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胃质子泵(H+/K+ATPase)是胃酸分泌的关键酶。本试验采用RACE和PCR方法从大菱鲆的胃组织中提取RNA克隆得到了H+/K+ATPaseα亚基cDNA全长序列。结果表明:大菱鲆H+/K+ATPaseα亚基序列全长3467 bp,开放阅读框为2964 bp,编码988个氨基酸。与GenBank上其它物种比对发现,大菱鲆H+/K+ATPaseα亚基与斑鳜同源性最高,为89%。进化树分析发现,H+/K+ATPase在进化上具有物种特异性。经RT-PCR和荧光定量PCR检测,大菱鲆H+/K+ATPase在胚胎孵化后22d开始表达,晚于大菱鲆胃腺出现的时间(16 d),说明大菱鲆胃腺的发育完成并不代表胃功能的完善。另外,大菱鲆H+/K+ATPase除了在胃中大量表达之外,在食道中的表达量也很高。结合组织学观察,作者认为,大菱鲆H+/K+ATPase在食道中大量表达是因为在发育上食道是胃的前体,因此保留了分泌H+/K+ATPase的能力。同时通过整体原位杂交试验表明:大菱鲆H+/K+ATPase会首先在食道的末端和胃的贲门处表达。本研究为进一步了解海水鱼类的消化机制提供了理论基础。
Stomach proton pump (H + / K + ATPase) is the key enzyme in gastric acid secretion. In this study, the full-length cDNA of H + / K + ATPase alpha subunit was obtained by RNA extraction from gastric tissues of turbot by RACE and PCR. The results showed that the H + / K + ATPase α subunit of Scophthalmus maximus was 3467 bp in length and the open reading frame was 2964 bp, encoding 988 amino acids. Compared with other species in GenBank, the homology of H + / K + ATPase alpha subunit of Scophthalmus maximus was the highest (89%). Phylogenetic tree analysis revealed that H + / K + ATPase is evolutionarily species-specific. The expression of H + / K + ATPase of turbot was expressed 22 days after embryo hatch, which was later than that of turbot (12 days) after RT-PCR and real-time quantitative PCR, indicating that the development of turbot stomach was not completed On behalf of the improvement of gastric function. In addition, turbot H + / K + ATPase in addition to the large number of expression in the stomach, the expression in the esophagus is also high. Combined with histological observations, the authors suggest that the abundant expression of turbot H + / K + ATPase in the esophagus is due to the development of the esophagus as a precursor to the stomach, thus preserving the ability to secrete H + / K + ATPase. At the same time, the whole in situ hybridization test showed that H + / K + ATPase of turbot first expressed at the end of the esophagus and the cardia of the stomach. This study provides a theoretical basis for further understanding of the digestive mechanism of marine fish.