摘要
IKKε(inhibitor of NF-κB kinasesε)是NF-κB(nucleur factorκB)信号通路中的关键成员,参与调控细胞的分化、发育、凋亡及免疫反应。本研究根据马氏珠母贝转录组数据库中注释为IKKε的unigene序列设计引物,应用cDNA末端快速扩增(RACE)技术克隆获得了马氏珠母贝(Pinctada martensii)IKKε基因cDNA的全长序列(PmIKKε)并对其序列特征进行分析;同时利用荧光定量PCR(Real-time PCR)技术检测了马氏珠母贝六个组织中PmIKKε基因mRNA的表达。结果表明:PmIKKε基因cDNA全长2 843 bp,其中5'UTR为116 bp,3'UTR为516 bp,含有27 bp polyA,开放阅读框为2 211 bp,编码737个氨基酸残基。预测其分子量为85.07 kD,等电点为6.17。多序列比对显示PmIKKε与其它物种IKKε有较高的同源性,与牡蛎的序列相似性有45%。软件分析结果显示PmIKKε的N端含有一个蛋白激酶功能结构域和一个MAPKK(mitogen-activated protein kinase kinase)的激活位点,C端含有一个亮氨酸拉链(leucin zipper,LZ)和一个螺旋-环-螺旋结构(helix-loop-helix,HLH)。荧光定量PCR分析表明PmIKKε在马氏珠母贝肝胰脏、性腺、血淋巴、鳃、外套膜、闭壳肌六个组织中均有表达,肝胰脏中表达量最高。本研究为进一步阐述PmIKKε在马氏珠母贝生长、发育及先天性免疫中的作用提供理论基础。
IKKε（inhibitor of NF-κB kinases ε） is an important component in the NF-κB（nucleur factor κB） signal pathway and plays crucial roles in the differentiation, development, apoptosis and immune response. In this study, based on the unigene sequence annotated as IKKε in the transcriptome database of Pinctada martensii, using rapid amplification of cDNA ends technology, the full length of IKKε gene was obtained from Pinctada martensii（PmIKKε）. The obtained full length of PmIKKε cDNA was 2 843 bp, containing a 5＇ untranslated region（UTR） of 116 bp and a 3＇ UTR of 516 bp with 27 bp polyA tail. The open reading frame（ORF） was 2 211 bp encoding 737 amino acid residues. The predicted molecular weight was 85.07 kD, isoelectric point was 6.17. Multiple sequence alignment indicated that IKKε was highly conservative among species and PmIKKε had 45% sequence identity with that from Crassostrea gigas. The N-terminal region contained a kinase domain and a mitogen-activated protein kinase kinase（MAPK）, The C-terminal region contained a leucine zipper（LZ） domain and a helix-loop-helix（HLH） motif. Real-time PCR analysis demonstrated that PmIKKε was constitutively expressed in all studied tissues（aductor muscle, gonad, hepatopancreas, mantle, hemocytes, gill） in P. martensii with the most abundant mRNA in the hep atopancreas. These studies provide the basis for the further study of the function of PmIKKε in growth, development and innate immunity in mollusk.
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2014年第2期266-272,共7页
Genomics and Applied Biology
基金
国家自然科学基金(31272635
41206141
31372526)
广东省自然科学基金(S2012040008042)
广东省教育厅育苗工程(2012LYM_0074)
广东海洋大学博士启动项目(1212318)共同资助
