三疣梭子蟹UHRF1基因在胚胎、幼体和性腺发育过程中的表达分析
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摘要
为探讨泛素样含PHD和环指域蛋白1(UHRF1)基因在三疣梭子蟹(Portunus trituberculatus)发育过程中的作用,实验采用SMART RACE方法,克隆了三疣梭子蟹UHRF1(PtUHRF1)基因。该基因cDNA全长为2849 bp,开放阅读框(ORF)为2298 bp,预测其编码1个含有765个氨基酸的蛋白质。结构域分析显示,该蛋白质包含UBL、PHD、TTD、SRA、RING finger 5个功能结构域。同源分析表明,三疣梭子蟹PtUHRF1的氨基酸序列与其他物种有较高的同源性。qRT-PCR结果显示,PtUHRF1基因在三疣梭子蟹所有组织中均有表达,但在精巢中表达量显著高于其他组织。该基因在胚胎和幼体发育不同时期表达差异显著,在受精卵中的表达量最高,并显著高于胚胎发育其他时期,是多细胞时期表达量的2.5倍。PtUHRF1基因在性腺发育不同时期表达存在显著差异,在卵巢II期表达量达到峰值,之后逐渐下降;在精巢Ⅰ期的表达量最高,随着精巢发育逐渐下降。实验结果表明,PtUHRF1参与了三疣梭子蟹胚胎、幼体和性腺发育调控,为进一步深入研究该基因在三疣梭子蟹及甲壳动物生长发育中的作用提供参考。
This study cloned the full-length cDNA of UHRF1(PtUHRF1) from the swimming crab Portunus trituberculatus using rapid amplification of cDNA ends(RACE).The length of PtUHRF1 cDNA was 2849 bp,which contained a 2298 bp open reading frame to encode 765 amino acids.Structure analysis revealed that PtUHRF1 contained several conserved domains,including UBL,PHD,TTD,SRA and RING.Homology analysis revealed that PtUHRF1 exhibited high identity with other species.The expression of PtUHRF1 at mRNA level was detected in all tested tissues with the highest level in testes.PtUHRF1 showed stage-specific expression during embryonic and larval development with the highest expression in fertilized egg.There were significant differences in PtUHRF1 expression at different ovary and testis stages.In ovary,its expression reached the highest value at stage II,and then decreased gradually,while in testis,the highest expression was detected at stage I,and then decreased as testis developed.These results indicated that PtUHRF1 is important in regulating embryonic,larval and gonadal development in the swimming crab,and provided useful reference for further studies on the roles of this gene in growth and development of P.trituberculatus and crustaceans.
This study cloned the full-length cDNA of UHRF1(PtUHRF1) from the swimming crab Portunus trituberculatus using rapid amplification of cDNA ends(RACE).The length of PtUHRF1 cDNA was 2849 bp,which contained a 2298 bp open reading frame to encode 765 amino acids.Structure analysis revealed that PtUHRF1 contained several conserved domains,including UBL,PHD,TTD,SRA and RING.Homology analysis revealed that PtUHRF1 exhibited high identity with other species.The expression of PtUHRF1 at mRNA level was detected in all tested tissues with the highest level in testes.PtUHRF1 showed stage-specific expression during embryonic and larval development with the highest expression in fertilized egg.There were significant differences in PtUHRF1 expression at different ovary and testis stages.In ovary,its expression reached the highest value at stage II,and then decreased gradually,while in testis,the highest expression was detected at stage I,and then decreased as testis developed.These results indicated that PtUHRF1 is important in regulating embryonic,larval and gonadal development in the swimming crab,and provided useful reference for further studies on the roles of this gene in growth and development of P.trituberculatus and crustaceans.
引文
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[4]Kim J K,Esteve P O,Jacobsen S E,et al.UHRF1 binds G9a and participates in p21 transcriptional regulation in mammalian cells[J].Nucleic Acids Research,2009,37(2):493-505
[5]Arita K,Ariyoshi M,Tochio H,et al.Recognition of hemimethylated DNA by the SRA protein UHRF1 by a base-flipping mechanism[J].Nature,2008,455(7217):818-839
[6]Chu J,Loughlin E A,Gaur N A,et al.UHRF1 phosphorylation by cyclin A2/cyclin dependent kinase 2 is required for zebrafish embryogenesis[J].Molecular Biology of the Cell,2012,23(1):59-70
[7]Kagiwada S,Kurimoto K,Hirota T,et al.Replicationcoupled passive DNA demethylation for the erasure of genome imprints in mice[J].The EMBO Journal,2013,32(3):340-353
[8]Maenohara S,Unoki M,Toh H,et al.Role of UHRF1 in de novo DNA methylation in oocytes and maintenance methylation in preimplantation embryos[J].PLoS Genetics,2017,13(10):e1007042
[9]Guan W B,Xuan F J,Chen H H,et al.Reproductive characteristics and condition status of adult swimming crab Portunus trituberculatus(Brachyura:Portunidae)in East China Sea[J].Marine Fisheries,2009,31(2):120-127[管卫兵,宣富君,陈辉辉,等.东海三疣梭子蟹成熟群体生殖特征和条件状况.海洋渔业,2009,31(2):120-127]
[10]Wu X G,Yao G G,Yang X Z,et al.A study on the ovarian development of Portunus trituberculatus in East China Sea during the first reproductive cycle[J].Acta Oceanologica Sinica,2007,29(4):120-127[吴旭干,姚桂桂,杨筱珍,等.东海三疣梭子蟹第一次卵巢发育规律的研究.海洋学报,2007,29(4):120-127]
[11]Ye H H,Li S Q,Huang H Y,et al.Histological studies on testes development of the mud crab,Scya serrate[J].Zoological Research,2002,23(2):141-144[叶海辉,李少菁,黄辉洋,等.锯缘青蟹精巢发育的组织学观察.动物学研究,2002,23(2):141-144]
[12]Hashimoto H,Horton JR,Zhang X,et al.UHRF1 a modular multi-domain protein,regulates replication-coupled crosstalk between DNA methylation and histone modifications[J].Epigenetics,2009,4(1):8-14
[13]Jenkins Y,Lang W,Sharma P,et al.Critical role of the ubiquitin ligase activity of UHRF1,a nuclear RING finger protein,in tumor cell growth[J].Molecular Biology of the Cell,2005,16(12):5628
[14]Yao CC,Liu Y,Sun M,et al.MicroRNAs and DNAmethylation as epigenetic regulators of mitosis,meiosis and spermiogenesis[J].Reproduction,2015,150(1):25-34
[15]KagiwadaS,KurimotoK,HirotaT,et al.Replicationcoupled passive DNA demethylation for the erasure of genome imprints in mice[J].Embo Journal,2013,32(3):340-353