黄鳝nanos3基因克隆鉴定及其组织表达分析

Cloning,identification and tissue expression analysis of nanos3 gene in Monopterus albus

  • 摘要: 【目的】明确nanos3基因在黄鳝卵巢发育和维持过程中的重要作用,为黄鳝PGCs可视化分子标记、生殖细胞移植及性腺发育分化机制研究打下基础。【方法】通过RACE克隆黄鳝nanos3基因cDNA序列,运用ProtParam、 SOMPA、 SWISS-MODEL、 SignalP、 TMHMM及NetPhos等在线软件进行nanos3蛋白生物信息学分析;采用实时荧光定量PCR检测nanos3基因在黄鳝不同组织中的表达情况,并以冰冻切片荧光原位杂交进行性腺组织定位。【结果】黄鳝nanos3基因cDNA序列全长1289 bp,包括147 bp的5'非编码区 (5'-UTR)、 531 bp的开放阅读框 (ORF)及611 bp的3'非编码区(3'-UTR),共编码176个氨基酸残基。黄鳝nanos3蛋白分子量为19.61 kD,理论等电点(pI)为9.07,为碱性蛋白;在黄鳝nanos3氨基酸序列第108~162位处存在2个锌指基序 “CCHC”结构域,无信号肽和跨膜结构域;蛋白二级结构以无规则卷曲的占比最高 (64.20%),其次是α-螺旋 (占24.43%),延伸链、 β-转角的占比分别为6.82%和4.55%。黄鳝与大刺鳅、射水鱼、斑马鱼等鱼类的nanos3氨基酸序列相似性分别为67.5%、 57.2%和50.3%,与中国林蛙、粗皮蛙等两栖类动物的相似性分别为39.3%和30.3%,与人类、小鼠等哺乳类动物的相似性分别为33.3%和30.9%。nanos3基因在黄鳝肌肉中不表达,在精巢、肾脏、脾脏、脑、肝脏、肠道、心脏和血液中的相对表达量较低,在卵巢中的相对表达量最高,极显著高于在其他组织中的相对表达量(P<0.01),呈明显的组织特异性表达模式;冰冻切片荧光原位杂交分析结果显示,黄鳝nanos3基因仅在生殖细胞中表达,支持细胞中未见表达。【结论】黄鳝nanos3基因进化相对保守,主要在卵巢的生殖细胞中表达,且以细胞质中的表达为主,与核遗传信息的传递相关,是黄鳝性腺发育及分化相关的功能基因。

     

    Abstract: 【Objective】To clarify the important role of nanos3 gene in development and maintenance of ovary in Monopterus albus,so as to lay the foundation for studies on visualized molecular marker of PGCs,germ cell transplantation and gonadal differentiation mechanism.【Method】 cDNA sequence of nanos3 gene was cloned by RACE.ProtParam,SOMPA,SWISS-MODEL,SignalP,TMHMM,NetPhos and other online software were used to conduct bioinformatics analysis of nanos3 protein.Expression of nanos3 gene in different tissues of M.albus was determined through quantitative real-time PCR(qRT-PCR)and frozen section fluorescence in situ hybridization method was used to detect cellular location of nanos3 in gonad tissue.【Result】Full length of cDNA sequence of nanos3 gene was 1289 bp,including 5'-UTR of 147 bp,open reading frame(ORF)of 531 bp and 3'-UTR of 611 bp,and 176 amino acid residues were encoded.Molecular weight of nanos3 protein in M.albus was 19.61 kD,with a oretical isoelectric point(pI)of 9.07,indicating it was alkaline protein.In the 108-162th position of amino acid sequence existed 2 zinc finger motif“CCHC”domains,without any signal peptide and transmembrane region.In the secondary structure of the protein,random coil(64.20%)accounted for the highest proportion,followed by α-helix(24.43%),and extension chain and β-turn accounted for 6.82% and 4.55%,respectively.nanos3 amino acid sequence similarity between M.albus and Mastacembelus armatusToxotes jaculatrix and Danio rerio was 67.5%,57.2% and 50.3%;similarity between M.albus and amphibian species of Rana chensinensis and Rana rugosa was 39.3% and 30.3%;similarity between M.albus and mammal species of humans and mice was 33.3% and 30.9%,respectively.nanos3 gene was not expressed in the muscle of M.albus,and its expression was low in testis,kidney,spleen,brain,liver,intestine,heart and blood and its expression in ovary was the highest,significantly higher than that in the tissues(P<0.01),showing tissue-specific expression patterns.Fluorescence in situ hybridization(FISH)analysis results of frozen sections showed that nanos3 gene in M.albus was only expressed in germ cells,not in somatic cells.【Conclusion】nanos3 gene evolutes conservatively,and it was mainly expressed in the germ cells of ovary,and mainly expressed in the cytoplasm,and this is related to the transmission of nuclear genetic information.nanos3 geneis the functional gene related to gonadal development and differentiation in M.albus.

     

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