猪苓菌丝形成菌核的MAPK基因克隆及表达分析

Molecular cloning and characterization of a MAPK gene from Polyporus umbellatus

【目的】克隆药用真菌猪苓MAPK基因并进行生物信息学分析及表达模式研究。【方法】利用5′-RACE-PCR技术从猪苓菌丝中克隆得到MAPK基因全长,利用生物信息学软件推测蛋白的理化性质、结构域;DNA Star对氨基酸进行多序列比对;用MEGA 5.0做进化关系分析;借助实时定量PCR检测基因表达模式。【结果】猪苓MAPK基因的全长c DNA为1 293 bp,其中编码区占1 161 bp,共编码386个氨基酸,推测分子量为43.872 k D,理论等电点为6.68。猪苓的MAPK有MAPK中ERK1/2类型的保守区。系统进化树结果显示猪苓MAPK蛋白属于担子菌类群。实时荧光定量PCR分析结果表明猪苓菌核形成初期,菌核中的MAPK表达量显著高于菌丝组织,随着菌核的快速生长而减少。【结论】猪苓MAPK基因Pu MAPK的分子特征为进一步研究其在猪苓菌丝形成菌核过程中的作用奠定基础。

[Objective] To clone the mitogen-activated protein kinase （MAPK） gene from Polyporus umbellatus and carry out the bioinformatics and expression mode analysis. [Methods] RACE technology was carried out to clone the full length eDNA of MAPK gene. The characteristics of physiochemical properties and conserved domains of the predicted MAPK protein were determined using bioinformatics tools. The analyses of multiple alignment and phylogenetie tree were performed using BioEditor and MEGA 5.0 software. Real time quantitative PCR was used for gene expression analysis. [Results] The full length eDNA of MAPK was 1 293 bp in length and encoded a 386-aa protein with a molecular weight of 43.872 kD and an isoelectrie point （pI） of 6.68. The PuMAPK clustered with Basidiomycete group according to the phylogenetic analysis. Real time quantitative PCR （qPCR） analysis revealed that transcripts were the most abundant in the beginning of selerotial formation （20-30 days） with 7.86 fold over that in the mycelium, but the transcripts decreased sharply with the sclerotial development. [Conclusion] Molecular characterization of PuMAPK will be usefulfor the further functional determination of the gene involving in the development of P. umbellatus sclerotium.