EsWnt-4基因的表达分析
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摘要
Wnt-4基因是Wnt基因家族重要的成员,在许多生物事件中都发挥着重要的作用。从中华绒螯蟹(Eriocheir sinensis)卵巢转录组数据库中筛选到EsWnt-4的cDNA序列1027 bp。RT-PCR检测发现EsWnt-4在神经组织、心脏和性腺中有表达,说明在中华绒螯蟹中EsWnt-4可能与多种组织细胞的活动有关。荧光定量检测发现,受精卵中EsWnt-4 mRNA的含量较成熟卵细胞和2、4、8细胞胚胎时期高,推测EsWnt-4基因有可能在受精卵中已经开始表达,而在之后的2、4、8细胞时期其表达又受到抑制。此外,EsWnt-4在16细胞期表达量再次升高,说明该时期可能有相关的生物学事件需要EsWnt-4基因的参与。在幼体阶段EsWnt-4在第二溞状幼体及第五溞状幼体阶段表达最高,说明EsWnt-4可能与幼体体节生成及变态发育有关。
Wnt-4 gene is an important member of the Wnt gene family and plays an important role in many biological events. The EsWnt-4 cDNA was obtained from the transcriptome data of Eriocheir sinensis ovary and the length of EsWnt-4 cDNA contains 1027 nucleotides,which encodes 263 deduced amino acid residues. the cDNA contains 1027 nucleotides. Multiple sequence alignment revealed that EsWnt-4 shared the highest similarity(98%) with that of the Euperipotaidei kanangrensis in conserved domains. Further,a phylogenetic tree of WNT amino acid sequences from various species was constructed. EsWnt-4 was clustered into one branch with Wnt-4 of other species. RT-PCR analysis showed that EsWnt-4 was expressed in neural organization,heart and gonadal,suggesting that EsWnt-4 was related to the activity of various of tissues.While EsWnt-4 expression was different among different tissues,it has the highest expression level in heart and brain,the lowest in thoracic ganglion. Interestingly,significantly higher expression level of EsWnt-4 was detected in fertilized eggs than in mature eggs,2-cell stage,4-cell stage,and 8-cell stage,suggesting that EsWnt-4 gene might have started expression in the spermatozoon,but the expression was suppressed in the later 2-cell,4-cell,8-cell stage. In addition,the EsWnt-4 increased in 8-16 cell stages,suggesting that EsWnt-4 might participate or play role in related biological events in this period. In the larval stage,significantly higher expression level of EsWnt-4 was detected in zoea-2 larvae and zoea-5 larvae,indicted that EsWnt-4 could be related to segment formation and metamorphosis.
Wnt-4 gene is an important member of the Wnt gene family and plays an important role in many biological events. The EsWnt-4 cDNA was obtained from the transcriptome data of Eriocheir sinensis ovary and the length of EsWnt-4 cDNA contains 1027 nucleotides,which encodes 263 deduced amino acid residues. the cDNA contains 1027 nucleotides. Multiple sequence alignment revealed that EsWnt-4 shared the highest similarity(98%) with that of the Euperipotaidei kanangrensis in conserved domains. Further,a phylogenetic tree of WNT amino acid sequences from various species was constructed. EsWnt-4 was clustered into one branch with Wnt-4 of other species. RT-PCR analysis showed that EsWnt-4 was expressed in neural organization,heart and gonadal,suggesting that EsWnt-4 was related to the activity of various of tissues.While EsWnt-4 expression was different among different tissues,it has the highest expression level in heart and brain,the lowest in thoracic ganglion. Interestingly,significantly higher expression level of EsWnt-4 was detected in fertilized eggs than in mature eggs,2-cell stage,4-cell stage,and 8-cell stage,suggesting that EsWnt-4 gene might have started expression in the spermatozoon,but the expression was suppressed in the later 2-cell,4-cell,8-cell stage. In addition,the EsWnt-4 increased in 8-16 cell stages,suggesting that EsWnt-4 might participate or play role in related biological events in this period. In the larval stage,significantly higher expression level of EsWnt-4 was detected in zoea-2 larvae and zoea-5 larvae,indicted that EsWnt-4 could be related to segment formation and metamorphosis.
引文
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[26]王武,李应森.河蟹生态养殖[M].北京:中国农业出版社,2010:12.WANG W,LI Y S.Eco-culturing of Eriocheir sinensis[M].Beijing:China Agricultural Press,2010:12.
[2]NUSSE R,VARMUS H E.Many tumors induced by the mouse mammary tumor virus contain a provirus integrated in the same region of the host genome[J].Cell,1982,31(1):99-109.
[3]RIJSEWIJK F,SCHUERMANN M,WAGENAARE,et al.The Drosophila homology of the mouse mammary oncogene int-1 is identical to the segment polarity gene wingless[J].Cell,1987,50(4):649-657.
[4]KRAUSS S,KORZH V,FJOSE A,et al.Expression of four zebrafish wnt-related genes during embryogenesis[J].Development,1992,116(1):249.
[5]MCGREW L L,OTTE A P,MOON R T.Analysis of Xwnt-4 in embryos of Xenopus laevis:A Wnt family member expressed in the brain and floor plate[J].Development,1992,115(2):463-73.
[6]VAINIO S,HEIKKIL M,KISPERT A,et al.Female development in mammals is regulated by Wnt-4signalling[J].Nature,1999,397(6718):405-9.
[7]JAMESON S A.LIN Y T,CAPEL B.Testis development requires the repression of Wnt4 by Fgf signaling[J].Developmental Biology,2012,370(1):24-32.
[8]TANDA N,KAWAKAMI Y,SAITO T,et al.Cloning and characterization of Wnt-4 and Wnt-11c DNAs from chick embryo[J].Dna Sequence,1995,5(5):277-281.
[9]HU Q,ZHU Y,LIU Y,et al.Cloning and characterization of wnt4a gene and evidence for positive selection in half-smooth tongue sole(Cynoglossus semilaevis)[J].Scientific Reports,2014,4(8):7167.
[10]姜开菊.Wnt家族基因Wnt4-like以及FLNα在荒漠沙蜥皮肤中的原位表达研究[D].兰州:兰州大学,2013.JIANG K J.Study on in situ expression of Wnt family Wnt-like and FLNαin skin of Phrynocehalus przewalskii[D].Lanzhou:Lanzhou University,2013.
[11]李海龙,刘建国,刘晓龙,等.栉孔扇贝wnt4基因c DNA克隆及表达分析[J].中国水产科学,2013,20(2):,260-268.LI H L,LIU J G,LIU X L,et al.Molecular cloning and expression analysis of wnt4 c DNA from the Zhikong scallop Chlamys farreri[J].Journal of Fishery Sciences of China,2013,20(2):,260-268.
[12]徐跃峰,李一峰,梁箫,等.厚壳贻贝Wnt4基因时空表达[J].水产学报,2016,40(10):1567-1575.XU Y F,LI Y F,LIANG X,et al.Temporal-spatial expression of Wnt4 gene in the mussel Mytilus coruscus[J].Journal of Fisheries of China,2016,40(10):1567-1575.
[13]HOLLYDAY M,MCMAHON J A,MCMAHON A P.Wnt expression patterns in chick embryo nervous system[J].Mechanisms of Development,1995,52(1):9-25.
[14]UNGAR A R,KELLY G M,MOON R T.Wnt4affects morphogenesis when misexpressed in the zebrafish embryo[J].Mechanisms of Development,1995,52(2-3):,153-164.
[15]SATO M,UMETSU D S,YASUGI T,et al.DWnt4regulates the dorsoventral specificity of retinal projections in the Drosophila melanogaster visual system[J].Nature Neuroscience,2006,9(1):67-75.
[16]PESSOT C A,BRITO M,FIGUEROA J,et al.Presence of RNA in the sperm nucleus[J].Biochemical&Biophysical Research Communications,1989,158(1):272-278.
[17]WYKES S M,MILLER D,KRAWETZ S A,et al.Mammalian spermatozoal mRNAs:Tools for the functional analysis of male gametes[J].Journal of Submicroscopic Cytology&Pathology,2000,32(1):77-81.
[18]WYKES S M,VISSCHER D W,KRAWETZ S A.Haploid transcripts persist in mature human spermatozoa[J].Molecular Human Reproduction,1997,3(1):15-19.
[19]FANG P,ZENG P,WANG Z,et al.Estimated diversity of messenger RNAs in each murine spermatozoa and their potential function during early zygotic development[J].Biology of Reproduction,2014,90(5):94,1-11.
[20]SOUSA P D,CAVENEY A,WESTHUSIN M,et al.Temporal patterns of embryonic gene expression and their dependence on oogenetic factors[J].Theriogenology,1998,49(49):115-128.
[21]TAYLOR K D,PIKóL.Expression of ribosomal protein genes in mouse oocytes and early embryos[J].Molecular Reproduction&Development,1992,31(3):182-188.
[22]JR D W,SOUSA P D,SCHULTZ R.Transient expression of translation initiation factor e IF-4Cduring the 2-cell stage of the preimplantation mouse embryo:Identification by mRNA differential display and the role of DNA replication in zygotic gene activation[J].Developmental Biology,1996,174(2):190-201.
[23]WHITE K P,RIFKIN S A,HURBAN P,et al.Microarray analysis of Drosophila development during metamorphosis[J].Science,1999,286(5447):2179-2184.
[24]PRATHER R S,BARNES F L,SIMS M M,et al.Nuclear transplantation in the bovine embryo:Assessment of donor nuclei and recipient oocyte[J].Biology of Reproduction,1987,37(4):859-866.
[25]CROSBY I M,GANDOLFI F,MOOR R M.Control of protein synthesis during early cleavage of sheep embryos[J].Journal of Reproduction and Fertility,1988,82(2):769-775.
[26]王武,李应森.河蟹生态养殖[M].北京:中国农业出版社,2010:12.WANG W,LI Y S.Eco-culturing of Eriocheir sinensis[M].Beijing:China Agricultural Press,2010:12.
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