鳜胰岛素样生长因子-Ⅱ cDNA基因的克隆与表达特征

Cloning and expression of full-length cDNA of insulin-like growth factor-Ⅱ in mandarin fish Siniperca chuatsi

为全面获得鳜Siniperca chuatsi GH-IGFs生长发育轴的调控因子特征,采用RT-PCR、cDNA末端快速扩增(RACE)技术成功克隆了肝组织胰岛素样生长因子-Ⅱ(IGF-Ⅱ)cDNA序列。结果表明:鳜IGF-ⅡcDNA全长为1 643 bp,包括5'端非翻译区103 bp、3'端非翻译区892 bp和开放阅读框648 bp,开放阅读框编码215个氨基酸;鳜IGF-Ⅱ前肽由信号肽、成熟肽和E肽3部分组成,其中信号肽为47个氨基酸,成熟肽为70个氨基酸,E肽为98个氨基酸;鳜IGF-Ⅱ氨基酸序列与其他脊椎动物的相似度为81%～98%,与鳜IGF-Ⅰ的相似度为62.5%。运用实时荧光定量PCR技术检测了鳜成鱼不同组织以及孵化后0～22 d仔稚鱼中IGF-ⅡmRNA的表达情况,结果表明:IGF-ⅡmRNA在鳜脑中表达量最高,在肌肉、心脏、胃、鳃、肾脏中表达量次之,在脾、前肠、后肠、性腺、肝脏中表达量较低;在孵化后早期发育阶段均检测到有IGF-ⅡmRNA表达,孵化当天(0 d)表达量最高,之后表达量有所降低。本研究结果可为鳜IGF-Ⅱ对生长发育的调控作用研究提供科学依据。

The complete cDNA sequence of insulin-like growth factor- Ⅱ （ IGF- Ⅱ ） was cloned from liver of man- darin fish Siniperca chuatsi by RT-PCR and rapid amplification of eDNA ends （RACE） to evaluate the regulating characteristics of HG- IGF- Ⅱ axis. A total of 1 643 bp IGF- Ⅱ eDNA sequence was found to be comprised of a 103 bp 5＇-untranslated region, 892 bp 3＇-untranslated region and 648 bp open reading frame （ORF） encoding 215 amino acids with a signal peptides of 47 amino acids, a mature pepfides of 70 amino acids and a E peptides of 98 amino acids. The mandarin fish IGF-]I amino acid sequence showed similarity of 81%-98% with other verte- brate IGF-Ⅱ s, but only similarity of 62.5% with the mandarin fish IGF- [ . The IGF-]] mRNA expression was detected in different adult tissues and larval development stages （0, 2, 4, 6, 8, 10, 12, 14, 16, 19,and 22 days posthatching） using real-time quantitative PCR technique. The maximal expression was observed in the brain, fol- lowed by the in muscle, heart, stomach, gill, and kidney, and the lower expression in the spleen, foregut, hind- gut, gonad, and liver. The IGF- Ⅱ mRNA was expressed during larval various development stages, the maximal expression in the newly hatched, and then reduced slightly. These findings may provide a molecular base for further knowledge of erowth regulation.