金钱鱼Zar1基因克隆及其表达分析
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摘要
【目的】分析金钱鱼(Scatophagus argus)卵巢发育过程重要基因——合子阻滞因子1(Zygote arrest 1,Zar1)组织表达及其在卵子成熟和胚胎发育过程中的表达。【方法】克隆金钱鱼Zar1基因,采用反转录-PCR(RT-PCR)检测其在各组织的分布情况,实时荧光定量PCR(q PCR)研究Zar1在不同卵巢发育时期和胚胎发育过程中的表达。【结果】金钱鱼Zar1的开放阅读框(ORF)序列全长为1008bp,编码335个氨基酸。序列分析发现,金钱鱼Zar1在蛋白C末端高度保守,具有非典型的PHD基序(Plant homeo domain)和锌指结构域。金钱鱼Zar1同源性分析发现,它与鲈形目物种相似度最高,其中大黄鱼(Larimichthys crocea)相似性最高,为85.1%。系统进化树分析显示,金钱鱼Zar1与大黄鱼亲缘关系最近,与其分类地位一致。组织分布发现,金钱鱼Zar1仅在卵巢中表达。Zar1在II、III和IV期卵巢表达呈现逐渐递增趋势,其中IV期表达水平显著高于II期卵巢。Zar1在胚胎发育早期表达较高,后期较低,其中在四细胞期表达水平最高,之后逐渐降低。【结论】金钱鱼Zar1在卵巢和胚胎期表达,在金钱鱼卵子发生和胚胎发育阶段发挥重要作用。
【Objective】Zygote arrest 1(Zar1), as an important gene in the ovarian development, the expression of tissue distribution, and its expression during ovarian maturation and embryonic development were analyzed in spotted scat(Scatophagus argus). 【Methods】In this study, we have cloned S. argus Zar1, and analyzed its tissue distribution by RT-PCR, and expression profiles at different ovary development stages and embryonic development by quantitative real-time PCR.【Results】The open reading frame(ORF) of the Zar1 was 100 8 bp in length and encoded 335 amino acids. Sequence analysis revealed that the S. argus Zar1 was extremely conserved at the C-terminus of the amino acid, and had an atypical PHD motif and a zinc finger structure. The homology analysis showed that the gene has highest identity with those of other perciformes fishes, and the similarity with the large yellow croaker(Larimichthys crocea) was up to 85.1%. Phylogenetic tree analysis showed that the Zar1 was closest to L. crocea, which indicated consistent with its taxonomy. The tissue distribution found that the Zar1 was only expressed in the ovary. In the stages of ovary development, stage II, III and IV showed an increasing expressed trend, and Zar1 expression level at stage IV was significantly higher than that of stage II. Zar1 expression was higher in the early stages of embryonic development stages and lower in the later stages. The expression reached the highest level at the four-cell stage, and then gradually decreased. 【Conclusion】The S. argus Zar1 is expressed in the ovary and embryonic stages, it may play an important role in oogenesis and embryonic development
【Objective】Zygote arrest 1(Zar1), as an important gene in the ovarian development, the expression of tissue distribution, and its expression during ovarian maturation and embryonic development were analyzed in spotted scat(Scatophagus argus). 【Methods】In this study, we have cloned S. argus Zar1, and analyzed its tissue distribution by RT-PCR, and expression profiles at different ovary development stages and embryonic development by quantitative real-time PCR.【Results】The open reading frame(ORF) of the Zar1 was 100 8 bp in length and encoded 335 amino acids. Sequence analysis revealed that the S. argus Zar1 was extremely conserved at the C-terminus of the amino acid, and had an atypical PHD motif and a zinc finger structure. The homology analysis showed that the gene has highest identity with those of other perciformes fishes, and the similarity with the large yellow croaker(Larimichthys crocea) was up to 85.1%. Phylogenetic tree analysis showed that the Zar1 was closest to L. crocea, which indicated consistent with its taxonomy. The tissue distribution found that the Zar1 was only expressed in the ovary. In the stages of ovary development, stage II, III and IV showed an increasing expressed trend, and Zar1 expression level at stage IV was significantly higher than that of stage II. Zar1 expression was higher in the early stages of embryonic development stages and lower in the later stages. The expression reached the highest level at the four-cell stage, and then gradually decreased. 【Conclusion】The S. argus Zar1 is expressed in the ovary and embryonic stages, it may play an important role in oogenesis and embryonic development
引文
[1]BEBBERE,DANIELA,LUISA B,et al.Expression pattern of zygote arrest 1(ZAR1),maternal antigen that embryo requires(MATER),growth differentiation factor 9(GDF9)and bone morphogenetic protein 15(BMP15)genes in ovine oocytes and in vitro-produced preimplantation embryos[J].Reproduction,Fertility and Development,2008,20(8):908-915.
[2]WANG D,XIE S Y,ZHANG W,et al.Cloning and expression analysis of zygote arrest 1(Zar1)in New Zealand white rabbits[J].Journal of Genetics,2017,96(1):3-8.
[3]江文波,赵冬梅,胡炜,等.斑马鱼Zar1和Zar1-like基因的克隆及其表达分析[J].水生生物学报,2011,35(04):572-577.
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[5]WU X,WANG P,BROWN C A,et al.Zygote arrest 1(Zar1)is an evolutionarily conserved gene expressed in vertebrate ovaries[J].Biology of Reproduction,2003,69(3):861-867.
[6]LIN C H.Molecular characterization of rainbow trout Zar1 and Zar1-like genes:their potential roles in egg quality[M].West Virginia University,2012.
[7]MIAO L,YUAN Y,CHENG F,et al.Translation repression by maternal RNA binding protein Zar1 is essential for early oogenesis in zebrafish[J].Development.2017,144(1):128-138.
[8]BARRY T P,FAST A W.Biology of the spotted scat(Scatophagus argus)in the Philippines[J].Asian Fisheries science,1992,5:163-179.
[9]GUPTA S.An overview on morphology,biology,and culture of spotted scat Scatophagus argus(Linnaeus1766)[J].Reviews in Fisheries Science&Aquaculture,2016,24(2):203-212.
[10]LI G L,ZHANG M Z,DENG S P,et al.Effects of temperature and fish oil supplementation on ovarian development and foxl2 mRNA expression in spotted scat Scatophagus argus[J].Fish Biol,2015,86(1):248-60.
[11]LIU H,MU X,GUI L,et al.Characterization and gonadal expression of FOXL2 relative to Cyp19a genes in spotted scat Scatophagus argus[J].Gene,2015,561(1):6-14.
[12]JIANG D N,LI J T,TAO Y X,et al.Effects of melanocortin-4 receptor agonists and antagonists on expression of genes related to reproduction in spotted scat,Scatophagus argus[J].Journal of Comparative Physiology B,2017,187(4):603-612.
[13]CHEN J,HE M,YAN B,et al.Molecular Characterization of dax1 and SF-1 and Their Expression Analysis During Sex Reversal in Spotted Scat,Scatophagus argus[J].Journal of the World Aquaculture Society,2015,46(1):1-19.
[14]CUI XF,ZHAO Y,CHEN H P,et al.Cloning,expression and functional characterization on vitellogenesis of estrogen receptors in Scatophagus argus[J].General and Comparative Endocrinology,2017,246:37-45.
[15]张克伟,陈华谱,江东能,等.金钱鱼IGF-1和IGF-2的克隆及其在胚胎发育过程的表达[J].广东海洋大学学报,2018,38(2):7-14.
[16]YANG W,CHEN H P,CUI X F,et al.Sequencing,de novo assembly and characterization of the spotted scat Scatophagus argus(Linnaeus 1766)transcriptome for discovery of reproduction related genes and SSRs[J].Chinese Journal of Oceanology and Limnology,2017(2):1-13.
[17]MICHAILIDIS G,ARGIRIOU A,AVDI M.Expression of chicken zygote arrest 1(Zar1)and Zar1-like genes during sexual maturation and embryogenesis[J].Veterinary Research Communications,2010,34(2):173-184.
[18]BIENZ M.The PHD finger,a nuclear protein-interaction domain[J].Trends in Biochemical Sciences,2006,31(1):35-40.
[19]SANGIORGIO L,STRUMBO B,BREVINi T A.Aputative protein structurally related to zygote arrest 1(Zar1),Zar1-like,is encoded by a novel gene conserved in the vertebrate lineage[J].Comparative Biochemistry and Physiology Part B:Biochemistry and Molecular Biology,2008,150(2):233-239.
[20]BOBE J,NGUYEN T,MAHéS,et al.In silico identification and molecular characterization of genes predominantly expressed in the fish oocyte[J].BMCgenomics,2008,9(1):499.
[21]UZBEKOVA S,ROY-SABAU M,DALBIèS-TRAN R,et al.Zygote arrest 1 gene in pig,cattle and human:evidence of different transcript variants in male and female germ cells[J].Reproductive Biology and Endocrinology,2006,4(1):12.
[22]YAMAMOTO T M,COOK J M,KOTTER C V,et al.Zar1 represses translation in Xenopus oocytes and binds to the TCS in maternal m RNAs with different characteristics than Zar2[J].Biochimica et Biophysica Acta(BBA)-Gene Regulatory Mechanisms,2013,1829(10):1034-1046.
[23]TADROS W,LIPSHITZ H D.The maternal-to-zygotic transition:a play in two acts[J].Development,2009,136(18):3033-3042.
[24]BREVINI T A,CILLO F,COLLEONI S,et al.Expression pattern of the maternal factor zygote arrest 1(Zar1)in bovine tissues,oocytes,and embryos[J].Molecular Reproduction and Development:Incorporating Gamete Research,2004,69(4):375-380
[2]WANG D,XIE S Y,ZHANG W,et al.Cloning and expression analysis of zygote arrest 1(Zar1)in New Zealand white rabbits[J].Journal of Genetics,2017,96(1):3-8.
[3]江文波,赵冬梅,胡炜,等.斑马鱼Zar1和Zar1-like基因的克隆及其表达分析[J].水生生物学报,2011,35(04):572-577.
[4]WU X M,VIVEIROS M M,EPPIG,J J,et al.Zygote arrest 1(Zar1)is a novel maternal-effect gene critical for the oocyte-to-embryo transition[J].Nature Genetics,2003,33(2):187.
[5]WU X,WANG P,BROWN C A,et al.Zygote arrest 1(Zar1)is an evolutionarily conserved gene expressed in vertebrate ovaries[J].Biology of Reproduction,2003,69(3):861-867.
[6]LIN C H.Molecular characterization of rainbow trout Zar1 and Zar1-like genes:their potential roles in egg quality[M].West Virginia University,2012.
[7]MIAO L,YUAN Y,CHENG F,et al.Translation repression by maternal RNA binding protein Zar1 is essential for early oogenesis in zebrafish[J].Development.2017,144(1):128-138.
[8]BARRY T P,FAST A W.Biology of the spotted scat(Scatophagus argus)in the Philippines[J].Asian Fisheries science,1992,5:163-179.
[9]GUPTA S.An overview on morphology,biology,and culture of spotted scat Scatophagus argus(Linnaeus1766)[J].Reviews in Fisheries Science&Aquaculture,2016,24(2):203-212.
[10]LI G L,ZHANG M Z,DENG S P,et al.Effects of temperature and fish oil supplementation on ovarian development and foxl2 mRNA expression in spotted scat Scatophagus argus[J].Fish Biol,2015,86(1):248-60.
[11]LIU H,MU X,GUI L,et al.Characterization and gonadal expression of FOXL2 relative to Cyp19a genes in spotted scat Scatophagus argus[J].Gene,2015,561(1):6-14.
[12]JIANG D N,LI J T,TAO Y X,et al.Effects of melanocortin-4 receptor agonists and antagonists on expression of genes related to reproduction in spotted scat,Scatophagus argus[J].Journal of Comparative Physiology B,2017,187(4):603-612.
[13]CHEN J,HE M,YAN B,et al.Molecular Characterization of dax1 and SF-1 and Their Expression Analysis During Sex Reversal in Spotted Scat,Scatophagus argus[J].Journal of the World Aquaculture Society,2015,46(1):1-19.
[14]CUI XF,ZHAO Y,CHEN H P,et al.Cloning,expression and functional characterization on vitellogenesis of estrogen receptors in Scatophagus argus[J].General and Comparative Endocrinology,2017,246:37-45.
[15]张克伟,陈华谱,江东能,等.金钱鱼IGF-1和IGF-2的克隆及其在胚胎发育过程的表达[J].广东海洋大学学报,2018,38(2):7-14.
[16]YANG W,CHEN H P,CUI X F,et al.Sequencing,de novo assembly and characterization of the spotted scat Scatophagus argus(Linnaeus 1766)transcriptome for discovery of reproduction related genes and SSRs[J].Chinese Journal of Oceanology and Limnology,2017(2):1-13.
[17]MICHAILIDIS G,ARGIRIOU A,AVDI M.Expression of chicken zygote arrest 1(Zar1)and Zar1-like genes during sexual maturation and embryogenesis[J].Veterinary Research Communications,2010,34(2):173-184.
[18]BIENZ M.The PHD finger,a nuclear protein-interaction domain[J].Trends in Biochemical Sciences,2006,31(1):35-40.
[19]SANGIORGIO L,STRUMBO B,BREVINi T A.Aputative protein structurally related to zygote arrest 1(Zar1),Zar1-like,is encoded by a novel gene conserved in the vertebrate lineage[J].Comparative Biochemistry and Physiology Part B:Biochemistry and Molecular Biology,2008,150(2):233-239.
[20]BOBE J,NGUYEN T,MAHéS,et al.In silico identification and molecular characterization of genes predominantly expressed in the fish oocyte[J].BMCgenomics,2008,9(1):499.
[21]UZBEKOVA S,ROY-SABAU M,DALBIèS-TRAN R,et al.Zygote arrest 1 gene in pig,cattle and human:evidence of different transcript variants in male and female germ cells[J].Reproductive Biology and Endocrinology,2006,4(1):12.
[22]YAMAMOTO T M,COOK J M,KOTTER C V,et al.Zar1 represses translation in Xenopus oocytes and binds to the TCS in maternal m RNAs with different characteristics than Zar2[J].Biochimica et Biophysica Acta(BBA)-Gene Regulatory Mechanisms,2013,1829(10):1034-1046.
[23]TADROS W,LIPSHITZ H D.The maternal-to-zygotic transition:a play in two acts[J].Development,2009,136(18):3033-3042.
[24]BREVINI T A,CILLO F,COLLEONI S,et al.Expression pattern of the maternal factor zygote arrest 1(Zar1)in bovine tissues,oocytes,and embryos[J].Molecular Reproduction and Development:Incorporating Gamete Research,2004,69(4):375-380
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