球孢白僵菌Hog1 MAPK同源基因BbHog1的克隆及特征分析

根据几种丝状真菌Hog1 MAPK的保守氨基酸序列设计简并引物,从昆虫病原真菌球孢白僵菌中扩增出MAPK同源基因的部分片段,然后利用YADE法延伸该片段的上、下游邻接序列,获得MAPK编码基因的全长序列,命名为BbHog1。序列分析表明,该基因编码358个氨基酸的多肽,推测分子量为40.99kDa,等电点为5.49。BbHog1含有MAPK保守的蛋白激酶激活域(TGY),序列与粗糙脉孢霉os-2(AF297032)、烟曲霉OSM1(XM_747571)、隐球酵母HOG1(AF243531)和酿酒酵母Hog1(Z73285)等Hog1 MAPK高度同源,相似性分别为94%、89%、83%和80%。系统聚类结果表明,BbHog1与酵母Hog1 MAPK同源。Southern杂交表明,BbHog1在球孢白僵菌基因组中以单拷贝形式存在。Northern分析表明,BbHog1在高渗、亚高温和营养胁迫等条件下的表达明显升高。由此推测,BbHog1基因可能与球孢白僵菌对逆境胁迫的适应性调节密切相关。

Cloning and characterization of a Hog1 MAPK homologous gene BbHog1 from Beauveria bassiana

Degenerate PCR primers were designed according to the conserved amino acid sequence of several filamentous fungus MAPKs which were homologous to yeast Hog1 MAPK, and partial DNA fragment encoding a MAPK was amplified from entomopathogenic fungus Beauveria bassiana using PCR. Thereafter, a whole DNA sequence encoding the MAPK, designated as BbHog1, was obtained from B. bassiana by extending upstream and downstream sequence of the amplified fragment using YADE method. Sequence analysis suggested that BbHog1 encoded a protein of 358 amino acids with Mr of 40.99KDa and PI of5.49.The BbHog1 contained the conserved TGY activation loop found in the stress-activated protein kinase subgroup of MAPKs and its amino acid sequence showed 94%,89%,83% and 80% identity to os-2(AF297032) from Neurospora crassa,OSM1 (XM_747571)from Aspergillus fumigatus,HOG1 (AF243531) from Cryptococcus neoformans var.neoformans and Hog1 (Z73285) from Saccharomyces cerevisiae,respectively.Phylogenetic clustering suggested that BbHog1 is homologous to yeast Hog1 MAPK.Southern analysis indicated that BbHog1 was present as a single copy in B.bassiana.Northern blot analysis showed that BbHog1 gene expression was obviously increased when the fungus was cultured under the condition of hyperosmotic stress,sub-high temperature and nutrition starvation,which suggested that BbHog1 gene was involved in the stress adaptation reguloation of Beauveria bassiana.