尼罗罗非鱼Ikaros基因的克隆及表达分析

MOLECULAR CLONING AND EXPRESSION ANALYSIS OF IKAROS GENE FROM NILE TILAPIA (OREOCHROMIS NILOTICUS)

  • 摘要: 为揭示尼罗罗非鱼Ikaros基因结构特征及其在抗病原感染中的免疫调控机制, 实验采用RT-PCR和RACE方法克隆了尼罗罗非鱼Ikaros的cDNA序列以及利用PCR和染色体步移技术克隆了Ikaros的基因组DNA序列, 通过荧光定量PCR分析了Ikaros mRNA的组织分布及其对无乳链球菌感染的响应。结果表明, 克隆的尼罗罗非鱼Ikaros基因组DNA为20454 bp, 包括7个内含子和8个外显子, 经可变剪接可形成6种不同的mRNA剪接异构体, 其编码的氨基酸序列均具有Ikaros家族典型的锌指结构域且与硬骨鱼类Ikaros氨基酸序列同源性较高(70.6%—93.7%)。Ikaros基因在尼罗罗非鱼各组织中均有表达, 在血液中的表达量最高, 其次为胸腺、脾脏和头肾。人工感染无乳链球菌后, 血液、胸腺、脾脏、头肾中Ikaros基因的相对表达量均显著上调, 并在48h达到峰值, 这表明Ikaros基因参与调控尼罗罗非鱼抵御无乳链球菌的免疫应答反应。研究可为进一步探索Ikaros基因在罗非鱼抗病原感染中的作用机制奠定理论基础。

     

    Abstract: Ikaros is a kind of transcription factor with zinc finger structure that is essential to the development of lymphocyte. In order to reveal the structure characteristic of Ikaros gene and the immune modulatory mechanisms of Ikaros gene on disease resistant, the cDNA of Ikaros were obtained from Oreochromis niloticus with RT-PCR and RACE methods, and the genomic DNA of Ikaros were obtained by using PCR and Genome Walking technique in this study. Quantitative real-time PCR was used for analyzing the organization distribution and the response of Ikaros to Streptococcus agalactiae infection. The genomic DNA is 20545 bp with 7 introns and 8 exons encoding 6 kinds of mRNA splicing isoform via alternative splicing. The deduced amino acid sequences of the six splicing isoforms with zinc finger domain have high homology (70.6%—93.7%) in other teleost fish. The Ikaros gene expressed in all tested tissues, which strongly expressed in blood, and moderately expressed in thymus, spleen and head kidney. After challenged with pathogenic bacteria Streptococcus agalactiae, the Ikaros gene expression levels rose in blood, thymus, spleen and head kidney, and then reached to the peak after 48 hours. The results suggested that Ikaros gene participated in the immune response of Nile tilapia against Streptococcus agalactiae infection. This research laid a foundation for further study on the disease resistant mechanism of Ikaros gene.

     

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