罗氏沼虾中性别发育相关基因Sxl和Dmrt基因的分子特征和功能研究
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摘要
罗氏沼虾(Macrobrachium rosenbergii)!一种重要的淡水经济虾类,广泛分布在热带及亚热带地区。该虾生长快,个体大且食性杂,具有很高的经济价值。目前有关罗氏沼虾的育种和养殖技术都有了长足的进步,但是对潜藏在雌雄个体的性别两态性下的分子机制知之甚少。本研究以罗氏沼虾为研究对象,从动物保守性别决定核心途径中选取了两种最为重要的关键基因Sxl和Dmrt,开展了结构解析及其在沼虾性别发育和生殖中重要作用的相关研究。
第一部分,罗氏沼虾Sxl基因的分子结构解析和功能分析。
首先,通过简并PCR的方法克隆得到了一个Sxl的片段,以这个片段为探针,筛选罗氏沼虾的神经节cDNA文库并测序,通过比对分析得到了四个异构体。基因全长分别为2054、1977、1897和1031bp,依次命名为MroSxl1、MroSxl2、 MroSxl3和MroSxl4。Northern blotting和异构体特异性的RT-PCR的结果都证明了这四个异构体在罗氏沼虾中的确实存在。采用Southern blotting的方法研究Sxl在基因组的拷贝情况,结果表明MroSxl基因在罗氏沼虾的基因组中只存在一个拷贝形式。
其次,通过异构体特异性RT-PCR和Western blotting在雌雄个体中检测四种异构体是否存在着雌雄差异性,RT-PCR结果表明四种MroSxl异构体在雌雄个体中同时存在,Western blotting的结果也证明了在蛋白水平上MroSXL在雌雄虾中都有表达,不存在性别特异性表达的MroSXL。其中MroSXL3/4在雌虾的神经节、肝胰脏和卵巢中特异性的高量表达。进一步对雌雄虾生殖腺的免疫组化证明：在雄虾精巢中该蛋白主要在精原细胞中表达,在精子中没有表达；在雌虾卵巢的卵子发生早期SXL蛋白主要在细胞质中表达,随着卵母细胞进入到卵黄发生阶段,蛋白主要集中在细胞核中。
第二部分,罗氏沼虾Dmrt基因的分子结构解析和功能分析。
首先,我们通过简并PCR和RACE的克隆方法得到了两个Dmrt基因,一个基因全长2073bp,包含了一个1614bp的开放阅读框,编码形成一个537aa的蛋白；另一个基因全长906bp,包含了一个618bp的开放阅读框,编码形成一个205aa的蛋白,通过对氨基酸序列的分析证明都包含了Dmrt家族特有的DM结构域—一个特殊的锌指结构。通过系统进化关系分析发现这两个基因分别属于Dmrt11E和Dmrt99B分支,因此分别命名为MroDmrt11E和MroDmrt99B。
其次,采用半定量RT-PCR的方法研究这两个基因在mRNA水平上的表达特征,结果表明罗氏沼虾中两个MroDmrt基因都表现出了明显的性别特异性：MroDmrt11E主要在精巢中有高量表达,在两性的神经节中有一定程度的表达,在卵巢表达极低(少于精巢的1/10,P<0.01)；而MroDmrt99B特异性的表达在精巢中且表达量较低。在胚胎发育过程中,两个基因都呈现随着胚胎发育的进行表达量随之升高,相对的MroDmrt99B有一个滞后性。
这些结果揭示了核心性别决定机制在甲壳类中的保守性和特殊性,为阐明经济虾蟹类为代表的甲壳类性别分化和调控分子机制提供了重要证据,为进一步应用于水产养殖中的性别人工控制提供了重要理论依据。
The giant freshwater prawn, Macrobrachium rosenbergii, is an important economic animal in aquaculture, and distributed widely in tropic and subtropic areas of the Pacific Ocean. The prawn has high commercial values because of its fast growth, great size and dietary omnivority. Although substantial progress has been achieved on breeding and culture technologies of prawn farming, the molecular mechanisms underlying the sexual phenotypic differentiation remain largely unclear. In the present study, we focus on two key genes of the most conservative and important sex determination pathway in metazoans, Sxl and Dmrt from the prawn Macrobrachium rosenbergii, reveal their structural properties and try to elucidate their potential function in the sexual development and reproduction in this economic species.
Part one, molecular cloning and functional characterization of the Sxl genes in M. rosenbergii. First, a partial Sxl fragment was amplified and determined with a degenerate nested PCR. With this fragment as a DNA probe, we screened a central nervous system cDNA-library of the prawn and sequenced all the positive clones. The sequencing revealed at least four Sxl isoforms existing in M. rosenbergii. The four structural variants were named as MroSxl1, MroSxl2, MroSxl3and MroSxl4with cDNA full lengths of2054,1977,1897and1031bp, respectively. Both results of Northern blotting and isoform-specific RT-PCR identified the authentically transcription of the four variants in the prawn. Southern blotting was performed to decide the copy number of MroSxl gene at the genomic level, where the result implicated that a single Sxl locus was present in the M. rosenbergii genome. Second, to identify whether the sex-specific expressing mechanism was applied to MroSxl, we performed isoform-specific RT-PCR and Western blotting. The results implied that each variant existed in both males and females. However, the expressed level of MroSXL3/4was much higher in ganglion, hepatopancreas and ovary of females. Further analysis of the protein distribution in bisexual gonads was performed with immunohistochemistry, where the results showed that the MroSXL protein was prominently localized in the spermatocyte but no expression in the sperm; while in the previtellogenic oocyte, MroSXL was prominently localized in the cytoplasm; when the oocyte differentiated into the vitellogenic stage the MroSXL protein was trans-imported into the nucleus.
Part two, molecular cloning and functional characterization of the Dmrt genes in M. rosenbergii. First, we performed degenerated RT-PCR and RACE method to obtain two entire cDNAs of Dmrt genes, where one (2073bp in length) contains a1614-bp open reading frame (ORP) encoding a537-aa protein, while the other906-bp cDNA contains a618-bp ORF that encodes a205-aa protein. Alignments of amino-acid sequences revealed that both the genes contained the DM domain, an unusual zinc finger module which is well conserved among all DMRT subfamilies. Further phylogenetic analysis undoubtedly placed the present two deduced M. rosenbergii DM domain proteins into two Arthropoda-specific subfamilies, DMRT11E and DMRT99B, according to which we named them MroDmrtllE and MroDmrt99B, respectively. Second, we used RT-PCR to investigate tissue distributions and temporal expression during embryogenesis of the two MroDmrt genes. The results implied a sexually dimorphic expression pattern of the two MroDmrt genes in the prawn:MroDmrt11E transcription was prominent in testis, moderate in bisexual ganglion, while much lower in ovary (over ten times lower than in testis, P<0.01). On the other hand, MroDmrt99B was exclusively expressed in testis but dispensable in ovary and other tissues, although its expression level in testis was relatively low. Additionally, during the embryogenesis, the expression pattern of two MroDmrt transcripts tended to higher levels, and MroDmrt99B exhibited a later transcription.
The present results reveal the conservation and specificity of the core sex determination pathway and its key molecules in crustaceans. Our study provides not only substantial evidence to elucidate the molecular mechanisms of sex differentiation and regulation in economic crustaceans like the prawn, but also important basic data for application on manual sex control in aquaculture.
第一部分,罗氏沼虾Sxl基因的分子结构解析和功能分析。
首先,通过简并PCR的方法克隆得到了一个Sxl的片段,以这个片段为探针,筛选罗氏沼虾的神经节cDNA文库并测序,通过比对分析得到了四个异构体。基因全长分别为2054、1977、1897和1031bp,依次命名为MroSxl1、MroSxl2、 MroSxl3和MroSxl4。Northern blotting和异构体特异性的RT-PCR的结果都证明了这四个异构体在罗氏沼虾中的确实存在。采用Southern blotting的方法研究Sxl在基因组的拷贝情况,结果表明MroSxl基因在罗氏沼虾的基因组中只存在一个拷贝形式。
其次,通过异构体特异性RT-PCR和Western blotting在雌雄个体中检测四种异构体是否存在着雌雄差异性,RT-PCR结果表明四种MroSxl异构体在雌雄个体中同时存在,Western blotting的结果也证明了在蛋白水平上MroSXL在雌雄虾中都有表达,不存在性别特异性表达的MroSXL。其中MroSXL3/4在雌虾的神经节、肝胰脏和卵巢中特异性的高量表达。进一步对雌雄虾生殖腺的免疫组化证明：在雄虾精巢中该蛋白主要在精原细胞中表达,在精子中没有表达；在雌虾卵巢的卵子发生早期SXL蛋白主要在细胞质中表达,随着卵母细胞进入到卵黄发生阶段,蛋白主要集中在细胞核中。
第二部分,罗氏沼虾Dmrt基因的分子结构解析和功能分析。
首先,我们通过简并PCR和RACE的克隆方法得到了两个Dmrt基因,一个基因全长2073bp,包含了一个1614bp的开放阅读框,编码形成一个537aa的蛋白；另一个基因全长906bp,包含了一个618bp的开放阅读框,编码形成一个205aa的蛋白,通过对氨基酸序列的分析证明都包含了Dmrt家族特有的DM结构域—一个特殊的锌指结构。通过系统进化关系分析发现这两个基因分别属于Dmrt11E和Dmrt99B分支,因此分别命名为MroDmrt11E和MroDmrt99B。
其次,采用半定量RT-PCR的方法研究这两个基因在mRNA水平上的表达特征,结果表明罗氏沼虾中两个MroDmrt基因都表现出了明显的性别特异性：MroDmrt11E主要在精巢中有高量表达,在两性的神经节中有一定程度的表达,在卵巢表达极低(少于精巢的1/10,P<0.01)；而MroDmrt99B特异性的表达在精巢中且表达量较低。在胚胎发育过程中,两个基因都呈现随着胚胎发育的进行表达量随之升高,相对的MroDmrt99B有一个滞后性。
这些结果揭示了核心性别决定机制在甲壳类中的保守性和特殊性,为阐明经济虾蟹类为代表的甲壳类性别分化和调控分子机制提供了重要证据,为进一步应用于水产养殖中的性别人工控制提供了重要理论依据。
The giant freshwater prawn, Macrobrachium rosenbergii, is an important economic animal in aquaculture, and distributed widely in tropic and subtropic areas of the Pacific Ocean. The prawn has high commercial values because of its fast growth, great size and dietary omnivority. Although substantial progress has been achieved on breeding and culture technologies of prawn farming, the molecular mechanisms underlying the sexual phenotypic differentiation remain largely unclear. In the present study, we focus on two key genes of the most conservative and important sex determination pathway in metazoans, Sxl and Dmrt from the prawn Macrobrachium rosenbergii, reveal their structural properties and try to elucidate their potential function in the sexual development and reproduction in this economic species.
Part one, molecular cloning and functional characterization of the Sxl genes in M. rosenbergii. First, a partial Sxl fragment was amplified and determined with a degenerate nested PCR. With this fragment as a DNA probe, we screened a central nervous system cDNA-library of the prawn and sequenced all the positive clones. The sequencing revealed at least four Sxl isoforms existing in M. rosenbergii. The four structural variants were named as MroSxl1, MroSxl2, MroSxl3and MroSxl4with cDNA full lengths of2054,1977,1897and1031bp, respectively. Both results of Northern blotting and isoform-specific RT-PCR identified the authentically transcription of the four variants in the prawn. Southern blotting was performed to decide the copy number of MroSxl gene at the genomic level, where the result implicated that a single Sxl locus was present in the M. rosenbergii genome. Second, to identify whether the sex-specific expressing mechanism was applied to MroSxl, we performed isoform-specific RT-PCR and Western blotting. The results implied that each variant existed in both males and females. However, the expressed level of MroSXL3/4was much higher in ganglion, hepatopancreas and ovary of females. Further analysis of the protein distribution in bisexual gonads was performed with immunohistochemistry, where the results showed that the MroSXL protein was prominently localized in the spermatocyte but no expression in the sperm; while in the previtellogenic oocyte, MroSXL was prominently localized in the cytoplasm; when the oocyte differentiated into the vitellogenic stage the MroSXL protein was trans-imported into the nucleus.
Part two, molecular cloning and functional characterization of the Dmrt genes in M. rosenbergii. First, we performed degenerated RT-PCR and RACE method to obtain two entire cDNAs of Dmrt genes, where one (2073bp in length) contains a1614-bp open reading frame (ORP) encoding a537-aa protein, while the other906-bp cDNA contains a618-bp ORF that encodes a205-aa protein. Alignments of amino-acid sequences revealed that both the genes contained the DM domain, an unusual zinc finger module which is well conserved among all DMRT subfamilies. Further phylogenetic analysis undoubtedly placed the present two deduced M. rosenbergii DM domain proteins into two Arthropoda-specific subfamilies, DMRT11E and DMRT99B, according to which we named them MroDmrtllE and MroDmrt99B, respectively. Second, we used RT-PCR to investigate tissue distributions and temporal expression during embryogenesis of the two MroDmrt genes. The results implied a sexually dimorphic expression pattern of the two MroDmrt genes in the prawn:MroDmrt11E transcription was prominent in testis, moderate in bisexual ganglion, while much lower in ovary (over ten times lower than in testis, P<0.01). On the other hand, MroDmrt99B was exclusively expressed in testis but dispensable in ovary and other tissues, although its expression level in testis was relatively low. Additionally, during the embryogenesis, the expression pattern of two MroDmrt transcripts tended to higher levels, and MroDmrt99B exhibited a later transcription.
The present results reveal the conservation and specificity of the core sex determination pathway and its key molecules in crustaceans. Our study provides not only substantial evidence to elucidate the molecular mechanisms of sex differentiation and regulation in economic crustaceans like the prawn, but also important basic data for application on manual sex control in aquaculture.
引文
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55.孙继贤,廖家遗(2003)罗氏沼虾胸神经节中促进卵母细胞发育的激素的初步分离《湖泊科学》15:63-68.
56.杜永成,林原声(2005)罗氏沼虾胸神经节性腺刺激激素的分离纯化与卵黄磷蛋白研究,硕士学位论文.
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60.2, Klug A, Schwabe JW (1995) Protein motifs 5. Zinc fingers. FASEB J 9:597-604.
61.3, Giese K, Cox J, Grosschedl R (1992) The HMG domain of lymphoid enhancer factor 1 bends DNA and facilitates assembly of functional nucleoprotein structures. Cell 69:185-195.
62.4, Haqq CM, King CY, Ukiyama E, Falsafi S, Haqq TN, Donahoe PK, Weiss MA (1994) Molecular basis of mammalian sexual determination:activation of Mullerian inhibiting substance gene expression by SRY. Science 266:1494-1500.
63.5, Van Houte L, Van Oers A, Van de Wetering M, Dooijes D, Kaptein R, Clevers H (1993) Aug 25;268(24):18083-7. The sequence-specific high mobility group 1 box of TCF-1 adopts a predominantly alpha-helical conformation in solution. J Biol Chem 268:18083-18087.
64.6, Ying M, Chen B, Tian Y, Hou Y, Li Q, Shang X, Sun J, Cheng H, Zhou R (2007) Nuclear import of human sexual regulator DMRT1 is mediated by importin-beta. Biochim Biophys Acta 1773:804-813.
65.7, McGuffin LJ, Bryson K, Jones DT (2000) The PSIPRED protein structure prediction server. Bioinformatics 16:404-405.
66.8, Zhang Y (2008) I-TASSER server for protein 3D structure prediction. BMC Bioinformatics 9:40.
67. Yang Y, Zhang W, Bayrer JR. Weiss MA (2008) Doublesex and the regulation of sexual dimorphism in Drosophila melanogaster:structure, function, and mutagenesis of a female-specific domain. J Biol Chem 283:7280-7092.
68. Zhang EF, Qiu GF (2010) A novel Dmrt gene is specifically expressed in the testis of Chinese mitten crab, Eriocheir sinensis. Dev Genes Evol 220:151-159.
69. Volff JN, Zarkower D, Bardwell VJ, Schartl M (2003) Evolutionary dynamics of the DM domain gene family in metazoans. J Mol Evol 57:241-249.
70. Yi W, Zarkower D (1999) Similarity of DNA binding and transcriptional regulation by Caenorhabditis elegans MAB-3 and Drosophila melanogaster DSX suggests conservation of sex determining mechanisms. Development 126:873-881.
71. Aoyama S, Shibata K, Tokunaga S, Takase M, Matsui K, Nakamura M (2003) Expression of Dmrtl protein in developing and in sex-reversed gonads of amphibians. Cytogenet Genome Res 101:295-301.
72. De Grandi A, Calvari V, Bertini V, Bulfone A. Peverali G, Camerino G, Borsani G, Guioli S (2000) The expression pattern of a mouse doublesex-related gene is consistent with a role in gonadal differentiation. Mech Dev 90:323-326.
73. Lu H, Huang X, Zhang L, Guo Y, Cheng H, Zhou R (2007) Multiple alternative splicing of mouse Dmrtl during gonadal differentiation. Biochem Biophys Res Commun 352:630-634.
74. Marchand O, Govoroun M, D'Cotta H, McMeel O, Lareyre JJ. Bernot A, Laudet V, Guiguen Y (2000) DMRT1 expression during gonadal differentiation and spermatogenesis in the rainbow trout, Oncorhynchus mykiss. Biochim Biophys Acta 1493:180-187.
75. Raymond CS, Kettlewell JR, Hirsch B, Bardwell VJ, Zarkower D (1999) Expression of Dmrtl in the genital ridge of mouse and chicken embryos suggests a role in vertebrate sexual development. Dev Biol 215:208-220.
76. Guan G, Kobayashi T. Nagahama Y (2000) Sexually dimorphic expression of two types of DM (Doublesex/Mab-3)-domain genes in a teleost fish, the Tilapia (Oreochromis niloticus). Biochem Biophys Res Commun 272:662-666.
77. Kobayashi T, Matsuda M, Kajiura-Kobayashi H, Suzuki A, Saito N, Nakamoto M, Shibata N, Nagahama Y (2004) Two DM domain genes, DMY and DMRT1, involved in testicular differentiation and development in the medaka, Oryzias latipes. Dev Dyn 231:518-526.
78. Smith CA. Katz M, Sinclair AH (2003) DMRT1 is upregulated in the gonads during female-to-male sex reversal in ZW chicken embryos. Biol Reprod 68:560-570.
79. Kato Y. Kobayashi K. Watanabe H, Iguchi T (2011) Environmental sex determination in the branchiopod crustacean Daphnia magna:deep conservation of a Doublesex gene in the sex-determining pathway. PLoS Genet 7:e1001345.
80. Kato Y. Kobayashi K, Oda S, Colbourn JK. Tatarazako N, Watanabe H. Iguchi T (2008) Molecular cloning and sexually dimorphic expression of DM-domain genes in Daphnia magna. Genomics 91:94-101.
81. Meng A. Moore B, Tang H. Yuan B, Lin S (1999) A Drosophila doublesex-related gene, terra, is involved in somitogenesis in vertebrates. Development 126:1259-1268.
82. Sato T, Rocancourt D. Marques L, Thorsteinsdottir S, Buckingham M (2010) A Pax3/Dmrt2/Myf5 regulatory cascade functions at the onset of myogenesis. PLoS Genet 6:el000897.
83. Zhou X, Li Q, Lu H, Chen H, Guo Y, Cheng H, Zhou R (2008) Fish specific duplication of Dmrt2:characterization of zebrafish Dmrt2b. Biochimie 90:878-887.
84. Brunner B, Hornung U. Shan Z, Nanda I, Kondo M, Zend-Ajusch E, Haaf T, Ropers HH. Shima A, Schmid M, Kalscheuer VM, Schartl M (2001) Genomic organization and expression of the doublesex-related gene cluster in vertebrates and detection of putative regulatory regions for DMRTl. Genomics 77:8-17.
85. El-Mogharbel N, Wakefield M. Deakin JE, Tsend-Ayush E, Grutzner F, Alsop A, Ezaz T, Marshall Graves JA (2007) DMRT gene cluster analysis in the platypus:new insights into genomic organization and regulatory regions. Genomics 89:10-21.
86. Yamaguchi A, Lee KH, Fujimoto H, Kadomura K. Yasumoto S, Matsuyama M (2006) Expression of the DMRT gene and its roles in early gonadal development of the Japanese pufferfish Takifugu rubripes. Comp Biochem Physiol Part D Genomics Proteomics 1:59-68.
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