摘要
目的克隆铁皮石斛(Dendrobium officinale)促分裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)基因DoMPK2并进行分子特征分析。方法采用反转录聚合酶链式反应(RT-PCR)、cDNA末端快速扩增(RACE)技术分离全长基因;生物信息学分析编码蛋白的理化特性、保守结构域等特征;用DNASTAR、MEGA软件分别进行氨基酸多序列比对和进化树构建分析;借助实时定量PCR技术检测基因组织表达模式。结果 DoMPK2基因cDNA全长1 585 bp(GenBank注册号JX297595),编码一条由369个氨基酸组成的肽链,相对分子质量42120,等电点6.56。DoMPK2蛋白包含MAPK家族保守的丝氨酸/苏氨酸蛋白激酶催化结构域(32^-319)和MAP激酶的保守位点(67~170)。DoMPK2与多种植物MAPK基因高度相似(56%~84%),与水稻、玉米等单子叶植物MAPK基因亲缘关系较近。DoMPK2为组成型表达,其转录本在石斛种子和根中的相对表达量较高,分别为叶中的9.696、2.327倍,茎和叶中的表达变化不显著。结论 DoMPK2基因的分子特征为进一步研究其在铁皮石斛生长发育、抗逆胁迫等生命活动过程中的生物学功能奠定基础。
OBJECTIVE To clone and characterize a mitogen-activated protein kinase （MAPK） gene DoMPK2 in Dendrobium officinale. METHODS RT-PCR and RACE technologies were used for gene clone. Characteristics including the physicochemical properties and conserved domain of the deduced DoMPK2 protein were determined using a series of bioinformatics tools. The analyses of muhiple alignment and phylogenetic tree were performed using DNASTAR and MEGA softwares, respectively, qPCR was employed to examine the tissue specific expression pattern of DoMPK2. RESULTS The full length cDNA of DoMPK2 was l 585 bp （ GenBank ac- cession No. JX297595） and encoded a 369-aa protein with a molecular weight of 42. 12 kD and an isoelectric point （pl） of 6. 54. The deduced DoMPK2 protein contained the conserved serine/threonine- protein kinase catalytic domain （32 -319 ） and MAP kinase signa- ture （67 - 170）. Multiple sequence alignment and phylogenetic analysis demonstrated that DoMPK2 was highly similar （56% -84% ） to MAPK genes from various plants and was closely related to rice and maize monocots. Real time quantitative PCR （qPCR） analysis revealed that DoMPK2 was constitutively expressed in the four included tissues. The transcripts were the most abundant in the seeds and roots, which were 9. 696 and 2. 327 fold over the leaves, and there had no significant expression in the stem and leaf samples. CONCLUSION Molecular characterization of DoMPK2 will be useful for further elucidation of the functions of this gene involving in the plant growth and development and stress response of D. officinale.
出处
《中国药学杂志》
CAS
CSCD
北大核心
2013年第19期1654-1659,共6页
Chinese Pharmaceutical Journal
基金
国家自然科学基金资助项目(31070300
31101608)
陕西省青年科技新星计划项目(2012KJXX-44)
陕西省教育厅专项科研计划项目(2013JK0829)
关键词
铁皮石斛
激酶
克隆
基因表达
实时定量PCR
Dendrobium officinale
kinase
cloning
gene expression
real time quantitative PCR
