团头鲂cdc20基因的序列特征和表达分析  

Sequence characteristics and expression analysis of cdc20 gene in Megalobrama amblycephala

作　　者：庞连慧 罗双双 石林林 陈天圣 刘红[1] 王焕岭[1] PANG Lianhui;LUO Shuangshuang;SHI Linlin;CHEN Tiansheng;LIU Hong;WANG Huanling(Lab of Freshwater Animal Breeding,Ministry of Agriculture and Rural Affairs/College of Fisheries,Huazhong Agriculture University,Wuhan 430070,China;Key Laboratory of Healthy Mariculture for the East China Sea,Ministry of Agriculture and Rural Affairs/College of Fisheries,J imei University,Xiamen 361021,China)

机构地区：[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

出　　处：《华中农业大学学报》2022年第4期226-232,共7页Journal of Huazhong Agricultural University

基　　金：国家自然科学基金项目(31872542)。

摘　　要：为研究鱼类细胞分裂周期蛋白20基因(cell division cycle 20 homolog,c dc20)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1482 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。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 1482 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-bladedβpropeller 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 stageⅡoocytes and the yolk granules of stageⅢandⅣoocytes.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.

关 键 词：团头鲂 cdc20基因 减数分裂 母源基因 表达模式 性腺成熟 

分 类 号：S917.4[农业科学—水产科学]