盐生杜氏藻MAPK基因的克隆及表达分析

Cloning and Expression Analysis of DsMAPK from Dunaliella salina

克隆盐藻MAPK基因(命名为DsMAPK),为研究盐藻耐盐分子机制奠定基础。采用RT-PCR和RACE技术克隆DsMAPKcDNA全长,对其进行生物信息学分析,并通过QRT-PCR方法检测盐胁迫下该基因的表达情况。DsMAPK的cDNA全长序列为1861bp(GenBank AccessionJQ782412),包含一个开放阅读框,长度为1407bp,编码468个氨基酸;DsMAPK蛋白为亲水性蛋白,α-螺旋和自由卷曲是其蛋白质二级结构的主要结构元件,伸展链散布其中。据氨基酸序列同源性分析表明,DsMAPK和衣藻、团藻的MAPK蛋白具有较近的亲缘关系。盐藻在高盐胁迫下,DsMAPK基因表达显著上调,1h后达到最大值,为正常对照组的5倍,差异极显著(P<0.01)。DsMAPK基因可能在盐藻对盐胁迫的反应中起重要作用,是一种参与盐藻耐盐反应的早期快速反应基因。

The aims were to clone Mitogen-activated protein kinase gene from Dunaliella salina （named DsMAPK） and provide a basis for research on the molecular mechanism of salt tolerance in D. salina. The complete DsMAPK cDNA sequences were obtained using RT-PCR and RACE methods. Its functional analysis was predicted by approaches of bioinformatics. The expression of DsMAPK gene in D.salina under salt stress was examined by real-time PCR. The total length of DsMAPK was 1861 bp. It contained a 1407 bp open reading frame （ORF） which encoded 468 amino acids. Hydrophobic analysis indicated that DsMAPK was a hydrophilic protein. The predicted secondary structure of the protein had mainly a-helix and random coil, also had extended strand among them. The homology analysis of amino acid sequence showed that DsMAPK had close relationships with MAPK proteins in Chlamydomonas reinhardtii and Volvox carteri f. nagariensis. DsMAPK gene expression was significantly up-regulated by high salt stress. Furthermore, 1 h later, high salt concentration led to the highest level of up-regulated DsMAPK expression in D. salina, The value was 5 times than that of the control group （P〈0.01）. DsMAPK gene probably played an important role in the response of salt stress in D.salina, which could served as a fast response component in the early reaction of salt tolerance.