Jiang Mei, Tu Dan-Dan, Gu Wen-Bin, Zhou Yi-Lian, Zhu Qi-Hui, Guo Xiao-Ling, Shu Miao-An
College of Animal Sciences, Zhejiang University, Hangzhou 310058, China.
College of Animal Sciences, Zhejiang University, Hangzhou 310058, China.
Dev Comp Immunol. 2018 Jul;84:199-212. doi: 10.1016/j.dci.2018.02.014. Epub 2018 Feb 15.
IKK (inhibitor of NF-κB kinase) is the critical regulator for NF-κB (nuclear factor-κB) pathway against pathogenic invasion in vertebrates or invertebrates. However, the IKK from crab species has not yet been identified. In the present study, three full-length cDNA sequences of IKKs from mud crab Scylla paramamosain, designated as SpIKKβ, SpIKKε1 and SpIKKε2, were firstly cloned through RT-PCR and RACE methods. This is also the first report about the identification of two IKKε genes in mud crab and even in crustaceans. The SpIKKβ cDNA was 2824 bp in length with an open reading frame (ORF) of 2382 bp, which encoded a putative protein of 793 amino acids (aa). The ORF of two SpIKKε isoforms, SpIKKε1 and SpIKKε2, were 2400 bp and 2331 bp in length encoding 799 aa and 776 aa, respectively. The crucial conserved residues and functional domains, including the kinase domains (KDs) and leucine zipper (LZ), were identified in all SpIKKs. Phylogenetic analysis suggested that SpIKKβ was classified into the IKKs class while SpIKKεs could be grouped into the IKK-related kinases class. The qRT-PCR analysis showed that three SpIKKs were constitutively expressed in all tested tissues and the highest expression levels of SpIKKβ and SpIKKεs were all in hemocyte. The gene expression profiles of SpIKKs were distinct when crabs suffered biotic and abiotic stresses including the exposures of Vibrio alginolyticus, poly (I:C), cadmium and air exposure, suggesting that the SpIKKs might play different roles in response to pathogens infections, heavy metal and air exposure. Moreover, IKKs from mud crab can significantly activate mammalian NF-κB pathway, suggesting the function of IKKs might be evolutionally well-conserved. Results of the RNAi experiments suggested that SpIKKs might regulate the immune signaling pathway when hemocytes were challenged with V. parahemolyticus or virus-analog poly (I:C). All of these results indicated that the obtained SpIKKs might be involved in stress responses against biotic or abiotic stresses, and it also highlighted their functional conservation in the innate immune system from crustaceans to mammals.
IKK（核因子κB激酶抑制剂）是脊椎动物或无脊椎动物中核因子κB（NF-κB）通路抵御病原体入侵的关键调节因子。然而，蟹类中的IKK尚未被鉴定出来。在本研究中，首先通过RT-PCR和RACE方法克隆了来自拟穴青蟹的3个IKK全长cDNA序列，分别命名为SpIKKβ、SpIKKε1和SpIKKε2。这也是关于在青蟹乃至甲壳类动物中鉴定出两个IKKε基因的首次报道。SpIKKβ cDNA长度为2824 bp，开放阅读框（ORF）为2382 bp，编码一个由793个氨基酸（aa）组成的推定蛋白。两个SpIKKε亚型SpIKKε1和SpIKKε2的ORF长度分别为2400 bp和2331 bp，分别编码799个aa和776个aa。在所有SpIKK中均鉴定出关键的保守残基和功能域，包括激酶结构域（KDs）和亮氨酸拉链（LZ）。系统发育分析表明，SpIKKβ被归类为IKK类，而SpIKKεs可归为IKK相关激酶类。qRT-PCR分析表明，3个SpIKK在所有测试组织中均组成性表达，SpIKKβ和SpIKKεs的最高表达水平均在血细胞中。当蟹受到生物和非生物胁迫，包括溶藻弧菌、聚肌胞苷酸（poly (I:C)）、镉暴露和空气暴露时，SpIKK的基因表达谱各不相同，这表明SpIKK在应对病原体感染、重金属和空气暴露时可能发挥不同作用。此外，青蟹的IKK可显著激活哺乳动物的NF-κB通路，表明IKK的功能可能在进化上得到了很好的保守。RNA干扰实验结果表明，当血细胞受到副溶血性弧菌或病毒类似物聚肌胞苷酸（poly (I:C)）攻击时，SpIKK可能调节免疫信号通路。所有这些结果表明，所获得的SpIKK可能参与了对生物或非生物胁迫的应激反应，也突出了它们在从甲壳类动物到哺乳动物的先天免疫系统中的功能保守性。
