斑鳜胃蛋白酶原C及其5′侧翼区的克隆及序列特征分析

Molecular cloning and characterization of pepsinogen C gene and its 5′ flanking region from mandarin fish Siniperca scherzeri

采用RT-PCR和RACE等技术克隆了斑鳜(Siniperca scherzeri)胃蛋白酶原C(pepsinogen C,PGC)的cDNA全长和部分DNA序列,结果表明:PGC cDNA序列全长1 505 bp,5′端非翻译区37 bp,3′端非翻译区304 bp,开放阅读框架(ORF)1 164 bp,共编码含有387个氨基酸的蛋白质,包括由16个氨基酸组成的信号肽、39个氨基酸的激活肽和332个氨基酸的成熟肽。斑鳜PGC氨基酸序列与斜带石斑鱼、伯氏豚虾虎鱼、狼鲈、美洲红点鲑4种鱼的相似度分别为92.0%、91.5%、90.2%、89.1%,与非洲爪蟾、鸡、人、兔、褐家鼠的序列相似度分别为76.8%、72.8%、72.5%、69.9%、67.3%,表明PGC基因在长期的进化中较为保守。PGC基因由9个外显子和8个内含子组成,内含子剪切位点符合GT-AG规则,在第7含子中发现(GACA)6、(CA)6类型的微卫星序列,第8内含子中发现(TG)5类型的微卫星序列。采用锚定PCR方法获得了PGC 5′侧翼区长1 924 bp的序列,启动子区域位于-40^+10 bp,包含TATA盒以及GATA-1、CdxA、Hand1/E47、AP-1、Nkx-2、S8、FOX J2等转录因子的结合位点。结果为进一步研究该基因的表达、功能及其转录调控特征奠定了分子基础。

The complete cDNA and genomic DNA sequence of Siniperca scherzeri pepsinogen C （PGC） was isolated from stomach and cloned by means of RT-PCR and rapid amplification of cDNA ends （RACE）. A 1 505 bp cDNA sequence contained a 37 bp 5＇-untranslated region, 304 bp 3＇-untranslated region and 1 164 bp open reading frame（ORF）, which encoded 378 amino acids with a signal peptides of 16 amino acids, a propeptide of 39 amino acids and an activation peptides of 332 amino acids. The amino acid sequence similarity of S. scherzeri PGC was 92.0% , 91.5% , 90.2% , 89.1% with Epinephelus coioides, Trematomus bernacchii, Dicentrarchus labrax, Salvelinus fontinalis, while 76.8% , 72.8% , 72.5%, 69.9% , 67.3% with Xenopus laevis, Gallus gallus, Homo sapiens, Oryctolagus cuniculus, Rattus norvegicus, respectively, which indicated that the PGC gene was relatively conserved in the progress of vertebrate evolution. Genomic PGC DNA consisted of nine exons interrupted by eight introns. The former seven introns splicing sites belonged to GT-AG type, except the 8th intron was GC-AG type, which may act as alternative splicing mode. Moreover, two GACA and CA repeat microsatellite sequences were found in the 7th intron and an TG repeat microsatellite sequence in the 8th intron. A 1 294 bp PGC 5＇ flanking region was isolated by anchored PCR. The inferred promoter sequence was located at -40 - ＋ 10 bp, such as TATA box was detected, but GC box and CCAAT box the basic transcriptional regulatory elements were not detected. The transcriptional factor binding sites such as GATA-1, CdxA, Handl/E47, AP-1, Nkx-2, $8 and FOX J2 existed in the upstream promoter sequence. These results may build the molecular base for further study on gene temporal expression at different developmental stages and various nutritional levels, gene function and its transcriptional regulation.