家蚕精氨酸激酶基因的克隆、基因结构与表达分析

cDNA Cloning, Genomic Structure and Expression of Arginine Kinase Gene from Bombyx mori (L.)

【目的】克隆家蚕精氨酸激酶基因,分析其基因结构与表达特性,为揭示无脊椎动物体内能量代谢调节规律提供重要基础。【方法】通过分析家蚕EST、利用RACE法和基因组文库筛选法克隆了家蚕精氨酸激酶(BmAK,Bombyx mori arginine kinase)基因,并对其基因结构和表达特性进行了分析。【结果】克隆了BmAK基因,cDNA全长为1 268bp,编码355个氨基酸,具有精氨酸激酶典型的酶活性部位氨基酸序列,酶活性中心位点氨基酸和能形成离子偶结构氨基酸;该基因由2个外显子和1个内含子组成;5′调控序列存在BRCZ、E74A、FTZ等多个潜在转录因子结合位点,但没有TATA盒启动子序列;该基因的表达在不同组织和不同发育时期存在明显差异。【结论】BmAK具有精氨酸激酶的典型特征,BmAK基因表达随发育时期不同而发生变化,基因的表达可能受蜕皮激素调控。

[Objective] In order to clarify regulation mechanisms of energy metabolism of invertebrates, the arginine kinase gene in the silkworm,Bombyx mori was cloned and analyzed. [ Method ] Based on the information of silkworm ESTs, the full-length cDNA of arginine ldnase gene was cloned using the strategy of rapid amplification of cDNA ends, and then the genomic structure of BreAK was analyzed. Further, the expression of BreAK was examined. [Results] BreAK cDNA has 1268 bp and contains an open reading frame encoding a 355 amino acid protein. BreAK contains the active sequence, the active site and a pair of highly conserved amino acids that form an ion pair. The BreAK gene contains only 2 exons and 1 intron. The BreAK promoter contains no TATA box, but potential binding sites for the transcription factors in both forward and reverse orientation：BRCZ, E74A, FTZ, etc. The expression level of BreAK in different stages and tissues was obviously different. [Conclusion] BreAK has typical character of arginine kinase. The analysis on the transcription factor binding sites and the expression pattern of BreAK showed that the developmental expression of BreAK gene might be under control of ecdysone.