加州鲈生长激素和胰岛素样生长因子I cDNA的克隆及序列分析

Cloning and Sequence Analysis of cDNA Encoding Growth Hormone and Insulin-Like Growth Factor I in Largemouth Bass(Micropterus salmoides)

GH-IGF-I轴是鱼体体内一个重要的内分泌生理轴,主要调控鱼体的生长发育。用RT-PCR方法从加州鲈脑垂体和肝脏组织中分别扩增出加州鲈GH和IGF-I cDNA,克隆到pMD19 T-Vector上进行序列测定和分析。结果表明:1)加州鲈GH cDNA开放阅读框长为615 bp,编码204个氨基酸,其中信号肽17个氨基酸,成熟肽187个氨基酸。成熟肽中有四个保守的半胱氨酸残基(分别位于69,177,193,202),可形成两对二硫键。加州鲈GH氨基酸序列与蓝太阳鱼、斜带石斑鱼、金头鲷、虹鳟、鲤鱼、斑马鱼相比较,同源性分别为100%、97%、94%、66%、56%、53%。2)加州鲈IGF-I cDNA开放阅读框长为561bp,编码包括信号肽和B、C、A、D、E五个区域的186个氨基酸,形成成熟肽时,信号肽和E区域被切除。成熟蛋白的氨基酸序列与GenBank中已知的河鲈、三角鲂、金头鲷、舌齿鲈、斑马鱼的相比较,结果发现四个区域的保守性有差异,A区和B区保守性较高,C区和D区保守性较差。加州鲈GH和IGF-I cDNA的获得为进一步研究鱼体GH-IGF-I轴对生长发育的调控机制奠定了基础。

cDNA encoding growth hormone（GH） and insulin-like growth factor I（IGF-I） were isolated from total RNA of pituitary and＇liver of largemouth bass（Micropterus salmoides） by RTPCR, respectively. Then they were cloned into pMD19-T-Vector for sequencing and analyzing. Results are as follows. First, GH open reading frame（ORF） contains a length of 615 bp and encodes 204 amino acids. Its preprotein consists of two domains： mature peptide and signal peptide of 17 amino acid residues. Four conservative cysteine residues（sited in 69,177,193,202） in mature peptide are proposed to form two disulphide bonds and play important roles in protein construction and biologic activity of GH. Comparing GH ORF of largemouth bass with those of green sunfish , estuary cod, gihhead seabream, rainbow trout, common carp, zebrafish, the amino acid homology is 100%,97%,94%,66%,56%and 53%, respectively; Second, IGF-I ORF contains a length of 561bp and encodes 186 amino acids, which consists of signal peptide and B, C , A, D, E domain. When mature peptide is processed, signal peptide and E domain are cut off. Comparing the amino acid sequence of IGF-I of largemouth bass with those of freshwater perch, black bream, gilthead seabream, seabass, zebrafish, great differences in homology are found among four domains. The sequence conservation of A and B domains is high, while low in C and D domains. Cloning of largemouth bass GH and IGF-I are helpful to the research of the GH-IGF-I axis regulating growth and development in fish.