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刘士力, 程顺, 蒋文枰, 迟美丽, 郑建波, 贾永义, 赵金良, 顾志敏. 翘嘴鲌胰岛素样生长因子-的克隆及序列分析[J]. 水生生物学报, 2019, 43(2): 272-281. DOI: 10.7541/2019.034
引用本文: 刘士力, 程顺, 蒋文枰, 迟美丽, 郑建波, 贾永义, 赵金良, 顾志敏. 翘嘴鲌胰岛素样生长因子-的克隆及序列分析[J]. 水生生物学报, 2019, 43(2): 272-281. DOI: 10.7541/2019.034
LIU Shi-Li, CHENG Shun, JIANG Wen-Ping, CHI Mei-Li, ZHENG Jian-Bo, JIA Yong-Yi, ZHAO Jin-Liang, GU Zhi-Min. CLONING AND SEQUENCE ANALYSIS OF INSULIN-LIKE GROWTH FACTOR-I GENE IN TOPMOUTH CULTER (CULTER ALBURNUS BASILEWSKY)[J]. ACTA HYDROBIOLOGICA SINICA, 2019, 43(2): 272-281. DOI: 10.7541/2019.034
Citation: LIU Shi-Li, CHENG Shun, JIANG Wen-Ping, CHI Mei-Li, ZHENG Jian-Bo, JIA Yong-Yi, ZHAO Jin-Liang, GU Zhi-Min. CLONING AND SEQUENCE ANALYSIS OF INSULIN-LIKE GROWTH FACTOR-I GENE IN TOPMOUTH CULTER (CULTER ALBURNUS BASILEWSKY)[J]. ACTA HYDROBIOLOGICA SINICA, 2019, 43(2): 272-281. DOI: 10.7541/2019.034

翘嘴鲌胰岛素样生长因子-的克隆及序列分析

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

  • 摘要: 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(PheB23-TyrB24-PheB25)。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-Ⅰ mRNA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。

     

    Abstract: In order to study the effect of IGF-Ⅰ gene on growth traits of topmouth culter (Culter alburnus Basilewsky), IGF-Ⅰ gene 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-Ⅰ were 298, 160, 182, 36, and 1360 bp long, respectively. The hypothetical open reading frame of the IGF-Ⅰ cDNA 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-Ⅰ receptor recognition sequence (PheB23-TyrB24-PheB25). Analysis of the E peptide showed that IGF-Ⅰ of the topmouth culter was an Ea-2 type. The IGF-Ⅰ amino acid sequence of the topmouth culter had high sequence identity with IGF-Ⅰ proteins from Cyprinidae fish, ranging from 94 to 100%. Phylogenetic analysis of IGF-Ⅰ amino acid sequences showed that IGF-Ⅰ of the topmouth culter does not cluster with the fish from subfamily Cultrinae. IGF-Ⅰ is 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-Ⅰ were 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-Ⅰ with (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 theIGF-Ⅰ gene.

     

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