鱼类ADAR1剪接异构体基因的鉴定及其转录调控

By converting the adenosine (A) to inosine (I), i.e., deamination of the adenosines, ADAR1 can edit the information and structure of exogenous or endogenous RNA and then regulate cellular physiology. Alternative splicing plays an extremely important role in regulating gene expression and expanding the protein diversity. The phenomenon is associated with the evolution, development and occurrence of immune system. The immune related genes would be apt to undergo alternative splicing and to form various splice isoforms. Similar to mamalian ADAR1, grass carp ADAR1 gene has at least three splice variant isoforms, i.e., type I, type II and type III. However, the genomic distribution of these three splice variant isoforms is different from that of mammalian. This fact implies that fish ADAR1 splice variants are of distinctly functional differentiation in the innate immunity. In order to study the molecular mechanisms of transcription regulation in fish ADAR1 splice variants and its latent function , we will carry out the following experiments: 1.Wondering how many splice variant isoforms of ADAR1 there are; Studying the expression pattern and the genomic distribution of grass carp ADAR1 splice variants. 2.Exploring the molecular mechanisms of transcription regulation in grass carp ADAR1 splice variants. 3.Searching for how to activate grass carp ADAR1 and the hidden molecular mechanisms. This project is of benefit to better understanding of fish innate immune factors and then provides theoretical basis for fish healthful aquaculture.

ADAR1能够将RNA的腺嘌呤经脱氨基作用转变成次黄嘌呤，从而改变其所携带的信息和结构，调控细胞生理功能。可变剪接是控制基因表达和产生蛋白质多样性的重要机制，它与宿主免疫系统及相关因子的进化、发育与发生密切相关。因此，免疫相关基因更易产生多变剪接并形成不同的剪接异构体。与哺乳类ADAR1基因相似，草鱼ADAR1起码存在3个剪接异构体基因，分别为Ⅰ型，Ⅱ型和Ⅲ型。这些剪接异构体基因在基因组排布和结构上都与哺乳类的有很大的不同，暗示鱼类ADAR1基因在先天免疫中存在明显的功能分化。为研究鱼类ADAR1剪接异构体基因转录的分子机制及其潜在的功能，本研究拟开展：1、鉴定草鱼ADAR1剪接异构体基因的种类，研究其表达图式及基因排布特征。2、草鱼ADAR1剪接异构体基因转录的分子机制。3、草鱼ADAR1激活的分子机理。本项目的实施，有助于我们进一步了解鱼类先天免疫因子，为鱼类的健康养殖提供理论依据。

ADAR1作为腺苷脱氨酶家族成员之一，能够通过催化双链RNA的A-to-I 编辑作用发挥抗病毒功能。本研究克隆与鉴定了草鱼ADAR1及其两种剪接异构体ADAR1-like、ADAR1a基因及其基因组序列。结果表明CiADAR1基因全长cDNA为4844bp，开放阅读框（ORF）为4179bp，蛋白结构分析显示，其多肽序列中含有2个Z-DNA结合域、3个dsRNA结合域和1个保守的脱氨酶催化区域；CiADAR1-like基因全长cDNA为3878bp，ORF为3324bp，其多肽序列中含有2个Z-DNA结合域、3个dsRNA结合域和1个较短的脱氨酶催化区域；CiADAR1a基因全长cDNA为3180bp，ORF为2592bp，其多肽序列中含有1个Z-DNA 结合域，3个dsRNA结合域和一个高度保守的水解脱氨酶激酶活性区。CiADAR1全基因组由16个外显子和15个内含子组成、CiADAR1-like由9个外显子和8个内含子组成、CiADAR1a则包含15个外显子和14个内含子。此外，荧光定量PCR与Western Blot分析了草鱼ADAR1、ADAR1-like、ADAR1a基因在poly I:C刺激下的表达图式，结果表明在草鱼各组织中ADAR1及ADAR1-like都能被诱导表达上调；在草鱼CIK cells中，CiADAR1表达明显上调，而CiADAR1a表达并不受poly I:C影响。进一步分析了CiADAR1及其剪接异构体转录调控的分子机制，非放射性凝胶阻滞实验结果表明草鱼IRF1 和 IRF3 对CiADAR1、CiADAR1-like、CiADAR1a启动子片段的迁移都有阻滞效果，草鱼IRF1和IRF3 对草鱼ADAR1的启动子近端和远端区域片段的迁移都具有阻滞效果。双荧光素酶实验结果表明，草鱼IRF3、IRF7都能够诱导上调CiADAR1启动子活性，CiADAR1a启动子并不受IRF及poly I:C诱导调控，且CiADAR1 启动子片段的近端区域对CiADAR1的转录调控起主要作用。另外，研究分析了草鱼ADAR1的定位，研究表明在正常情况下，CiADAR1a主要存在于细胞核而不是细胞质中，poly I:C刺激下，发现CiADAR1a逐渐从细胞核转运到细胞质中。此外，荧光显微镜观察结果表明CiADAR1定位于细胞核中。