太平洋鳕IRF3基因的克隆及绝对定量表达分析

Cloning and expression analysis of IRF3 in Gadus macrocephalus using absolute quantitative PCR

本研究通过克隆首次得到太平洋鳕(Gadus macrocephalus)干扰素调节因子3(interferon regulatory factor 3,IRF3)的部分序列。该序列长1878 bp,包含长1377 bp的完整开放阅读框(open reading frame,ORF),编码459个氨基酸,其编码的蛋白质分子量约为51 k D。经氨基酸序列比对发现,太平洋鳕IRF3的氨基酸序列包括1个含有5个保守色氨酸残基的DNA结合域(DNA binding domain,DBD)和1个保守的C端IRF关联域(IRF association domain,IAD);系统进化树分析表明,太平洋鳕IRF3与其他物种的IRF3亲缘关系较近。应用绝对荧光定量PCR方法对IRF3在不同组织和不同日龄(days post-hatching,dph)仔鱼的表达水平进行检测,并对干扰素在仔鱼期的免疫作用进行分析。组织表达绝对定量结果显示,性腺,肝和胸腺表达量高于其他组织。不同日龄仔鱼IRF3表达量结果显示,IRF3在受精卵就已经表达,在25 dph表达量大幅提高。本研究的结果为进一步研究太平洋鳕干扰素作用机制以及其在早期发育中的作用奠定了基础。

Gadus macrocephalus is a marine teleost that is economically important throughout the world. The species has a high nutritional value and is processed into a variety of products. As a result of the increase in fisheries targeting this species, the abundance of wild populations has declined. Our laboratory has successfully carried out artificial breeding and rearing of G. macrocephalus. However, we observed significant mortality as a result of immune system dysfunction. To improve artificial breeding techniques for G. macrocephalus, the molecular mechanisms and expression characteristics of immune-related genes needs to be understood. Interferon regulatory factor 3 （IRF3） is a member of the interferon regulatory factor family. It is an important transcription factor for the expression of the interferon α/βgene and plays an important role in the host antiviral response mechanism. We obtained a cDNA sequence of G. mac-rocephalus IRF3 gene for the first time using gene cloning. The sequence was 1878 bp in length with a whole open reading frame （ORF） of 1377 bp that encoded a 459 amino acid protein. The molecular weight of the encoded protein was^51 kDa. Amino acid sequence alignment revealed that the protein included a DNA binding domain （DBD） con-taining five conserved tryptophan residues and a conserved C-terminal IRF association domain （IAD）. The phylogenetic tree revealed that the IRF3 of G. macrocephalus was clustered with the IRF3 of other species, distant from IRF1 and IRF2. The tissue and age-specific expression of IRF3 was detected using absolute quantitative PCR. Additionally, the effect of interferon during the larval stages was analyzed. The expression levels were highest in the gonad, liver, and thymus. The copy number of IRF3 in the different tissues was： 231.244 copies/ng, 516.649 copies/ng, 695.158 cop-ies/ng, 2128.273 copies/ng, 198.548 copies/ng, 101.758 copies/ng, 419.927 copies/ng, 13.016 copies/ng, 1102.775 cop-ies/ng, and 13.016 copies/ng in the intestine, heart, thymus, gonad, gill, muscle, spleen, brain, liver, and kidney, respec-tively. The expression of IRF3 was detected in fertilized eggs and remained relatively constant to 5 d post-hatching （dph） but increased by 25 dph. The copy number of IRF3 at different ages was：6.189 copies/ng, 6.809 copies/ng, 8.066 cop-ies/ng, 5.009 copies/ng, 5.009 copies/ng, 10.390 copies/ng, and 10.390 copies/ng in the fertilized egg, 1 dph, 5 dph, 9 dph, 17 dph, 25 dph, or 33 dph, respectively. Our results suggest that the gonad, liver, and thymus are the primary or-gans for IRF3 expression. Additionally, our results suggest that interferon plays an antiviral role at 25 dph. The results of this study lay the foundation for further study of the mechanism of interferon action during the early development of G. macrocephalus and provide basic data for studies of the immune system function in G. Macrocephalus.