胡子鲇Dmrt1基因全长cDNA克隆及其表达分析

邓思平; 王静杰; 吴天利; 朱春华; 李广丽

doi:10.3724/SP.J.1035.2012.00610

胡子鲇Dmrt1基因全长cDNA克隆及其表达分析

邓思平^1,2,
王静杰^1,2,
吴天利^1,2,
朱春华^1,2,
李广丽^1,2, ,

广东海洋大学水产学院, 湛江524025

基金项目:

广西科学研究与技术开发计划重点项目(桂科合1140009-4)资助

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- 收稿日期: 2011-07-11
- 修回日期: 2012-04-10
- 发布日期: 2012-07-24

cDNA CLONING AND EXPRESSION ANALYSIS OF Dmrt1 IN CLARIAS FUSCUS

Fisheries College, Guangdong Ocean University, Zhanjiang 524025, China;

摘要

摘要: 以胡子鲇(Clarias fuscus)为研究对象, 利用RT-PCR技术和SMART RACE技术克隆获得Dmrt1基因cDNA全长, 并利用生物信息学分析其结构及功能; 利用半定量RT-PCR技术检测胡子鲇性腺(精巢/卵巢)、肌肉、肠、肝脏、心脏、头肾、鳃丝、脑和眼等10种组织以及Ⅱ-Ⅴ期精巢中Dmrt1基因表达。结果表明: 胡子鲇Dmrt1基因cDNA全长为1417 bp, 其中5非编码区(5-UTR)为35 bp, 3非编码区(3-UTR)为516 bp, 开放阅读框(ORF)包含864 bp, 编码287个氨基酸(aa), 预测所编码DMRT1为主要位于细胞核内的不稳定性亲水蛋白。氨基酸序列比对显示, 胡子鲇DMRT1与已公布的非洲胡子鲇、蟾胡子鲇、黄颡鱼等鲇形目鱼类的相似性为83.3%-96.1%。胡子鲇DMRT1中具有DMRT基因家族共有的、保守性很高的DM结构域, 此结构域具有典型的C2H2C4锌指结构, 与上述鲇形目鱼类的相似性达100%, 与斑马鱼、青鳉、虹鳟等鱼类的相似性为91.9%-97.3%, 而与鸡、鼠、猪人等的相似性达80%以上。组织表达显示, 胡子鲇Dmrt1基因仅在精巢中表达, 且Ⅱ期精巢(即精子发生期)中Dmrt1基因表达量显著高于Ⅲ、Ⅳ和Ⅴ期精巢(P0.05), 而卵巢及其他8种组织中均无表达, 表明Dmrt1是胡子鲇精巢特异性表达基因, 可能与胡子鲇的雄性性别决定、精子发生及精巢发育密切相关。
- 胡子鲇 /
- Dmrt1 /
- DM结构域 /
- 精子发生 /
- 表达分析
Abstract: Sex of fish is determined by both genetic substance and environment. The studies on the relationship between the sex and sex-related genes expression can not only reveal the mechanisms of sex determination, but also provide the theoretical basis to control the sex differentiation in artificial propagation in the fish. Catfish (Clarias fuscus) is a common freshwater fish in China. The Dmrt1 cDNA sequences were isolated from the testes of C. fuscus by RT-PCR and SMART RACE. Structure and function of the predicted DMRT1 protein were analyzed by bioinformatics. Expressions of Dmrt1 were examined using semi-quantitative RT-PCR in ten tissues (testes, ovaries, muscles, intestine, livers, heart, head kidney, gills, brains and eyes) and the testes from stageⅡ to stageⅤ. Results showed that the Dmrt1 complete cDNA was 1417 bp in length, and the 5 un-translated region (5-UTR) and the 3 un-translated region (3-UTR) were 35 bp and 516 bp, respectively. The open reading frame (ORF) was 864 bp, and a sequence of predicted 287 amino acids residues was encoded. The C. fuscus DMRT1 shared a higher amino acid identity of 83.3% to 96.1% with three Siluriformes fishes such as C. gariepinus, C. batrachus and Pelteobagrus fulvidraco. DMRT1 protein coded by Dmrt1 gene located mainly in nucleus, and worked as an unstable hydrophobic protein. A highly conserved DM domain was found in the DMRT1 protein, which was a common characteristic in DMRT clusters. The C. fuscus shared a higher DM domain identity of 100% with the above-mentioned Siluriformes fishes, 91.9% to 97.3% with Danio rerio, Oryzias latipes and Oncorhynchus mykiss, and over 80% with chicken, rat, pig and human. In addition, the DM domain of C. fuscus had a typical C2H2C4 zinc finger structure, which bound with the downstream DNA and control the gene transcription. Expression of Dmrt1 was found only in the testes, but not in ovaries, muscles, intestine, liver, heart, head kidney, gills, brain and eyes, which suggested that Dmrt1 was a testis-specific gene in C. fuscus. The expression of Dmrt1 in testes stageⅡ(spermatogenesis) was significantly higher compared with the testes from stageⅢ to stageⅤ(P0.05), which indicated that Dmrt1 was related to sex determination, spermatogenesis and testes development in male C. fuscus.
- Clarias fuscus /
- Dmrt1 /
- DM domain /
- Spermatogenesis /
- Expression analysis

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参考文献(20)

[1]	Villella A, Hall J C. Courtship anomalies caused by double sex mutations in Drosophila melanogaster [J]. Genetics, 1996, 143(1): 331-344
[2]	Aoyama S, Shibata K, Tokunaga S, et al. Expression of Dmrt1 protein in developing and in sex-reversed gonads of amphibians [J]. Cytogenetic Genome Research, 2003, 101 (3-4): 295-301
[3]	Craig A. Smith, Kelly N. Rosezler, Thomas Ohnesorg, et al. The conserved Z-linked gene, DMRT1, is required for testicular morphogenesis in the chicken embryo [J]. Developmental Biology, 2010, 344(1): 485-486
[4]	Raymond C S, Shamu C E, Shen M M, et al. Evidence for evolutionary conservation of sex-determining genes [J]. Nature, 1998, 391(6668): 691-695
[5]	Kobayashi T, Kajiura-Kobayashi H, Guan G. Sexual dimorphic expression of DMRT1 and Sox9a during gonadal differentiation and hormone-induced sex reversal in the teleost fish Nile tilapia (Oreochromis niloticus) [J]. Developmental Dynamics, 2008, 237(1): 297-306
[6]	[6] Chen Y, Wang Y L, Tian J, et al. Expression analysis of genes which related to sex determination of zebrafish[J]. Journal of Hydroecology, 2010, 3(5): 10-16[陈芸, 王艺磊, 田佳, 等. 斑马鱼性别决定相关基因的表达分析. 水生态学杂志, 2010, 3(5): 10-16] Chen Y, Wang Y L, Tian J, et al. Expression analysis of genes which related to sex determination of zebrafish[J]. Journal of Hydroecology, 2010, 3(5): 10-16[陈芸, 王艺磊, 田佳, 等. 斑马鱼性别决定相关基因的表达分析. 水生态学杂志, 2010, 3(5): 10-16]
[7]	Raghuveer K, Senthilkumaran B. Identi?cation of multiple dmrt1s in cat?sh: localization, dimorphic expression pattern, changes during testicular cycle and after methyl testosterone treatment [J]. Journal of Molecular Endocrinology, 2009, 42(5): 437-448
[8]	[8] Liu X S, Liang B, Zhang S Y. cDNA cloning, tissue distribution and mrna transcription of dmrt1 gene in the Protandrous black porgy Acanthopagrus schlegeli[J].Zoological Research, 2004, 25(2): 158-161[刘绪生, 梁冰, 张树义. 黑鲷dmrt1基因cDNA的克隆、组织表达谱及在性别逆转前后性腺中的表达. 动物学研究, 2004, 25(2): 158-161] Liu X S, Liang B, Zhang S Y. cDNA cloning, tissue distribution and mrna transcription of dmrt1 gene in the Protandrous black porgy Acanthopagrus schlegeli[J].Zoological Research, 2004, 25(2): 158-161[刘绪生, 梁冰, 张树义. 黑鲷dmrt1基因cDNA的克隆、组织表达谱及在性别逆转前后性腺中的表达. 动物学研究, 2004, 25(2): 158-161]
[9]	Raymond C S, Kettlewell J R, Hirsch B, et al. Expression of Dmrt1 in the genital ridge of mouse and chicken embryos suggests a role in vertebrate sexual development [J]. Developmental Biology, 1999, 215(2): 208-220
[10]	Kim S, Bardwell V J, Zarkower D. Cell type-autonomous and non-autonomous requirements for Dmrt1 in postnatal testis differentiation [J]. Developmental Biology, 2007, 307(2): 314-327
[11]	Xia W, Zhou L, Yao B, et al. Differential and spermatogenic cell-specific expression of DMRT1 during sex reversal in protogynous hermaphroditic groupers[J]. Molecular and Cellular Endocrinology, 2007, 263(1-2): 156-172
[12]	[12] Deng S P, Chen S L, Tian Y S, et al. Gonadal differentiation and effects of temperature on sex determination in half- smooth tongue-sole, Cynoglossus semilaevis [J].Journal of Fishery Sciences of China, 2007, 14(5): 714-719 [邓思平, 陈松林, 田永胜, 等. 半滑舌鳎的性腺分化和温度对性别决定的影响. 中国水产科学, 2007, 14(5): 714-719] Deng S P, Chen S L, Tian Y S, et al. Gonadal differentiation and effects of temperature on sex determination in half- smooth tongue-sole, Cynoglossus semilaevis [J].Journal of Fishery Sciences of China, 2007, 14(5): 714-719 [邓思平, 陈松林, 田永胜, 等. 半滑舌鳎的性腺分化和温度对性别决定的影响. 中国水产科学, 2007, 14(5): 714-719]
[13]	Kitano T, Takamune K, Kobayashi T, et al. Suppression of P450 aromatase gene expression in sex-reversed males produced by rearing genetically female larvae at a high water temperature during a period of sex differentiation in the Japanese flounder (Paralichthys olivaceus) [J]. Journal of Molecular Endocrinology, 1999, 23(2): 167-176
[14]	[14] Nan P, Du Q Y, Yan S G, et al. Effects of temperature on sex differentiation of gonads and the cloning and expression of CYP19a in two species of loaches [J]. Journal of Fishery Sciences of China, 2005, 12(5): 407-413 [南平, 杜启艳, 燕帅国, 等. 温度对泥鳅和大鳞副泥鳅性腺分化的影响和CYP19a基因的克隆与时空表达. 中国水产科学, 2005, 12(5): 407-413] Nan P, Du Q Y, Yan S G, et al. Effects of temperature on sex differentiation of gonads and the cloning and expression of CYP19a in two species of loaches [J]. Journal of Fishery Sciences of China, 2005, 12(5): 407-413 [南平, 杜启艳, 燕帅国, 等. 温度对泥鳅和大鳞副泥鳅性腺分化的影响和CYP19a基因的克隆与时空表达. 中国水产科学, 2005, 12(5): 407-413]
[15]	[15] Cao J L, Chen J J, Wu T T. Structural and functional prediction of Dmet1 deduced protein in Oreochromis aureus[J].Journal of Shanghai Ocean University, 2009, 18(4): 403-408 [曹谨玲, 陈剑杰, 吴婷婷. 奥利亚罗非鱼Dmet1基因推导蛋白的结构和功能预测. 上海海洋大学学报, 2009, 18(4): 403-408] Cao J L, Chen J J, Wu T T. Structural and functional prediction of Dmet1 deduced protein in Oreochromis aureus[J].Journal of Shanghai Ocean University, 2009, 18(4): 403-408 [曹谨玲, 陈剑杰, 吴婷婷. 奥利亚罗非鱼Dmet1基因推导蛋白的结构和功能预测. 上海海洋大学学报, 2009, 18(4): 403-408]
[16]	Sinclair A H, Berta P, Palmer M S, et al. A gene from the human sex-determining region encodes a protein with homology to a conserved DNA-binding motif [J]. Nature, 1990, 346(6281): 240-244
[17]	Kobayashi T, Kajiura-Kobayashi H, Guan G. Sexual dimorphic expression of DMRT1 and Sox9a during gonadal differentiation and hormone-induced sexreversal in the teleost ?sh Nile tilapia (Oreochromis niloticus) [J]. Developmental Dynamics, 2008, 237: 297-306
[18]	Matson C, Murphy M, Griswol M. The mammalian doublesex homolog DMRT1 is a transcriptional gatekeeper that controls the mitosis versus meiosis decision in male germ cells [J]. Developmental Cell, 2010, 19(4): 612-624
[19]	Guo Y Q, Cheng H H, Huang X, et al. Gene structure, multiple alternative splicing, and expression in gonads of zebra?sh Dmrt1 [J]. Biochemistry Biophysics Research Communication, 2005, 330(3): 950-957
[20]	Fernandino J I, Guilgur L G, Somoza G M. Dmrt1 expression analysis during spermato genesis in pejerrey, Odontesthes bonariensis [J]. Fish Physiology Biochemistry, 2006, 32(3): 231-240Marchand O, Govoroun M, Cotta H D, et al. DMRT1 expression during gonadal differentiation and spermatogenesis in the rainbow trout, Oncorhynchus mykiss [J]. Biochimica et Biophysica Acta, 2000, 1493(1-2): 180-187

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胡子鲇Dmrt1基因全长cDNA克隆及其表达分析

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cDNA CLONING AND EXPRESSION ANALYSIS OF Dmrt1 IN CLARIAS FUSCUS

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