翘嘴鲌胰岛素样生长因子-Ⅰ的克隆及序列分析

CLONING AND SEQUENCE ANALYSIS OF INSULIN-LIKE GROWTH FACTORI GENE IN TOPMOUTH CULTER(CULTER ALBURNUS BASILEWSKY)

为探讨胰岛素样生长因子-Ⅰ(Insulin like growth factor-Ⅰ,IGF-Ⅰ)对于翘嘴鲌(Culter alburnus)生长性状的影响,对其DNA序列进行了克隆。翘嘴鲌IGF-Ⅰ全长14567 bp,由5个外显子和4个内含子组成。其5个外显子长度分别为298、160、182、36和1360 bp。推测的阅读框为486 bp,编码由161个氨基酸组成的IGF-Ⅰ前体蛋白。前体肽由信号肽、成熟肽、E肽三部分组成,其中信号肽44个氨基酸,成熟肽70个氨基酸,E肽47个氨基酸。成熟肽由B、C、A、D四个区域组成,其中B结构域和A结构域的保守性最高,在这2个区域包含由6个半胱氨酸残基形成的3个二硫键。翘嘴鲌B区域还包含保守的IGF-Ⅰ受体识别序列(Phe B23-Tyr B24-Phe B25)。E肽的长度表明翘嘴鲌IGF-Ⅰ属Ea-2型。同源性分析表明翘嘴鲌与鲤科鱼类的IGF-Ⅰ编码氨基酸同源性较高,为94%—100%,但在聚类分析中翘嘴鲌并不是首先和鲌亚科的鱼类聚集在一起。Real-time qPCR组织特异性表达结果显示IGF-ⅠmRNA在肝脏组织中的表达量最高,脾、心脏、精巢、脑次之,肾、鳃、胃和卵巢中表达量较低。翘嘴鲌IGF-Ⅰ基因4个内含子长度分别为1170、9364、251和1746 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。翘嘴鲌IGF-Ⅰ基因中包含6个微卫星,(GATG)5AATAT(ATAG)11位于第一内含子中,(CT)8、(TTA)5、(AC)13、(TG)12和(ATT)5位于第二内含子中。其中4个微卫星位点具有多态性,将它们在120尾同塘养殖的翘嘴鲌中进行基因型与生长性状的关联性分析,均未达到显著水平(P>0.05)。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。

In order to study the effect of IGF-Ⅰgene on growth traits of topmouth culter(Culter alburnus Basilewsky),IGF-Ⅰgene was cloned from the genomic DNA of topmouth culter in this study.The gene spanned 14567 bp and comprised five exons and five introns.The five exons of IGF-Ⅰwere 298,160,182,36,and 1360 bp long,respectively.The hypothetical open reading frame of the IGF-ⅠcDNA precursor was 486 bp,encoding a putative protein of 161 amino acids.The precursor peptide included a signal peptide(44 amino acids),a mature peptide(70 amino acids),and an E peptide(47 amino acids).The mature peptide comprised four regions:B,C,A,and D.The A and B domains were conserved with six cysteine residues in these two regions to form three disulfide bonds.The B region contained a conserved IGF-Ⅰreceptor recognition sequence(PheB23-TyrB24-PheB25).Analysis of the E peptide showed that IGF-Ⅰof the topmouth culter was an Ea-2 type.The IGF-Ⅰamino acid sequence of the topmouth culter had high sequence identity with IGF-Ⅰproteins from Cyprinidae fish,ranging from 94 to 100%.Phylogenetic analysis of IGF-Ⅰamino acid sequences showed that IGF-Ⅰof the topmouth culter does not cluster with the fish from subfamily Cultrinae.IGF-Ⅰis expressed in all 10 tested tissues with the highest mRNA level in liver,modest level in spleen,heart,testis,and brain,and low level in kidney,gill,stomach,and ovary.The lengths of the four introns of topmouth culter IGF-Ⅰwere 1170,9364,251,and 1746 bp,respectively.The variation in the sequence of the introns among species was greater than that of the exons with the highest variation in the third intron.Six microsatellite loci were found in the introns of IGF-Ⅰwith(GATG)5AATAT(ATAG)11 in intron 1,and(CT)8,(TTA)5,(AC)13,(TG)12,and(ATT)5 in intron 2.Four of 6 microsatellite loci were polymorphic,and the genotypes of the four microsatellite loci had no significant correlation with the growth traits of 120 topmouth culter cultured in the same pond(P>0.05).These results provide molecular basis for studying the expression pattern,function and transcriptional regulation of the IGF-Ⅰgene.