusion, the two genes (CgCalbin, CgTyrosinase) associated withneuromodulation and prodissconchⅠformation, have been characterized and analyzed respectively. The investigation will be helpful for studying the molecular mechanism ofgrowth and development in bivalve larvae.
引文
[1]李晔，苏秀榕，李太武，宋林生.贝类等无脊椎动物抗菌肽的研究进展[J].水产科学, 2004, 23(9):40-43.
[2]楼永明.牡蛎形态学的研究与比较[J].海峡药学, 2003, 15(6): 51-53.
[3]徐凤山.中国海双壳类软体动物[M].科学出版社, 1997.
[4]胡国成，许木启，戴家银，曹宏.我国外来水生生物入侵现状及其影响[J].生物学通报, 2007, 08:1-3.
[5]李树国，张少杰.水产动物育苗与营养的关系[J].饲料工业, 2006, 27(16): 28-29.
[6]王国栋.调控文蛤幼虫变态的肾上腺素能受体类型及幼虫发育中神经和肌肉的变化[D].中国科学院研究生院, 2006.
[7] Cannuel R, Beninger PG. Gill development, functional and evolutionary implications in the Pacificoyster Crassostrea gigas (Bivalvia: Ostreidae)[J]. Mar. Biol, 2006, 149: 547-563.
[8] De la Roche JP, Marin B, Freites L, et al. Embryonic development and larval and post-larval growthof the tropical scallop Nodipecten (= Lyropecten) nodosus (L. 1758) (Mollusca: Pectinidae). Aquac.Res, 2002, 33: 819-827.
[9] Beninger PG, Dwiono SAP, Le Pennec M. Early development of the gill and implications forfeeding in Pecten maximus (Bivalvia: Pectinidae)[J]. Mar. Biol. 1994, 119: 405-412.
[10] Beaumont AR, Abdul-Matin AKM, Seed R. Early development, survival and growth in pure andhybrid larvae of Mytilus edulis and M. Galloprovincialis[J]. J. Molluscan Stud, 1993, 59: 120-123.
[11] Gustafson RG, Lutz RA. Larval and early post-larval development of the protobranch bivalveSolemya velum (Mollusca: Bivalvia)[J]. J. Mar. Biol. Assoc. U.K., 1992, 72: 383-402.
[12] Morse MP. Comparative functional morphology of the bivalve excretory system[J]. Integr. Comp.Biol., 1987, 27: 737-746.
[13] Lelong C, Mathieu M, Favrel P. Structure and expression of mGDF, a new member of thetransforming growth factor-beta superfamily in the bivalve mollusc Crassostrea gigas[J]. Eur. J.Biochem, 2000, 267: 3986-3993.
[14] Herpin A, Lelong C, Becker T, et al. Structural and functional evidence for a singular repertoire ofBMP receptor signal transducing proteins in the lophotrochozoan Crassostrea gigas suggests ashared ancestral BMP/activin pathway[J]. FEBS J., 2005, 272: 3424-3440.
[15] Wang GD, Liu BZ, Tang BJ, et al. Pharmacological and immunocytochemical investigation of therole of catecholamines on larval metamorphosis byβ-adrenergic-like receptor in the bivalveMeretrix meretrix[J]. Aquaculture, 2006, 258: 611-618.
[16]王晓梅，刘保忠，相建海.文蛤(Meretrix meretrix)幼虫发育过程中细胞凋亡和Caspase功能分析[J].海洋与湖沼, 2009, 40(2): 181-186.
[17] Mou?za M, Gros O, Frenkiel L. Embryonic, larval and postlarval development of the tropical clam,Anomalocardia brasiliana (Bivalvia, Veneridae)[J]. J. Molluscan Stud. 1999, 65,: 73-88.
[18] Doroudi MS, Southgate PC. Embryonic and larval development of Pinctada margaritifera(Linnaeus, 1758)[R]. Molluscan Research, 2003, 23.
[19] Arkett S.Ciliary arrest controlled by identified central neurons in a urochordate (Ascidiacea)[J].Journal of Comparative Physiology A: Neuroethology, Sensory, Neural, and Behavioral Physiology,1987, 161: 837-847.
[20] Croll R, Dickinson A. Form and function of the larval nervous system in molluscs[J]. InvertebrReprod Dev, 2004, 46: 173-187.
[21]王宜艳，孙虎山.中国蛤蜊神经系统的初步观察[J].生物学通报, 1997, 32(4): 41-42.
[22]姚丹，汝少国.海胆早期血清素能神经系统的发育及功能研究进展[J].中国海洋大学学报,2011, 41(1/2): 93-96.
[23] Wanninger A, Koop D, Bromham L, et al. Nervous and muscle system development in Phascolionstrombus (Sipuncula)[J]. Dev. Genes Evol, 2005, 215: 509-518.
[24]牟洪善，李金萍，王琨.软体动物神经系统的研究进展[J].生命科学仪器, 2008(6): 6-8.
[25]刘鹏超.长牡蛎早期胚胎发育中生化成分和能量变化的研究[D].中国科学院研究生院, 2010.
[26] Marin F, Luquet G. Molluscan shell proteins[J]. Comptes Rendus Palevol, 2004, 3(6-7): 469-492.
[27] Gardner Luke David. Investigation of molecular mechanisms regulating biomineralization of pearloyster Pinctada maxinma[D]. Queensland University of Technology, 2008.
[28] Zhang Y, Meng Q X, Jiang TM, et al. A novel ferritin subunit involved in shell formation from thepearl oyster (Pinctada fucata)[J]. Comp. Biochem. Physiol. B-Biochem. Mol. Biol, 2003, 135:43-54.
[29] Zhang C, Xie L, Huang J, et al. A novel putative CgTyrosinase involved in periostracum formationfrom the pearl oyster(Pinctada fucata)[J]. Biochemical and biophysical research communications,2006, 342: 632-639.
[30] Meenakshi VR, Hare PE, Watabe N, et al. The chemical composition of the periostracum of themolluscan shell[J]. Comparative Biochemistry and Physiology, 1969, 29(2): 611-620.
[31] Waite JH, Saleuddin ASM, Andersen SO. Periostracin - a soluble precursor of sclerotizedperiostracum in Mytilus edulis[J]. L. J Comp Physiol, 1979, 130: 301-307.
[32] Giusti AF, Hinman VF, Degnan SM, et al. Expression of a Scr/Hox5 gene in the larval centralnervous system of the gastropod Haliotis, a non-segmented spiralian lophotrochozoan[J]. Evol. Dev.,2000, 2: 294-302.
[33] Morse DE, Duncan H, Hooker N, et al. GABA induces behavioral and developmentalmetamorphosis in planktonic molluscan larvae[J]. Fed. Proc., 1980, 39: 3237-3241.
[34]潘天虹，周孝达.钙结合蛋白及其神经保护作用[J].中风与神经疾病杂志, 1999, 16(3): 191-192.
[35] Naaby-Hansen S, Diekman A, Shetty J, et al. Identification of calcium-binding proteins associatedwith the human sperm plasma membrane[J]. Teproductive Biology and Endocrinology, 2010, 8: 6.
[36] Luan J, Zhang SC, Liu ZH, et al. Characterization, evolution and tissue-specific expression ofAmphiCalbin, a novel gene encoding EF-hand Calcium-binding protein in AmphioxusBranchiostoma belcheri[J]. Acta Biochimica et Biophysica Sinica. 2007, 39(11): 891-900.
[37]朱运峰.钙离子结合蛋白及其在神经系统疾病中的作用[J].中国生物化学与分子生物学报,2008, 24(5): 413-418.
[38] Katsutoshi Mizuno, Potturi Padma, Aru Konno, et al. A novel neuronal calcium sensor familyprotein, calbin, is a potential Ca2+-dependent regulator for theouter arm dynein of metazoan ciliaand flagella[J]. Biol. Cell, 2009, 101: 91-103.
[39] Kovalick GE, Beckingham K. Calmodulin transcription is limited to the nervous system duringDrosophila embryogenesis[J]. Dev. Biol., 1992, 150(1): 33-46.
[40] Di Gregorio A, Villani MG, Locascio A, et al. Developmental regulation and tissue-specificlocalization of calmodulin mRNA in the protochordate Ciona intestinalis[J]. Dev Growth Differ,1998, 40: 387-394.
[41] Manickam Sugumaran. Comparative Biochemistry of Eumelanogenesis and the Protective Roles ofPhenoloxidase and Melanin in Insects[J]. Pigment Cell Research, 2002, 15(1): 2-9.
[42] Vass E, Nappi AJ, Carton Y. Alterations in the activities of CgTyrosinase, N-acetyltransferase, andtyrosine aminotransferase in immune reactive larvae of Drosophila melanogaster[J]. Developmentaland comparative immunology, 1993, 17(2): 109-118.
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[2]楼永明.牡蛎形态学的研究与比较[J].海峡药学, 2003, 15(6): 51-53.
[3]徐凤山.中国海双壳类软体动物[M].科学出版社, 1997.
[4]胡国成，许木启，戴家银，曹宏.我国外来水生生物入侵现状及其影响[J].生物学通报, 2007, 08:1-3.
[5]李树国，张少杰.水产动物育苗与营养的关系[J].饲料工业, 2006, 27(16): 28-29.
[6]王国栋.调控文蛤幼虫变态的肾上腺素能受体类型及幼虫发育中神经和肌肉的变化[D].中国科学院研究生院, 2006.
[7] Cannuel R, Beninger PG. Gill development, functional and evolutionary implications in the Pacificoyster Crassostrea gigas (Bivalvia: Ostreidae)[J]. Mar. Biol, 2006, 149: 547-563.
[8] De la Roche JP, Marin B, Freites L, et al. Embryonic development and larval and post-larval growthof the tropical scallop Nodipecten (= Lyropecten) nodosus (L. 1758) (Mollusca: Pectinidae). Aquac.Res, 2002, 33: 819-827.
[9] Beninger PG, Dwiono SAP, Le Pennec M. Early development of the gill and implications forfeeding in Pecten maximus (Bivalvia: Pectinidae)[J]. Mar. Biol. 1994, 119: 405-412.
[10] Beaumont AR, Abdul-Matin AKM, Seed R. Early development, survival and growth in pure andhybrid larvae of Mytilus edulis and M. Galloprovincialis[J]. J. Molluscan Stud, 1993, 59: 120-123.
[11] Gustafson RG, Lutz RA. Larval and early post-larval development of the protobranch bivalveSolemya velum (Mollusca: Bivalvia)[J]. J. Mar. Biol. Assoc. U.K., 1992, 72: 383-402.
[12] Morse MP. Comparative functional morphology of the bivalve excretory system[J]. Integr. Comp.Biol., 1987, 27: 737-746.
[13] Lelong C, Mathieu M, Favrel P. Structure and expression of mGDF, a new member of thetransforming growth factor-beta superfamily in the bivalve mollusc Crassostrea gigas[J]. Eur. J.Biochem, 2000, 267: 3986-3993.
[14] Herpin A, Lelong C, Becker T, et al. Structural and functional evidence for a singular repertoire ofBMP receptor signal transducing proteins in the lophotrochozoan Crassostrea gigas suggests ashared ancestral BMP/activin pathway[J]. FEBS J., 2005, 272: 3424-3440.
[15] Wang GD, Liu BZ, Tang BJ, et al. Pharmacological and immunocytochemical investigation of therole of catecholamines on larval metamorphosis byβ-adrenergic-like receptor in the bivalveMeretrix meretrix[J]. Aquaculture, 2006, 258: 611-618.
[16]王晓梅，刘保忠，相建海.文蛤(Meretrix meretrix)幼虫发育过程中细胞凋亡和Caspase功能分析[J].海洋与湖沼, 2009, 40(2): 181-186.
[17] Mou?za M, Gros O, Frenkiel L. Embryonic, larval and postlarval development of the tropical clam,Anomalocardia brasiliana (Bivalvia, Veneridae)[J]. J. Molluscan Stud. 1999, 65,: 73-88.
[18] Doroudi MS, Southgate PC. Embryonic and larval development of Pinctada margaritifera(Linnaeus, 1758)[R]. Molluscan Research, 2003, 23.
[19] Arkett S.Ciliary arrest controlled by identified central neurons in a urochordate (Ascidiacea)[J].Journal of Comparative Physiology A: Neuroethology, Sensory, Neural, and Behavioral Physiology,1987, 161: 837-847.
[20] Croll R, Dickinson A. Form and function of the larval nervous system in molluscs[J]. InvertebrReprod Dev, 2004, 46: 173-187.
[21]王宜艳，孙虎山.中国蛤蜊神经系统的初步观察[J].生物学通报, 1997, 32(4): 41-42.
[22]姚丹，汝少国.海胆早期血清素能神经系统的发育及功能研究进展[J].中国海洋大学学报,2011, 41(1/2): 93-96.
[23] Wanninger A, Koop D, Bromham L, et al. Nervous and muscle system development in Phascolionstrombus (Sipuncula)[J]. Dev. Genes Evol, 2005, 215: 509-518.
[24]牟洪善，李金萍，王琨.软体动物神经系统的研究进展[J].生命科学仪器, 2008(6): 6-8.
[25]刘鹏超.长牡蛎早期胚胎发育中生化成分和能量变化的研究[D].中国科学院研究生院, 2010.
[26] Marin F, Luquet G. Molluscan shell proteins[J]. Comptes Rendus Palevol, 2004, 3(6-7): 469-492.
[27] Gardner Luke David. Investigation of molecular mechanisms regulating biomineralization of pearloyster Pinctada maxinma[D]. Queensland University of Technology, 2008.
[28] Zhang Y, Meng Q X, Jiang TM, et al. A novel ferritin subunit involved in shell formation from thepearl oyster (Pinctada fucata)[J]. Comp. Biochem. Physiol. B-Biochem. Mol. Biol, 2003, 135:43-54.
[29] Zhang C, Xie L, Huang J, et al. A novel putative CgTyrosinase involved in periostracum formationfrom the pearl oyster(Pinctada fucata)[J]. Biochemical and biophysical research communications,2006, 342: 632-639.
[30] Meenakshi VR, Hare PE, Watabe N, et al. The chemical composition of the periostracum of themolluscan shell[J]. Comparative Biochemistry and Physiology, 1969, 29(2): 611-620.
[31] Waite JH, Saleuddin ASM, Andersen SO. Periostracin - a soluble precursor of sclerotizedperiostracum in Mytilus edulis[J]. L. J Comp Physiol, 1979, 130: 301-307.
[32] Giusti AF, Hinman VF, Degnan SM, et al. Expression of a Scr/Hox5 gene in the larval centralnervous system of the gastropod Haliotis, a non-segmented spiralian lophotrochozoan[J]. Evol. Dev.,2000, 2: 294-302.
[33] Morse DE, Duncan H, Hooker N, et al. GABA induces behavioral and developmentalmetamorphosis in planktonic molluscan larvae[J]. Fed. Proc., 1980, 39: 3237-3241.
[34]潘天虹，周孝达.钙结合蛋白及其神经保护作用[J].中风与神经疾病杂志, 1999, 16(3): 191-192.
[35] Naaby-Hansen S, Diekman A, Shetty J, et al. Identification of calcium-binding proteins associatedwith the human sperm plasma membrane[J]. Teproductive Biology and Endocrinology, 2010, 8: 6.
[36] Luan J, Zhang SC, Liu ZH, et al. Characterization, evolution and tissue-specific expression ofAmphiCalbin, a novel gene encoding EF-hand Calcium-binding protein in AmphioxusBranchiostoma belcheri[J]. Acta Biochimica et Biophysica Sinica. 2007, 39(11): 891-900.
[37]朱运峰.钙离子结合蛋白及其在神经系统疾病中的作用[J].中国生物化学与分子生物学报,2008, 24(5): 413-418.
[38] Katsutoshi Mizuno, Potturi Padma, Aru Konno, et al. A novel neuronal calcium sensor familyprotein, calbin, is a potential Ca2+-dependent regulator for theouter arm dynein of metazoan ciliaand flagella[J]. Biol. Cell, 2009, 101: 91-103.
[39] Kovalick GE, Beckingham K. Calmodulin transcription is limited to the nervous system duringDrosophila embryogenesis[J]. Dev. Biol., 1992, 150(1): 33-46.
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