铁皮石斛DoMAPK5基因的克隆及表达特征分析

Cloning and expression analysis of DoMAPK5 in Dendrobiumofficinale

目的克隆及分析铁皮石斛DoMAPK5基因的表达特征。方法利用cDNA末端快速克隆技术(RACE),首次从铁皮石斛接菌共生萌发种子中分离得到1个新的MAPK基因,对其编码蛋白的理化性质、保守结构域等特征及进行了分析。并应用荧光定量PCR对DoMAPK5基因在石斛不同组织中表达模式分析。结果 DoMAPK5基因(Gen Bank注册号KJ472788),全长为2184bp、编码一条597个氨基酸的肽链,预测分子量为67.39KDa,等电点为9.28,编码的蛋白均不含信号肽,在210-232位具有23个氨基酸的跨膜域,具有MAPK蛋白家族保守的丝氨酸/苏氨酸蛋白激酶的结构域及MAPK位点。序列比对及系统发育树分析结果表明DoMAPK5基因与葡萄MAPK基因均具有很高的相似性(82%),与水稻MAPK10亲缘关系最近。应用荧光定量PCR对DoMAPK5基因在石斛不同组织中表达模式分析显示,该基因在未接菌的植物组织中属组成型表达,在接菌共生萌发的植物组织中显著上调,为未萌发种子的10.49倍。结论 DoMAPK5在植物组织中属于组成型表达,具有受菌根真菌诱导表达的特征。

Objective Clone and analyze the expression characteristics of Mitogen- activated protein kinase（ MAPK） gene in Dedrobium officinale. Methods The full length cDNA of DoMAPK5（ Gen Bank NO KJ472788） gene was cloned from symbiotic germinated seeds of an endangered medicinal orchid species Dendrobium officinale,using the rapid amplification of cDNA ends（ RACE）- PCR technique. Real- time q PCR was performed to detect the gene expression. Results The full length cDNA was2184 bp,encoding a 597 amino acid（ aa） protein with a molecular mass of 67. 39 k Da and a theoretical isoelectric point of 9. 28.The deduced DoMAPK5 protein,without signal peptide,had a typical serine / threonine- protein kinase domain and a conserved MAP kinase site. Multiple sequence alignments and phylogentic analysis showed that DoMAPK5 has a highly similarity（ 82%）to Vitis vinifera MAPK and Musa acuminate MAPK,and highly homologous to Oryza sativa MAPK. Real time quantitative PCR（ q PCR） analysis revealed that DoMAPK5 was consitutively expressed in leaves,stems,roots and seeds. Furthermore,DoMAPK5 transcript was markedly induced in symbiotic geminated seeds, with 10. 49 fold compared to un- germinated seeds.Conclusion The expression of DoMAPK5 fluctuated in response to mycorrhizal fungal infection and indicating their possible involvements in symbiotic seed germination of D. officinale.