彩鲫hira基因片段的克隆及表达分析

SEQUENCE CLONING AND EXPRESSION ANALYSIS OF HIRA GENE IN COLOR CRUCIAN CARP CARASSIUS AURATUS

hir/hira基因最先作为组蛋白基因表达的一种负调节因子从酵母中被鉴定出来。现已证实,HIRA包含一组保守的蛋白家族,广泛存在于低等真核生物、无脊椎动物和脊椎动物等多种生物体当中,为生命发育所必需。关于hira基因在动物发育过程中的具体作用研究还不是很多,为了进一步探讨hira基因在鱼类发育过程中的作用,作者根据已知hira基因保守序列设计一对简并引物,分别以彩鲫基因组DNA和卵巢cDNA为模板克隆了彩鲫hira基因(Cahira)片段,该片段基因组序列为2181bp,基因组片段含有6个内含子,长度分别为118bp、275bp、372bp、84bp、472bp、86bp;cDNA序列为759bp,编码253个氨基酸,具有5个WD结构域。对其氨基酸序列进行比较,结果表明,彩鲫hira基因的氨基酸序列与河、爪蟾、鸡、小鼠、人的hira基因氨基酸序列具有非常高的同源性,分别高达92%、89%、89%、87%和88%。组织特异性表达分析表明,所检测的彩鲫组织除了精巢和肌肉中未检测到hira基因表达外,其余组织均有表达。其中卵巢和肝脏中表达很强,而在脑、心、脾、肾中表达较弱,说明该基因可能在维持卵巢和肝脏组织的功能方面起一定作用。

Hir/hira （histone regulation） genes were first identified in yeast Saccharomyces cerevisiae as negative regulators of histone gene expression. They represent a conserved protein family found in various organisms, including Drosophila, pufferfish, Xenopus, chicken, mouse and human. The HIRA complex in D. melanogater fulfilled both protamine removal and replication-independent deposition of H3.3-H4 tetramers in the male nucleus after sperm entry, thus making the sperm decondensation and forming male pronucleus to participate in the formation of the diploid zygote. In order to further analyse the function of hira gene in fish development and in gynogenesis, we cloned a segment of hira gene from colored crucian carp Carassius auratus （named Cahira） which shows high sequence similarity with vertebrate hira genes. The incomplete genomic sequence of Cahira gene is 2181bp including 6 introns, and the introns length are 118bp, 275bp, 372bp, 84bp, 472bp and 86bp, respectively. Its opening reading-frame （ORF） is 759bp, and encodes a 253 amino acid peptide. The peptide derived from the gene sequence contains WD-repeat domains in their amino-terminus, which is consistent with the characteristic of HIRA proteins. RT-PCR analysis showed that higher level of Cahira mRNA could be detected in ovary and liver, while lower expression level were present in brain, heart, spleen and kidney. No transcripts were detected in testis and muscle. This indicates that hira gene may play a definitive role in ovary and liver in colored crucian carp. The cloning of HIRA homologue from this carp and its expression analysis may provide basic documents for further study on the role of hira gene in fishes.