三疣梭子蟹F型ATP酶β亚基(F-ATPaseβ)基因的克隆、组织表达及在家系近交中的变化

cDNA Cloning and Expression Analysis of F-ATPaseβ Subunit Gene in Portunus trituberculatus and Its Variation in Family Inbreeding

中文关键词: 三疣梭子蟹  F-ATPaseβ  基因克隆  表达分析  近交

英文关键词: Portunus trituberculatus  ptF-ATPaseβ  Gene cloning  Expression analysis  Inbreeding

中文摘要:

      采用RACE技术(cDNA末端快速扩增技术)克隆获得三疣梭子蟹(Portunus trituberculatus) F型ATP酶b亚基(F-ATPaseβ)基因，命名为ptF-ATPaseβ。该基因cDNA全长为1965 bp，5¢和3¢非编码区分别为571 bp和341 bp，开放阅读框为1053 bp，推测编码350个氨基酸，预测分子量为 37.9 kDa，理论等电点为4.86。ptF-ATPaseβ氨基酸序列含有F1-ATPaseβ标志性蛋白结构域、AAA结构域和ATP-synt-ab-C结构域。同源性及系统分析显示，ptF-ATPaseβ氨基酸序列与斑节对虾(Penaeus monodon)、凡纳滨对虾(Litopenaeus vannamei)同源性高达89%。实时荧光定量PCR显示，ptF-ATPaseβ基因在肝胰腺、肌肉、心脏、鳃、胃、肠、精巢和卵巢中均有表达，其中，在肝胰腺和心脏中表达量最高，在肠中最少。随着近交系数的增加，各代ptF-ATPaseβ基因的表达量在肝胰腺和心脏中均下降且显著低于F0代(P<0.05)。酶活检测结果显示，心脏中的ATP合酶活性从F6代开始出现下降且显著低于F0代(P<0.05)，但肝胰腺中ATP合酶活性无显著变化。本研究结果表明，近交造成了三疣梭子蟹ptF-ATPaseβ基因表达及ATP合酶活力的衰退。

英文摘要:

      A full-length cDNA sequence of F-ATPase β subunit gene from Portunus trituberculatus (ptF-ATPaseβ) was cloned by rapid amplification of cDNA ends (RACE). The sequence of ptF-ATPaseβ was 1965 bp, containing a 571 bp 5' UTR, 341 bp 3' UTR, and a 1053 bp open reading frame (ORF) that encodes 350 amino acids polypeptides. The isoelectric point (pI) was 4.86 and the molecular mass was 37.9 kDa. The amino acid sequence analysis demonstrated that ptF-ATPaseβ has an F1-ATPaseβ domain, an AAA domain, and an ATP-synt-ab-C domain. Homology and phylogenetic analysis revealed that the amino acid sequence of ptF-ATPaseβ shared a high similarity (89%) with Penaeus monodon and Litopenaeus vannamei. ptF-ATPaseβ mRNA level was detected in all tested tissues including the hepatopancreas, muscle, heart, gill, stomach, intestine, testis, and ovary. The ptF-ATPaseβ had the highest level in the hepatopancreas and heart, and the lowest expression in the intestine. With the increase of inbreeding coefficient, ptF-ATPaseβ expression decreased significantly in the hepatopancreas and heart (P<0.05). The ATP synthase activity in the heart began to fall from F6 generation and was significantly lower than F0 generation (P<0.05), but there was no significant change in the hepatopancreas. The results illustrate that inbreeding gradually reduces the expression of ptF-ATPaseβ and the ATP synthase activity in P. trituberculatus.