异齿裂腹鱼CCK基因的cDNA克隆及其摄食功能

旨在研究异齿裂腹鱼(Schizothorax o’connori)缩胆囊素(cholecystokinin, CCK)基因的摄食功能。本研究克隆得到了异齿裂腹鱼CCK基因的cDNA全长,通过生物信息学分析发现其属于CCK-1亚型。异齿裂腹鱼CCK的cDNA全长为773bp,其中开放阅读框(ORF)为372bp,可以编码123个氨基酸。异齿裂腹鱼CCK由1个信号肽和1个典型的CCK-8肽保守结构域组成,为亲水性蛋白,但没有跨膜结构。运用实时荧光定量PCR(Real-timePCR)检测异齿裂腹鱼CCK基因在组织中的分布情况,以及餐前餐后和禁食复喂对其表达量的影响。结果表明,异齿裂腹鱼CCK在各组织中均有表达,其中在脑中表达量最高,在肠道、心脏、肝、脾、肾、皮肤、鳃、眼和鳔中表达量相对较高,在肌肉中表达量最低。餐后CCK基因的表达量显著升高,禁食使异齿裂腹鱼CCK基因的表达量显著下降,而复喂使CCK基因的表达量显著上升,表明CCK基因既是异齿裂腹鱼的餐后饱感信号因子,又是长期调控摄食因子。本研究为异齿裂腹鱼的人工饲养和品种保护等提供了理论依据。

Molecular cloning and appetite regulation of CCK in Schizothorax o'connori

The CCK (cholecystokinin) gene is a peptide substance which mainly distributed in the brain and intestine in a variety of macromolecules. Cholecystokinin plays an important role in the functions of regulating food intake and gastrointestinal motility, promoting pancreatic secretion and gallbladder contraction, etc. However, there are differences in the regulation modes among different species. In order to study the appetite regulation of CCK in Schizothorax o''connori. The cDNA full-length of the cholecystokinin gene from Schizothorax o''connori was cloned using RACE and RT-PCR methods, and it belonged to the CCK-1 subtype by bioinformatics analysis. The CCK mRNA levels in major tissues, before and after feeding, fasting and refeeding were detected and analyzed using quantitative real-time PCR. The results showed that the full-length cDNA of CCK gene was 773 bp, the open reading frame (ORF) was 372 bp and can encode 123 amino acids. The CCK was composed of a signal peptide and a conserved domain of the typical CCK-8 peptide, which was a hydrophilic protein but had no transmembrane structure. Sequence homologous analysis of amino acids showed that the CCK of Schizothorax o''connori had high homology with those of the fish belonging to Cypriniformes-teleosts, and low homology with mammals and amphibians. Tissue distribution analysis showed that the expression of CCK was highest in brain and was high in intestines, heart, liver, spleen, kidney, skin, gill, eyes, maw, while it was lowest in muscle. The CCK mRNA expression was highly elevated after feeding, and decreased after fasting and re-increased after refeeding. The results indicated that the CCK gene was both the postprandial satiety signal and the long-term appetite regulation factor in Schizothorax o''connori.