摘要
为了阐明MAPKs级联反应在盐藻耐盐机制中发挥的作用,需要克隆MAPKs信号转导途径中的成员之一MAPKKK基因并研究其功能。采用RT-PCR与RACE技术从盐藻中首次克隆到一个盐藻MAPKK激酶基因,将其命名为DsMAPKKK(GenBank Accession No.KF366904)并进行了生物信息学分析。结果表明,DsMAPKKK基因的cDNA全长为1460 bp,开放阅读框为879 bp,编码292个氨基酸;该蛋白无信号肽,无跨膜域,是定位于细胞质基质的亲水性不稳定蛋白质;蛋白质序列中包含一个24—45位的蛋白激酶ATP结合区和一个137—149位的丝氨酸/苏氨酸蛋白激酶活性区;发现了多个潜在的磷酸化位点和一个重要的催化活性位点Asp141;蛋白质二级结构的主要组成元件为α-螺旋和自由卷曲;成功构建了蛋白质三级结构立体模型;系统进化分析表明该蛋白质与团藻、衣藻的相应蛋白进化关系最近。
To illustrate the function of MAPKs cascade reaction in salt-tolerance mechanism of Dunaliella salina, it was necessary to clone a MAPKKK gene which was a member of MAPKs signal transduction pathway and study its function. A MAPKKK gene was firstly cloned from Dunaliella salina by RT-PCR and RACE technology and named as DsMAPKKK（GenBank Accession No.KF366904） and further bioinformatics-analysis was conducted. The result showed that, full length of cDNA for DsMAPKKK gene was 1460 bp with 879 bp open reading frame coding 292 amino acids. This protein was unstable, hydrophilic and located in cytoplasmic matrix without signal peptide and transmembrane region. The protein sequence contained a protein kinases ATP-binding region spanning from 24rd to 45rd amino acid and a Serine/Threonine protein kinase regions panning from 137rd to 149 rd amino acids. Several potential phosphorylation sites and an important catalytic active site Asp141 were found in this protein. The main components of protein secondary structure were α-helix and random coil. A 3D model of protein tertiary structure was built successfully. Phylogenic analysis showed this protein had the closest evolutionary relationship with the corresponding proteins of Volvox carteri f. Nagariensis and Chlamydomonas reinhardtii.
出处
《中国农学通报》
CSCD
2014年第15期274-281,共8页
Chinese Agricultural Science Bulletin
基金
国家自然科学基金项目"盐藻中磷酸化转录因子对盐胁迫的分子响应"(30972240)
辽宁省教育厅科技研究项目"胰蛋白酶抑制剂基因在盐藻中的高效表达"(2008T023)
