布氏鲳鲹CCK基因的克隆及表达分析

Cloning and Expression Analysis of the Cholecystokinin Gene from Snubnose Pompano(Trachinotus blochii)

缩胆囊素(cholecystokinin, CCK)已被证实是生物体中一类能抑制食欲的重要的细胞因子,为进一步补充鱼类CCK基因相关的生物信息,本研究利用RACE技术和PCR方法对布氏鲳鲹(Trachinotus blochii)CCK基因进行了克隆和组织表达模式研究。结果表明:布氏鲳鲹CCK基因cDNA序列全长822 bp,5'端、3'端非翻译区(untranslated regions, UTR)长度分别为70 bp和340 bp,开放阅读框(open reading frame, ORF)412 bp,共编码137个氨基酸,其中包括一个信号肽和一个CCK-8肽。预测的蛋白分子量为14.79 kD,理论等电点是5.89。氨基酸序列同源性比对及系统进化树表明,布氏鲳鲹CCK基因与其与同属鲈形目的鰤鱼的同源性最高(91.9%),并且与其聚为一支。实时荧光定量PCR (real time-PCR, RT-PCR)分析显示CCK在布氏鲳鲹的端脑高表达,垂体、肌肉和下丘脑相对表达丰富,肾脏、脾脏、肠和脂肪中低表达,证实了CCK在布氏鲳鲹的神经和消化系统中可能具有重要的调节功能。本研究结果可为今后进一步探讨布氏鲳鲹CCK基因的摄食调控功能提供理论参考。

Cholecystokinin(CCK) has been well-established as a class of important cytokines that inhibits appetite of biological body. In order to further supplement the related biological information of the CCK gene in fish, we used rapid amplification of complementary DNA(cDNA) ends(RACE method) and PCR to clone and carry out expression analysis from snubnose pompano(Trachinotus blochii). The results showed that the full length of CCK gene cDNA in Trachinotus blochii was 822 bp, including a 5’ untranslated regions(UTR) of 70 bp and a 3’ UTR of 340 bp. The open reading frame(ORF) was 412 bp, which encoded a putative protein of 137 amino acids, including a signal peptide and a CCK-8 peptide. The predicted molecular weight was 14.79 kD and the theoretical isoelectric point was 5.89. The homology comparison of amino acid sequences and phylogenetic tree indicated that CCK gene from Trachinotus blochii had the highest homology(reaching 91.9%) with the same genus of Perciformes yellowtails(Seriola quinqueradiata), and they were clustered into one branch. Real-time quantitative PCR(RT-PCR) analysis revealed that CCK was expressed highly in the telencephalon, and relatively high in the pituitary, muscle, and hypothalamus, whereas it was expressed at low levels in the kidney, spleen, intestine and adipose. The results demonstrated that CCK might play an important regulatory role in the nerves and digestive systems of Snubnose pompano. The study might provide a theoretical reference for further research on the appetite regulation of CCK gene in snubnose pompano(Trachinotus blochii).