张岗, 李依民, 胡本祥, 张大为, 郭顺星. 铁皮石斛促分裂原活化蛋白激酶基因DoMPK4的分离和差异表达分析J. 药学学报, 2014,49(7): 1076-1083.
引用本文: 张岗, 李依民, 胡本祥, 张大为, 郭顺星. 铁皮石斛促分裂原活化蛋白激酶基因DoMPK4的分离和差异表达分析J. 药学学报, 2014,49(7): 1076-1083.
ZHANG Gang, LI Yi-min, HU Ben-xiang, ZHANG Da-wei, GUO Shun-xing. Isolation and differential expression of a novel MAP kinase gene DoMPK4 in Dendrobium officinaleJ. Acta Pharmaceutica Sinica, 2014,49(7): 1076-1083.
Citation: ZHANG Gang, LI Yi-min, HU Ben-xiang, ZHANG Da-wei, GUO Shun-xing. Isolation and differential expression of a novel MAP kinase gene DoMPK4 in Dendrobium officinaleJ. Acta Pharmaceutica Sinica, 2014,49(7): 1076-1083.

铁皮石斛促分裂原活化蛋白激酶基因DoMPK4的分离和差异表达分析

Isolation and differential expression of a novel MAP kinase gene DoMPK4 in Dendrobium officinale

  • 摘要: 促分裂原活化蛋白激酶 (mitogen-activated protein kinases,MAPKs) 是所有真核生物中普遍存在的一种重要的信号转导组分,在植物生理代谢、生长发育和激素反应等多项途径中起重要调控作用。本研究利用RT-PCR、RACE方法,从珍稀濒危兰科药用铁皮石斛中分离到一个新的促分裂原活化蛋白激酶基因,命名为DoMPK4 (GenBank注册号JX297597)。DoMPK4基因cDNA全长1 518 bp,编码一条由369个氨基酸组成的肽链,相对分子质量42.42 kD,等电点为5.55。推定的DoMPK4蛋白不含信号肽,具有一个跨膜域 (214~232),包含MAPK蛋白家族保守的丝氨酸/苏氨酸蛋白激酶活性位点 (158~170) 和MAP激酶的保守位点 (71~174)。多序列比对表明,DoMPK4与多种植物MAPKs蛋白一致性较高 (74.9%~80.6%)。进化树结果显示,DoMPK4隶属于植物MAPKs蛋白的Group A分支,与单子叶植物亲缘关系较近。实时定量PCR分析表明,DoMPK4在石斛根、茎、叶、种子和原球茎等5种器官中的相对表达量有显著差异。DoMPK4转录本在原球茎中的表达量为叶中的17.65倍,种子、根和茎等次之,分别为5.84、2.28和1.64倍。DoMPK4在原球茎较萌发种子中的高表达量特征,提示该基因在原球茎发育过程中起一定作用。

     

    Abstract: Mitogen-activated protein kinases (MAPKs) are important signaling transduction components well conserved in eukaryotes and play essential roles in various physiological, developmental and hormonal responses in plant. In the present study, a MAPK gene, designated as DoMPK4 (GenBank accession No. JX297597), is identified from a rare endangered medicinal orchid species D. officinale using the reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. The full length cDNA of DoMPK4 is 1 518 bp in length and encoded a 369 aa protein with a molecular weight of 42.42 kD and an isoelectric point of 5.55. DoMPK4 protein contained a serine/threonine protein kinase active site (158-170), a MAP kinase site (71-174), and eight conserved motifs. DoMPK4 had a transmembrane (214-232) but no signal peptide. Multiple sequence alignment showed that DoMPK4 shared high identities (74.9%-80.6%) with MAPK proteins from various plants. Phylogenetic analysis demonstrated that DoMPK4 belonged to group A of the MAPK evolutionary tree, and is closely related to monocots. Real time quantitative PCR (qPCR) analysis revealed that DoMPK4 is differentially expressed among the five organs including leaf, stem, root, seed, and protocorm-like body (PLB). The transcription level of DoMPK4 is the highest in the PLBs with 17.65 fold, followed by seeds, roots, and stems with 5.84, 2.28, and 1.64 fold, respectively. The progressive enhancement of DoMPK4 transcripts in the developing PLBs compared to that in the germinating seeds, suggests a role of DoMPK4 during the development of embryogenic PLBs formation in D. officinale.

     

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