球孢白僵菌FUS3/KSS1类MAPK同源基因(BbMPK1)的克隆及特征分析  

CLONING AND CHARACTERIZATION OF A HOMOLOGOUS GENE OF YEAST FUS3/KSS1 MAPK FROM BEAUVERIA BASSIANA

作　　者：张永军[1] 方卫国[1] 金凯[1] 罗志兵[1] 周永洪[1] 肖月华[1] 裴炎[1] 

机构地区：[1]农业部生物技术与作物品质改良重点实验室重庆市农业生物技术重点实验室西南农业大学生物技术中心,重庆400716

出　　处：《菌物学报》2005年第1期104-111,共8页Mycosystema

基　　金：国家重点基础研究计划(973)项目(2003CB114203);国家高技术研究发展计划(863)项目(2002AA214051);重庆市教委项目(20204)

摘　　要：根据几种丝状真菌FUS3/KSS1类MAPK的保守序列设计简并引物,从昆虫病原真菌球孢白僵菌中扩增出MAPK基因的部分片段,进而利用YADE法延伸该片段的上、下游邻接序列,获得MAPK基因的全长序列,命名为BbMPK1。用3′RACE扩增出BbMPK1的全长cDNA序列,该序列含有一个1071bp的ORF,编码356个氨基酸的多肽,推测分子量为41.2kDa,等电点为6.61。BbMPK1含有11个MAPK共有的蛋白激酶区域和1个MAPK激酶作用的磷酸化位点区域(TEY),其氨基酸序列与丝状真菌的TMKA、PMK1、CMK1、FMK1和BMP1等MAPK高度同源。系统聚类结果表明,BbMPK1属于酵母FUS3/KSS1类MAPK。Southern杂交表明,BbMPK1在球孢白僵菌基因组中以单拷贝形式存在。RT-PCR分析表明BbMPK1在分生孢子休眠阶段、萌发阶段和菌丝生长时期均表达。研究结果为阐明酵母FUS3/KSS1类MAPK同源基因在球孢白僵菌中的作用奠定了基础。In this paper, degenerate PCR primers were designed according to the conserved amino acid sequence of several filamentous fungus MAPKs which were homologous to yeast FUS3/KSS1 MAPK, and partial MAPK DNA fragment was amplified from entomopathogenic fungus Beauveria bassiana using PCR. Thereafter, a whole DNA sequence of MAPK, designated as BbMPK1, was obtained from B. bassiana by extending upstream and downstream sequence of the amplified fragment using YADE method. The cDNA of BbMPK1 was cloned by 3′RACE according to the sequence of BbMPK1, which contained a ORF of 1071 bp and encoded a protein of 356 amino acids with Mr = 41.2 kDa and PI = 6.61. The BbMPK1 contained 11 protein kinase domains and 1 domain of phosphorylation for MAPK kinase (TEY) and its amino acid sequence showed 93%, 92%, 92%, 93% and 90% similarity to TMKA, PMK1, CMK1, FMK1 and BMP1, respectively. Phylogenetic clustering suggested that BbMPK1 belonged to homologs of yeast FUS3/KSS1. Southern analysis indicated that BbMPK1 was present as a single copy in B. bassiana. The result of RT-PCR showed that BbMPK1 was all transcripted during conidial dormancy, conidial germination and hyphal growth.

关 键 词：MAPK 扩增 基因 研究结果 蛋白激酶 序列 表达 球孢白僵菌 菌球 昆虫病原真菌 

分 类 号：Q785[生物学—分子生物学]