铁皮石斛促分裂原活化蛋白激酶基因DoMPK1的克隆及特征分析  

Molecular characterization of a mitogen-activated protein kinase gene DoMPK1 in Dendrobium officinale

作　　者：张岗[1,2] 赵明明[1,3] 宋超[1] 张大为[1] 李标[1] 郭顺星[1] 

机构地区：[1]中国医学科学院、北京协和医学院药用植物研究所,北京100193 [2]陕西中医学院药学院/陕西省中药基础与新药研究重点实验室,陕西西安712046 [3]北华大学医学部,吉林吉林132013

出　　处：《药学学报》2012年第12期1703-1709,共7页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金资助项目(31070300;31101608;31170314);陕西省科技计划青年科技新星项目(2012KJXX-44);陕西省教育厅专项科研计划项目(2010JK496);陕西省自然科学基础研究计划项目(2011JQ4016);教育部科学技术研究重点项目(211177)

摘　　要：促分裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)及其级联途径在根瘤、从枝菌根宿主植物共生体系中起重要调控作用。然而,MAPK在兰科菌根共生体系中的作用机制尚不明确。本研究利用RT-PCR、RACE方法,首次从小菇真菌(Mycena sp.)侵染的铁皮石斛根中克隆到一个促分裂原活化蛋白激酶基因,命名为DoMPK1(GenBank注册号JX297594)。DoMPK1基因cDNA全长1 632 bp,编码一条由372个氨基酸组成的肽链,分子量42.61 kD,等电点6.07。DoMPK1蛋白包含MAPK蛋白家族保守的丝氨酸/苏氨酸蛋白激酶催化结构域(39 325)和MAP激酶的保守位点(77-177)。DoMPK1与多种植物MAPK基因高度同源(71%～85%),与单子叶植物MAPK基因亲缘关系较近。DoMPK1为组成型表达,其转录本在石斛根、茎、叶和种子等4种组织中的相对表达量差异不显著。在小菇真菌侵染30天的根中,DoMPK1显著上调,为对照根中的7.91倍,表明DoMPK1可能在小菇真菌铁皮石斛菌根共生早期互作过程中起作用。本研究为进一步解析DoMPK1在小菇真菌铁皮石斛菌根共生中的分子作用奠定基础。The mitogen-activated protein kinase （MAPK） cascade, composed of MAPK kinase kinase （MAP3K）, MAPK kinase （MAP2K）, and MAPK, is abundantly conserved in all eukaryotes. MAPK along with MAPK cascade plays a vital regulatory role in the plant-arbuscular mycorrhiza/rhizobium nodule symbioses. However, the biological function of MAPK in orchid mycorrhiza （OM） symbiosis remains elusive. In the present study, a MAPK gene, designated as DoMPK1 （GenBank accession No. JX297594）, was identified from D. officinale roots infected by an OM fungus-Mycena sp. using the reverse transcription polymerase chain reaction （RT-PCR） and rapid amplification of cDNA ends （RACE） methods. The full length cDNA of DoMPK1 was 1 263 bp and encoded a 372 aa protein with a molecular weight of 42.61 kD and an isoelectric point （pI） of 6.07. The deduced DoMPK1 protein contained the conserved serine/threonine-protein kinase catalytic domain （39-325） and MAP kinase signature （77-177）. Multiple sequence alignment and phylogenetic analysis demonstrated that DoMPK1 was highly homologous （71%-85%） to MAPK genes from various plant species and was closely related to those from monocots. Real time quantitative PCR （qPCR） analysis revealed that DoMPK1 was constitutively expressed in leaves, stems, roots and seeds, and the transcript abundance was not significantly different in the four included tissues. Furthermore, DoMPK1 transcript was markedly induced in roots at 30 d after fungal infection, with 7.91 fold compared to that of the mock inoculated roots, suggesting implication of DoMPK1 in the early D. officinale and Mycena sp. interaction and an essential role in the symbiosis. Our study characterized a MAPK gene associated with OM symbiosis for the first time, and will be helpful for further functional elucidation of DoMPK1 involving in D. officinale and Mycena sp. symbiotic interaction.

关 键 词：铁皮石斛 兰科菌根 激酶 基因表达 共生 

分 类 号：R931[医药卫生—生药学]