翘嘴鲌生长激素(GH)基因与侧翼区的克隆及分析

Cloning and sequence analysis of growth hormone gene and its flanking region in topmouth culter(Culter alburnus Basilewsky)

采用保守引物进行PCR成功扩增了编码翘嘴鲌生长激素的基因,该基因全长5 966 bp,其中转录单元长1 648 bp,由5个外显子和4个内含子组成。5'端和3'端侧翼序列长度分别为2 282 bp和2 036 bp,分别包含(AAT)8和(TTC)5T(TAA)8的微卫星序列。上游区域包含TATA框,还有一些重要的转录因子结合位点,如Pit-1、Pit-1a、CREB、AP1、GR、HNF-3、HNF-3B等转录因子。翘嘴鲌的5个外显子长度分别为64、140、117、162和255 bp。推测的阅读框为603 bp,编码由22个氨基酸的信号肽和178个氨基酸成熟肽组成的多肽。在这个多肽中发现了5个保守的半胱氨酸残基(Cys^(71)、Cys^(135)、Cys^(173)、Cys^(190)和Cys^(198))和2个可能的N-糖基化位点(145th和197th)。翘嘴鲌GH氨基酸序列与团头鲂完全相同,与草鱼只有一个氨基酸残基的差异,构建的鱼类进化树符合基本分类地位。翘嘴鲌生长激素基因4个内含子长度分别为229、103、565和103 bp。相对外显子来说,种间内含子变异较大,其中第三内含子变异最大。该结果为进一步研究翘嘴鲌GH基因的表达、功能及其转录调控特征奠定了分子基础。

Using conserved primers and PCR,a gene encoding topmouth culter（Culter alburnus Basilewsky） growth hormone（CaGH） was amplified and sequenced.The gene spans 5 966 bp,including 2 282 bp of 5＇-and 2 036 bp of 3＇-flanking sequences and a 1.7-kb transcription unit comprised of five exons and five introns.（AAT）_8 and （TTC）_5T（TAA）_8 microsatellite sequences were found in the 5＇-and 3＇-flanking regions,respectively.The upstream region contains TATA boxes,and binding sites of important transcription factors such as Pit-1,Pit-1a,CREB,AP1,GR,HNF-3,and HNF-3B.The five exons in Culter alburnus were 64 bp,140 bp,117 bp,162 bp and 255 bp in length,respectively.The complete coding sequence was 603 bp and encodes a protein with a 22 amino acid signal peptide and a 178 amino acid mature peptide.Five conserved Cys residues（Cys^（71）,Cys^（135）,Cys^（173）,Cys^（190）,and Cys^（198）） and two possible sites of N-glycosylation（residues 145 and 197） were detected in the GH polypeptide.The amino acid sequence of GH in Culter alburnus was identical to that in Megalobrama amblycephala,and only one amino acid residue differs from Ctenpharyngodon idellus.The phylogenetic relationships among the GH amino acid sequences in fish were in accord with traditional classification.The lengths of the four introns in Culter alburnus GH gene were 229 bp,103 bp,565 bp and 103 bp,respectively.The variation of the introns among species was greater than that of the exons,and the variation of the third intron is the highest.These results provided the molecular basis for study of function and transcriptional regulation of the GH gene in C.alburnus,as well as the temporal expression in different developmental stages and at various nutritional levels.