团头鲂cdc20基因的序列特征和表达分析
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作者:
作者单位:

1.华中农业大学水产学院/农业农村部淡水生物繁育重点实验室,武汉 430070;2.集美大学水产学院/农业农村部东海海水健康养殖重点实验室,厦门 361021

作者简介:

庞连慧,E-mail:819555965@qq.com

通讯作者:

王焕岭,E-mail: hbauwhl@hotmail.com

中图分类号:

S917.4

基金项目:

国家自然科学基金项目(31872542)


Sequence characteristics and expression analysis of cdc20 gene in Megalobrama amblycephala
Author:
Affiliation:

1.Lab of Freshwater Animal Breeding,Ministry of Agriculture and Rural Affairs/ College of Fisheries,Huazhong Agriculture University,Wuhan 430070,China;2.Key Laboratory of Healthy Mariculture for the East China Sea,Ministry of Agriculture and Rural Affairs/College of Fisheries,Jimei University,Xiamen 361021,China

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    摘要:

    为研究鱼类细胞分裂周期蛋白20基因(cell division cycle 20 homolog,cdc20)的功能,以团头鲂为试验对象,经PCR扩增获得了团头鲂cdc20基因的CDS,基于多序列比对和系统进化树分析该基因的结构和不同物种间的进化关系,采用半定量PCR(semi-quantitative reverse transcription PCR,sqRT-PCR)、荧光定量PCR(quantitative real-time PCR,qRT-PCR)和切片原位杂交的方法检测了该基因的时空表达模式。结果显示,该基因编码区所包含的序列长度为1 482 bp,编码493 aa。团头鲂Cdc20氨基酸序列在进化上十分保守,具有7个WD40基序形成的稳定7叶β螺旋桨状结构,与鲤和斑马鱼的相似性最高。qRT-PCR结果显示团头鲂cdc20基因在早期胚胎中的表达量最高,随后呈下降趋势。sqRT-PCR检测发现cdc20基因主要在性腺中表达。性腺切片原位杂交结果显示cdc20在团头鲂各时期卵母细胞中均有表达,主要集中在Ⅱ期卵母细胞的胞质和Ⅲ、Ⅵ期卵母细胞的卵黄颗粒中;另外也发现该基因在精巢的精原细胞和生精小管的生殖上皮中表达量最高,在精子中的表达较低。以上结果表明团头鲂cdc20基因是一个母源基因,可能参与生殖细胞的发育。

    Abstract:

    In order to investigate function of cell division cycle 20 homolog (Cdc20),in the present study,the coding sequence (CDS) of Megalobrama amblycephala cdc20 gene was cloned,and the domains and evolutionary relationship of Cdc20 were analyzed based on multiple sequence alignment and phylogenetic analysis.The temporal and spatial expression patterns of cdc20 were detected by semi-quantitative reverse transcription PCR (sqRT-PCR),quantitative real-time PCR (qRT-PCR) and in situ hybridization.The results showed that the cdc20 CDS of M. amblycephala is 1 482 bp in length and encodes 493 aa.Multiple sequence alignments and phylogenetic analysis revealed that amino acids sequences of Cdc20 were very conserved through evolution and consisted of seven-bladed β propeller structure,which was formed by seven WD40 motifs.The similarity of M. amblycephala Cdc20 was the highest with those of Danio rerio and Cyprinus carpio.Based on qRT-PCR,the mRNA levels of cdc20 were the highest at the zygote stage,and gradually decreased with the development of embryos.Additionally,the cdc20 gene also exhibited tissue-specific expression pattern,which was abundant in the gonads based on sqRT-PCR.The results of in situ hybridization showed that the cdc20 mRNA was widely expressed in all stages of M. amblycephala oocytes,mainly in the cytoplasm of stage Ⅱ oocytes and the yolk granules of stage Ⅲ and Ⅳ oocytes. In the testis,the cdc20 expression was the highest in spermatogonia and reproductive epithelium,but low in sperm.In conclusion,the results suggested that the M. amblycephala cdc20 may be a maternal gene and play a vital role in the development of germ cells.

    表 1 本研究所用引物Table 1 All primers used in this study
    图1 团头鲂Cdc20蛋白的多序列比对Fig.1 Multiple sequence alignment of Cdc20 proteins of Megalobrama amblycephala
    图2 团头鲂Cdc20蛋白预测的三级结构Fig.2 Predicted structure model of M. amblycephala Cdc20 protein
    图3 脊椎动物CDC20蛋白的系统进化树Fig.3 Phylogenetic analysis of the protein sequences of CDC20 in vertebrates
    图4 团头鲂cdc20基因在成鱼各组织中的表达Fig.4 Expression of cdc20 gene in different tissues of M. amblycephala
    图5 团头鲂cdc20基因在胚胎发育各时期中的表达Fig.5 Expression of cdc20 gene at different embryonic development stages of M. amblycephala
    图6 cdc20基因在团头鲂不同发育时期卵母细胞中的表达Fig.6 Expression of cdc20 gene at differentdevelopment stages of M. amblycephala oocytes
    图7 团头鲂cdc20基因在精巢中的表达Fig.7 Expression of cdc20 gene in testis of M. amblycephala
    参考文献
    [1] BAUDAT F,IMAI Y,DE MASSY B.Meiotic recombination in mammals: localization and regulation[J].Nature reviews genetics,2013,14(11):794-806.
    [2] HADJIHANNAS M V,BERNKOPF D B,BRüCKNER M,et al.Cell cycle control of Wnt/β-catenin signalling by conductin/axin2 through CDC20[J].EMBO reports,2012,13(4):347-354.
    [3] YU H T.Cdc20: a WD40 activator for a cell cycle degradation machine [J].Molecular cell,2007,27(1):3-16.
    [4] JIN F,HAMADA M,MALUREANU L,et al.Cdc20 is critical for meiosis I and fertility of female mice[J/OL].PLoS genetics,2010,6(9):e1001147[2022-04-01].https://doi.org/10.1371/journal.pgen.1001147.
    [5] LI M,SHIN Y H,HOU L F,et al.The adaptor protein of the anaphase promoting complex Cdh1 is essential in maintaining replicative lifespan and in learning and memory[J].Nature cell biology,2008,10(9):1083-1089.
    [6] ZHAO L,XUE S G,YAO Z Y,et al.Biallelic mutations in CDC20 cause female infertility characterized by abnormalities in oocyte maturation and early embryonic development[J].Protein & cell,2020,11(12):921-927.
    [7] LI L W,FAN L Q,PENG N N,et al.R383C mutation of human CDC20 results in idiopathic non-obstructive azoospermia[J].Oncotarget,2017,8(59):99816-99824.
    [8] PETERS J M.The anaphase promoting complex/cyclosome: a machine designed to destroy[J].Nature reviews molecular cell biology,2006,7(9):644-656.
    [9] GOTO M,EDDY E M.Speriolin is a novel spermatogenic cell-specific centrosomal protein associated with the seventh WD motif of Cdc20[J].Journal of biology chemistry,2004,279(40):42128-42138.
    [10] FONG H K,HURLEY J B,HOPKINS R S,et al.Repetitive segmental structure of the transducin beta subunit: homology with the CDC4 gene and identification of related mRNAs[J].PNAS,1986,83(7):2162-2166.
    [11] LAMBRIGHT D G,SONDEK J,BOHM A,et al.The 2.0 ? crystal structure of a heterotrimeric G protein [J].Nature,1996,379(6563):311-319.
    [12] STIRNIMANN C U,PETSALAKI E,RUSSELL R B,et al.WD40 proteins propel cellular networks[J].Trends in biochemical sciences,2010,35(10):565-574.
    [13] GROH K J,SCH?NENBERGER R,EGGEN R I L,et al.Analysis of protein expression in zebrafish during gonad differentiation by targeted proteomics[J].General and comparative endocrinology,2013,193:210-220.
    [14] YAMAMURO T,KANO K,NAITO K.Functions of FZR1 and CDC20,activators of the anaphase-promoting complex,during meiotic maturation of swine oocytes[J].Biology of reproduction,2008,79(6):1202-1209.
    [15] 胥鹏飞,孟安明.斑马鱼母源因子在胚胎发育中的作用[J].中国科学:生命科学,2014,44(10):954-964.XU P F,MENG A M.Maternal factors regulate embryonic development in zebrafish [J].Scientia sinica,2014,44(10):954-964(in Chinese with English abstract).
    [16] HU Y B,HU X W,LI D C,et al.The APC/C FZY-1/Cdc20 complex coordinates with OMA-1 to regulate the oocyte-to-embryo transition in Caenorhabditis elegans[J/OL].Frontiers in cell and developmental biology,2021,9:749654[2022-04-01].https://doi.org/10.3389/fcell.2021.749654.
    [17] VERLHAC M H,TERRET M E,PINTARD L.Control of the oocyte-to-embryo transition by the ubiquitin-proteolytic system in mouse and C. elegans[J].Current opinion in cell biology,2010,22(6):758-763.
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庞连慧,罗双双,石林林,陈天圣,刘红,王焕岭.团头鲂cdc20基因的序列特征和表达分析[J].华中农业大学学报,2022,41(4):226-232

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  • 收稿日期:2022-04-01
  • 在线发布日期: 2022-10-12
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