鳜胃泌素与胆囊收缩素基因cDNA全长的克隆与表达特征

为探明鱼类消化液分泌的相关调控因子,采用RACE技术分别克隆了鳜(Siniperca chuatsi)两种肠道激素——胃泌素(gastrin,GAS)与胆囊收缩素(cholecystokinin,CCK)基因的cDNA序列。鳜GAS基因cDNA全长581 bp,5′非翻译区长100 bp,3′非翻译区长145 bp,开放阅读框长336 bp,编码111个氨基酸;鳜CCK具有2种类型:CCK1与CCK2,CCK1 cDNA全长为843 bp,5′非翻译区长60 bp,3′非翻译区长369 bp,开放阅读框414 bp,编码137个氨基酸;CCK2 cDNA全长846 bp,5′非翻译区长112 bp,3′非翻译区长332 bp,开放阅读框403 bp,编码134个氨基酸。鳜GAS与CCK成熟肽C末端具有相似的八肽结构(DYQGWVDF/DYLGWMDF),仅在C末端第3位和第6位氨基酸发生替换。荧光定量PCR分析表明,鳜GAS mRNA主要表达于肠和幽门垂,CCK1与CCK2 mRNA在脑中表达量最高,在肠和幽门垂也有较高表达,表明GAS与CCK同为消化调控因子,而CCK还是神经分泌因子。鳜GAS与CCK mRNA表达贯穿于整个幼体早期发育阶段(孵化后0～22 d),前期表达水平较高,后期表达水平较低,并趋于平稳,GAS与CCK mRNA发育表达水平变化可能与这一时期消化道生长发育旺盛有关。

Full-length cDNAs cloning and expression characteristics of gastrin and cholecystokinin in the mandarin fish (Siniperca chuatsi)

We evaluated the factors regulating the secretion of digestive juices in fish.We cloned two intestinal hormone genes,gastrin(GAS) and cholecystokinin(CCK),from mandarin fish(Siniperca chuatsi).The GAS cDNA consists of 581 bp nucleotides,including a 100 bp 5′ untranslated region(UTR),a 336 bp open reading frame(ORF) encoding 111 amino-acid residues,and a 145 bp 3′ UTR.The CCK gene has two isoforms,CCK1 and CCK2,consisting of 843 bp and 846 bp nucleotides,respectively.The CCK1 gene consists of a 60 bp 5′ untrans-lated region(UTR),a 414 bp ORF encoding a polypeptide of 137 amino-acid residues and a 369 bp 3′ UTR,while the CCK2 gene consists of a 112 bp 5′ UTR,a 403 bp ORF encoding a polypeptide of 134 amino-acid residues and a 332 bp 3′ UTR.The C-terminal of the GAS polypeptide has a similar octapeptide structure(DYQGWVDF/ DYLGWMDF) to that of CCKs’.The only differences occur in the third and the sixth amino acid in the C-terminal.Val3(GAS) and Glu6(GAS) are replaced by Met3(CCK) and Leu6(CCK),respectively.Real-time PCR analysis revealed that GAS mRNA was primarily expressed in the intestine and pyloric caecum.The expression of CCK1 and CCK2 mRNA was highest in the brain,but expression was also detected in intestine and pyloric caecum.Our results suggest that GAS and CCKs may be involved in regulating digestion,while CCKs appear to be associated with digestive and neurosecretory processes.The GAS and CCK genes are constitutively expressed during larval development(0?22 days post hatching,DPH).Expression was higher during the early stage(4–8 DPH for GAS and 2–4 DPH for CCK),possibly linked to development and growth of the digestive tract,then gradually de-creased to a stable level during the later stages of development.