Microsoft Word - 5--杨梅_new_.doc

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1 46 1 Vol.46, No OCEANOLOGIA ET LIMNOLOGIA SINICA Jan., 2015 (Crassostrea gigas)wnt4 cdna * 1, 2 1 1, ( ; ) Wnt4 Wnt, RACE Wnt4 cdna, 1999bp, 1068bp, 355, (Homo sapiens) (Platynereis dumerilii) (Chlamys farreri) Wnt4 44% 48% 46% RT-PCR Wnt4, Wnt4, (), Wnt4 ; Wnt4 (, ),, Wnt4 ; Wnt4 ; ; Q78 doi: /hyhz Wnt Wnt (Smolich et al,1993) Wnt Nusse 1982 (mouse mammary tumor vious, MMTV), int-1, 1973 wingless, Wnt(Nusse et al, 1992), (Parr et al, 1994; Bergstaina et al, 1997) Wnt Wnt, (, 2008) Wnt,, (Klaus et al, 2008;, 2011) Wnt4 Wnt,, (Mus musculus) (Daniorerio) (Platynereis dumerilii) (Drosophila melanogaster) (Chlamys farreri) (Schistosoma japonicum),wnt4 ;, Wnt4, 24 (, 2007);, Sato (2006), Wnt4, ; Wnt4 * (973 ), 2010CB ;, ; (863 ), 2012AA10A405 ;, CARS-48 ;,, yangmeiquietsnow@163.com :,,, gfzhang@qdio.ac.cn : , :

2 36 46 (, 2007); Wnt4 (,,,, ), (, 2013), Wnt4, (, 2008), (Hedgecock et al, 2005) RACE (Crassostrea gigas) Wnt4 cdna, RT-PCR, Wnt4, Wnt 1 1.1, 10L ; 90μm, 25μm, 5L /ml ,, 26ºC 25m 3, 30, (,,,,, ),, D,,, ( 1), 5 (, ), 80 C, RNA 1.2 Wnt4 Trizol RNA NanoDrop 2000(Thermo Scientific) RNA ; 1.5% RNA, PrimeScript II 1st Strand cdna Synthesis Kit (TaKaRa, Japan) RACE cdna 表 1 长牡蛎幼虫不同发育时期样品取样 Tab.1 Sampling of C. gigas different developmental stages Egg Egg E 0:00 Cleavage Two cells TC 1:20 Early morula stage EM 2:15 Morula stage MS 3:30 Embryogenesis Blastula stage BS 4:30 Early gastrula stage EG 6:30 Gastrula stage GS 7:30 Trochophore 1 Trochophore1 T1 9:30 2 Trochophore2 T2 11:30 D D-shaped larvae D 1 D-shaped larva1 D1 16:30 D 2 D-shaped larva2 D2 2d(day) D 3 D-shaped larva3 D3 4d Umbo larvae 1 Umbo larva1 U1 10d 2 Umbo larva2 U2 16d 3 Umbo larva3 U3 24d Pediveliger Pediveliger P 38d Spat 1 Spat1 S1 40d 2 Spat2 S2 44d 3 Spat3 S3 49d Juvenile Juvenile J 83d GenBank Wnt4,, Wnt4, Wnt4,

3 1 : (Crassostrea gigas)wnt4 cdna 37, RACE, PCR Wnt4, 2 表 2 长牡蛎 Wnt4 基因引物信息 Tab.2 The information of C.gigasWnt4 primers (5-3 ) Wnt4-F ATCAGTTTGCCAATCGTC Wnt4 Wnt4-R CACATCAGGTTACACCCATC Wnt4 Wnt4-U5R1 TTCTTCACCGATGTCTG Wnt4 5 Wnt4-U5R2 ATCGGCAGTGATAAGTCC Wnt4 5 Wnt4-U3F1 GTATGTAAATGTCACGGAGTC Wnt4 3 Wnt4-U3F2 TGGGTGTAACCTGATGTG Wnt4 3 dg-ap GGCCACGCGTCGACTAGTACG Wnt4 5 GGGGGGGGG dt-ap GGCCACGCGTCGACTAGTAC TTTTTTTTTTTTTTTTT Wnt4 3 AP GGCCACGCGTCGACTAGTAC Wnt4 5 /3 1.5% PCR DNA (Axygen), peasy-t1 ( ) DH5α ( ),, 37 C,, PCR, 1.3 Wnt4 Lasergene, Mw(Molecular weight) pi (Isoelectric point) Expasycompute pi/mw( web.expasy. org/compute_pi/), Clustal X DNAMAN, SignalP 4.1( SignalP/), phyml3.0( RT-PCR, (,,,,, ),, D,,,, (, ), cdna cdna Wnt4 Wnt4-real F(5 AAGAACGCCGCCTACCCA3 ), Wnt4-real R(5 ACA GCCACAGTTGTCCAG3 )( 239bp), 18S rrna(rs18) (RS18-real F: 5 GCCATCAAGGGTATCGGTAGAC 3 ; RS18-real R: 5 CTGCCTGTTAAGGAACCAGTCAG 3 ; 168bp)(Du et al, 2013) ABI7500 Fast PCR Wnt4, 3 RT-PCR : 94 C 30s, 1 ; 94 C 5s, 60 C 30s, %, ABI7500 Fast SDS software V2.0, 2 Ct, SPSS Wnt4 cdna Wnt, , 23 24,, Porcupine (Bergsteina et al, 2006) Wnt Wnt Frizzled (Mikels et al, 2006), Wnt4 cdna 1999bp, 5 (Untranslated region, 5'UTR)466bp, 3 UTR465bp, (open reading frame, ORF) 1068bp, 355 ( 1) Wnt kD, Wnt4 ; 24 Wnt, ( 1; 2), 22 Wnt4 (Homo sapiens), (Branchiostoma floridae), (Platynereis dumerilii) (Capitella teleta) (Euprymna scolopes) (Chlamys farreri)wnt4 44%, 43%, 48%, 45%, 42% 46%( 2) phyml 3.0 Wnt4, Wnt4, ( 3),

4 38 海图1 Fig.1 洋与湖沼 46 卷长牡蛎 Wnt4 基因完整 cdna 序列与推导的氨基酸序列 The cdna sequence of C. gigas Wnt4 gene and the deduced amino acids sequence 图中方框区域代表起始密码子和终止密码子; 波浪下划线代表信号肽; 双下划线代表保守的半胱氨酸残基(C) 2.2 长牡蛎 Wnt4 基因在成体各组织及不同幼体发在外套膜鳃唇瓣消化腺雄性性腺雌性性腺育阶段中的定量表达分析荧光定量RT-PCR结果表明, 长牡蛎Wnt4mRNA 中均有表达, 但各组织间存在差异, 在唇瓣鳃和消化腺中的表达量相对较高, 同时在雌性性腺中的表

5 1 : (Crassostrea gigas)wnt4 cdna 39 2 Wnt4 Fig.2 Multiple alignment of Wnt4 proteins from different species 100% 75% : (Homo sapiens, Hs), (Branchiostoma floridae, Bf), (Platynereis dumerilii, Pd), (Capitella teleta, Ct), (Chlamys farreri, Cf), (Euprymnas colopes, Es), (Nematostella vectensis, Nv), (Crassostrea gigas, Cg) ( 4), Wnt4 (E) (TC) (EM), (MS), (BS), 3 (P<0.05); (GS), (T1, T2) D (D1,D2,D3), (U1, U2, U3) (S1, S2, S3),, 20%, (J) ( 5)

6 (ML) Wnt4 Fig.3 Molecular phylogenetic analysis of Wnt4amino acids sequences in C. gigas based on maximum likelyhood(ml) method 4 Wnt4 Fig.4 Relative expression of the Wnt4 gene in different tissues of C.gigas Man: mantle ; Amu:adductor muscle ; Gil: gills ; Lpa: labial palp ; Dgl: digestive gland ; Hem: hemocyte ; Fgo: female gonad ; Mgo:male gonad Wnt4 Yu (2006), Wnt4 ; 鳉 Wnt4, Wnt4a Wnt4b(, 2012; Nicol et al, 2012) Wnt4a Wnt4 80%, mrna, Ⅱ ; Wnt4b Wnt4 60%, (Nicol et al, 2012) (2013) RT-PCR, Wnt4 mrna, 5 Wnt4 Fig.5 Relative expression of the Wnt4 gene in different developmental stages of C.gigas. E: egg ; TC: two cells ; EM:early morula ; MS: morula stage ; B:blastula stage ; EG: early gastrula stage ; G:gastrula stage ; T1:trochophore 1 1; T2:trochophore 2 2; D1:D-shaped larva 1D 1; D2:D-shaped larva 2D 2; D3: D-shaped larva 3D 3; U1: umbo larva 1 1; U2: umbo larva 2 2; U3: umbo larva 3 3; P: pediveliger ; S1: spat1 1; S2: spat2 2; S3: spat3 3; J: juvenile * (P<0.05), Wnt4, ( ), 17% ( 4), Wnt4

7 1 : (Crassostrea gigas)wnt4 cdna 41, Wnt4 (Bernard et al, 2007) Wnt4mRNA,,, (Barrionuevo et al, 2006) Wnt4, 1.5, (, 2012), Wnt4mRNA, 1.2, Wnt4,, (, 2013),, Wnt4 (Yuki et al, 2005), Wnt4,, Wnt4mRNA,,, Wnt4,,, Wnt4,, (Bernardet al, 2007) Wnt, Frizzled, glycogen synthase kinase 3β(GSK-3β), Dishevelled, β- catenin T-cell factor (TCF) (Yang et al, 2014),,, (, 2011) 3 Wnt4 Wnt, Wnt4,, Wnt4 Wnt4,,,,, :, ,, Wnt :., 33(2): ,,, Wnt., 27(2): ,,, 鳉 Wnt4., 36(5): ,,, Wnt4 cdna., 20(2): :, 63,, Wnt., 33(7): ,,, Sjwnt-4., 23(3): ,, Wnt., 16(3): Banziger C, Soldini D, Schutt C et al, Wntless, a conserved membrane protein dedicated to the secretion of Wnt proteins from signaling cells. Cell, 125(3): Barrionuevo F, Bagheri-Fam S, Klattig J et al, Homozygous inactivation of Sox9 causes complete XY sex reversal in mice. Biology of Reproduction, 74(1): Bergsteina I, Eisenbergb L M, Bhaleraoc J et al, Isolation of two novel WNT genes, WNT14 and WNT15, one of which (WNT15) is closely linked to WNT3 on human chromosome 17q21. Genomics, 46(3): Bernard P, Harley V R, Wnt4 action in gonadal development and sex determination. International journal of biochemistry & cell biology, 39(1): Du Y S, Zhang L L, Xu F et al, Validation of housekeeping genes as internal controls for studyinggene expression during Pacific oyster (Crassostreagigas) development by quantitative real-time PCR. Fish and Shellfish Immunology, 34(3): Hedgecock D, Gaffney P M, Goulletquer P et al, The case for sequencing the Pacific oyster genome. Journal of Shellfish Research, 24(2): Klaus A, Birchmeier W, Wntsignalling and its impact on development and cancer. Nature Reviews Cancer, 8(5): McElroy M D, Elrefaei M, Jones N et al, Coinfection with Schistosomamansoniis associated with decreased HIV- Specific cytolysis and increased IL-10 production. Journal of Immunology, 174(8):

8 42 46 Mikels A J, Nusse R, Wnts as ligands: processing, secretion and reception. Oncogene, 25: Nicol B, Guerin A, Fostier A et al, Ovary-predominant wnt4 expression during gonadal differentiation is not conserved in the rainbow trout (Oncorhynchus mykiss). Molecular Reproduction and Development, 79(1): Nusse R, Varmus H E, Many tumors induced by the mouse mammary tumor virus contain a provious integrated in the same region of the host genome. Cell, 31(1): Nusse R, Varmus H E, Wnt genes. Cell, 69(7): Oshima Y, Hayashi T, Tokunaga S et al, Wnt4 Expression in the differentiating gonad of the frog Rana rugosa. Zoological Science, 22(6): Parr B A, McMahon A P, Wnt genes and vertebrate development. Current Opinion in Genetics & Development, 4(4): Sato M, Umetsu D, Murakami S et al, DWnt4 regulates thedorsoventral specificity of retinal projections in the Drosophilamelanogaster visual system. Nature Neuroscience, 9(1): Smolich B D, McMahon J A, McMahon A P et al, 1993.Wnt family proteins are secreted and associated with the cell surface. Molecular Biology of The Cell, 4(12): Ungar A R, Kelly G M, Moon R T, Wnt4 affects morphogenesis when misexpressed in the zebrafish embryo. Mechanism of Development, 52(2 3): Yang M, Xu F, Liu J et al, 2015 Identification and expression patterns of Wnt signaling pathway-related genes in Crassostrea gigas. Unpublished Yu H S, Pask A J, Shaw G et al, Differential expression of Wnt4 in testicular and ovarian development in a marsupial. BMC Developmental Biology, 6(44): 1 13 Yuki O, Toshiyuki H, Shuichi T et al, Wnt4 expression in the differentiating gonad of the frog Rana rugosa. Zoological Science, 22(6): MOLECULAR CLONING AND EXPRESSION OF WNT4 GENE IN PACIFIC OYSTER CRASSOSTREA GIGAS YANG Mei 1, 2, XU Fei 1, LIU Jun 1, 2, LI Li 1, ZHANG Guo-Fan 1 (1. National & Local Joint Engineering Laboratory of Ecological Mariculture, Institute of Oceanology, Chinese Academy of Sciences, Qingdao , China; 2. University of Chinese Academy of Sciences, Beijing , China) Abstract As an important member of the Wnt gene family, Wnt4 plays a pivotal role in development processes of many animals. Using the RACE technique, we obtained a complete cdna sequence of Wnt4 gene in Pacific oyster (Crassostrea gigas). The full length of this gene was 1999 bpencoding 355 amino acids. Homologous analysis on the amino acids showed that C. gigas Wnt4 was over 40% similar to the most species of Wnt4, including Homo sapiens, Platynereis dumerilii, and Chlamys farreri. QRT-PCR results showed that C. gigas Wnt4 gene was expressed in multiple tissues (mantle, gills, labial palp, digestive gland, male gonad, and female gonad), implying that C. gigas Wnt4 plays various biological roles. Among different developmental stages in C. gigas, the early embryogenesis showed high Wnt4 mrna expression level (highest in the morula stage, followed by gastrula stage), and lowest in the juvenile stage. Therefore, C. gigas Wnt4 may be involved in the formation of certain organs in early developmental stages. Key words Crassostrea gigas; Wnt4 gene; cloning; expression analysis

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Microsoft Word - 5--杨梅_new_.doc