剑尾鱼卵黄蛋白原C(Vg C)全长cDNA的克隆及表达

The full length cDNA cloning and expression profile of vitellogenin C(Vg C)gene from swordtail fish Xiphophorus helleri

利用逆转录聚合酶链式反应(RT-PCR)和cDNA末端快速扩增(RACE)等方法,克隆获得剑尾鱼卵黄蛋白原C(Vg C)基因的全长cDNA序列。剑尾鱼Vg C基因cDNA序列全长4 011 bp,其5'非编码区包含12 bp和3'非编码区包含246 bp;含有一个3 753 bp的开放阅读框(ORF),编码1 250个氨基酸,推测其编码氨基酸分子量大小为141.7 ku,编码氨基酸序列与其他鱼类卵黄白原C编码氨基酸序列相似性在44%～85%。荧光定量PCR结果显示,Vg C在剑尾鱼肝脏中表达量最高,脾、肾、卵巢中有微量表达,脑、肌肉、鳃中几乎没有检测到表达;对不同时间暴露在雌激素中剑尾鱼肝脏进行实时荧光定量PCR表达分析的结果表明,Vg C在剑尾鱼肝脏中第5天表达量最高,随后降低,第9天后维持相对较低的表达量。研究首次克隆了剑尾鱼Vg C基因全长cDNA序列,并对Vg C在剑尾鱼体内表达组织器官分布及雌激素诱导后不同时间表达谱进行了初步研究,为剑尾鱼生殖生理及环境污染物监测应用等不同领域研究打下重要基础。

Endocrine disruption seems to be related to high vitellogenin（Vg）levels in male fish and has emerged as a useful biomarker of exposure to estrogenic substances.In this study,we show full-length cDNA sequence of swordtail fish vitellogenin C（Vg C）to provide a basis for assessing estrogenic effects of endocrine disrupting compounds in aquatic environment.Full-length Vg C gene cDNA of swordtail fish were obtained using reverse transcription polymerase chain reaction（RT-PCR）and rapid amplification of cDNA ends（RACE）techniques with total RNA extracted from liver of a swordtail fish after treatment with 17β-estradiol（E2）.The result of sequence analysis indicated that the full length of swordtail fish＇s Vg C cDNA is 4 011 bp,containing an open reading frame（ORF）of 3 753 bp which encodes a 1 250 amino acids protein with deduced protein molecular weight of 141.7 ku.The comparation results showed that the nucleotide homology of Vg C was 57%-92% and amino acid homology of Vg C was 44%-85% between swordtail fish and other ten teleosts.Furthermore,expression profile of Vg C mRNA in different tissues and time intervals of swordtail fish was determined by real-time fluorescent quantitative PCR.The result showed the swordtail fish＇s Vg C mRNA was mainly detected in liver and weakly in spleen,kidney,ovary.After exposure to E2,Vg C mRNA expression quantified in liver of swordtail fish had reached a peak on the fifth day,and then the expression level declined gradually.The data obtained from present study provided valuable information for research on reproduction and physiology and application of environmental pollution monitoring of swordtail fish.