鲫鱼Vitellogenin基因的克隆与组织表达分析

Cloning and Tissue Expression Analysis of Vitellogenin Genes in Carassius auratus

为研究鲫鱼不同卵黄蛋白原(Vtg)亚型基因生物学功能及其分子机制,克隆鉴定了鲫鱼中Vtg同源基因的2种亚型,分别命名为Ca-VtgAo1和Ca-VtgC。Ca-VtgAo1和Ca-VtgC cDNA序列全长分别为4241 bp和4207 bp,开放阅读框(ORF)分别为3873 bp和3612 bp,5′非编码区(UTR)分别为192 bp和150 bp,3′UTR分别为176 bp和445 bp,分别编码1290个和1203个氨基酸残基,预测蛋白质分子质量分别为140.6 ku和134.8 ku。结构域分析结果显示,Ca-VtgAo1和Ca-VtgC都具有LPD_N(Lipoprotein N-terminal domain)、DUF1943和DUF1944保守结构域,而都不具有β′组分卵黄蛋白(β′-component yolk protein,β′-C)和C-末端组分卵黄蛋白(Carboxy-terminal component yolk protein,CT)结构域,且Ca-VtgAo1富含磷酸化多聚丝氨酸(Phosvitin,Pv)结构域,Ca-VtgC没有该结构域。进化分析结果表明,VtgC先与尖端单鳍七鳃鳗(Ichthyomyzon unicuspis)VtgABCD聚为一支,再与VtgAa、VtgAb、VtgAe、VtgAo聚为一支,Ca-VtgAo1由于存在Pv结构域,其进化速率比Ca-VtgC快。实时荧光定量PCR分析结果表明,Ca-VtgAo1 mRNA在检测组织中均有表达,其中在肝脏组织中的相对表达量最高,且雌性肝脏中Ca-VtgAo1 mRNA相对表达量是雄性肝脏中的50倍,推测鲫鱼Vtg来源主要是肝脏外源性合成,但依然保留卵巢内源性合成Vtg的古老方式,Ca-VtgAo1基因可作为分子标记应用于鲫鱼性别鉴定。

In order to study the biological functions and molecular mechanisms of different Vitellogenin(Vtg)subtype genes in Carassius auratus,two subtypes of Vtg homologous genes were cloned and identified,named Ca-VtgAo1 and Ca-VtgC,respectively.The full-length cDNA sequences of Ca-VtgAo1 and Ca-VtgC were 4241 bp and 4207 bp,the open reading frames(ORF)were 3873 bp and 3612 bp,and the 5′untranslated regions(UTR)were 192 bp and 150 bp,3′UTR were 176 bp and 445 bp,encoding 1290 and 1203 amino acid residues,and the predicted protein molecular weight were about 140.6 ku and 134.8 ku,respectively.The domain analysis results showed that both Ca-VtgAo1 and Ca-VtgC had the conserved domains of LPD_N(lipoprotein N-terminal domain),DUF1943 and DUF1944,but neither hadβ′-component yolk protein(β′-C)and the carboxy-terminal component yolk protein(CT)domain,and Ca-VtgAo1 had the phosvitin(Pv)domain,Ca-VtgC did not have this domain.The results of evolutionary analysis showed that VtgC first clustered with VtgABCD in Ichthyomyzon unicuspis,and then clustered with VtgAa,VtgAb,VtgAe and VtgAo.Ca-VtgAo1,due to the Pv domain,evolved faster than Ca-VtgC.Quantitative real-time PCR analysis results showed that Ca-VtgAo1 mRNA was expressed in the tested tissues,of which the relative expression level in liver tissue reached the highest,and the relative expression level of Ca-VtgAo1 mRNA in female liver was fifty times of that in male liver.The relative expression level suggests that the source of Vtg is mainly exogenous synthesis in the liver,but the old way of endogenous synthesis of Vtg in the ovary is still retained.The Ca-VtgAo1 gene can be used as a molecular marker for sex identification in C.auratus.