Identification and expression characterization of pepsinogen A in orange‐spotted grouper, Epinephelus coioides
Identification and expression characterization of pepsinogen A in orange‐spotted grouper, Epinephelus coioides
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斜带石斑鱼胃蛋白酶原A的鉴定及表达特征
DOI：
10.1111/j.1095-8649.2008.01999.x
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发表时间：
2008
影响因子：
2
通讯作者：
Hao
中科院分区：
文献类型：
--
作者：
Shaozhen Feng;Weizhong Li;Hao
为了解斜带石斑鱼胃消化生理的分子机制，构建了斜带石斑鱼（Epinealphanus coioides）的cDNA文库，获得了331个表达序列标签（expressed sequence tags，EST）。在226条EST中，有37.17%的EST可能与消化酶有关。首次在大肠杆菌中鉴定出两种等电点不同的A型胃蛋白酶原。coioides。其cDNA全长分别为1312和1314 bp，推导的多肽序列分别由376和377个氨基酸组成。这两个基因都由8个不同长度的内含子组成。根据胃蛋白酶氨基酸序列的比对和系统发育树分析表明，胃蛋白酶A1和A2可分为两个不同的亚型。北方印迹杂交显示，胃蛋白酶原A1有两种形式的转录本，而胃蛋白酶原A2有三种形式的转录本。逆转录-PCR（RT-PCR）和Southern印迹分析表明，两种A型胃蛋白酶原mRNA存在于成年胃组织中，胃蛋白酶原A2也可在中肠中检测到。此外，胃蛋白酶原A1的转录本在孵化后29天首次检测到，而胃蛋白酶原A2在孵化至成鱼期间持续表达。这些结果表明，这两种A型胃蛋白酶原的转录调控和生理功能可能是不同的。
To understand the molecular mechanisms associated with digestive physiology of the stomach of orange-spotted grouper Epinephelus coioides, a cDNA library was constructed and 331 expressed sequence tags (EST) were generated and annotated. About 37·17% of 226 identified ESTs were possibly related to digestive enzymes. Two type-A pepsinogens differing in putative isoelectric point were identified and characterized for the first time in E. coioides. The complete cDNAs were 1312 and 1314 bp in length, and the deduced polypeptide sequences comprised 376 and 377 amino acids, respectively. Both genes consisted of eight introns of various lengths. Alignment and a phylogenetic tree based on the amino acid sequences of pepsins indicated that pepsin A1 and A2 could be classified into two different subtypes. Northern blot hybridization showed that there were two forms of transcripts for pepsinogen A1 and three for pepsinogen A2. Reverse transcription-PCR (RT-PCR) followed by Southern blot analysis revealed that two type-A pepsinogen mRNAs were present in the adult stomach tissue, and pepsinogen A2 was also detectable in the mid-intestine. Furthermore, pepsinogen A1 transcript was first detected at 29 days post-hatching, while pepsinogen A2 was continuously expressed through hatching to adult fish. These results imply that transcriptional regulation and physiological function of the two type-A pepsinogens may be different.