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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 the DoMPK2 gene of Dendrobium officinale mitogen-activated protein kinase (MAPK) and analyze its molecular characteristics. Methods RT-PCR and rapid amplification of cDNA ends (RACE) were used to isolate full-length genes. The bioinformatics analysis was used to analyze the physicochemical properties and conserved domains of the proteins. DNASTAR, MEGA Multiple amino acid sequence alignment and phylogenetic tree analysis were performed by software. Genomic expression patterns were detected by real-time quantitative PCR. Results The full-length cDNA of DoMPK2 gene was 1 585 bp (GenBank accession number JX297595) and encoded a peptide chain consisting of 369 amino acids with a molecular weight of 42120 and an isoelectric point of 6.56. The DoMPK2 protein contains the conserved serine / threonine protein kinase catalytic domain (32-319) of the MAPK family and the conserved site of MAP kinase (67-170). DoMPK2 is highly similar to many plant MAPK genes (56% -84%) and has a close genetic relationship with the MAPK gene of monocotyledons such as rice and maize. DoMPK2 is constitutively expressed, and its transcripts are relatively highly expressed in seeds and roots of Dendrobium, which are respectively 9.696 and 2.327 times higher in leaves than in leaves. The expression of DoMPK2 is not significantly changed in stems and leaves. Conclusion The molecular characteristics of DoMPK2 gene lay a foundation for further study on the biological function of DoMPK2 in the growth and development of Dendrobium candidum and its anti-stress.