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目的:克隆高良姜1-脱氧-D-木酮糖5-磷酸还原异构酶(DXR)的全长cDNA,分析其组织表达模式及茉莉酸甲酯(MeJA)的调控模式,为高良姜有效成分的基因调控及基因工程育种奠定基础。方法:应用简并引物RT-PCR和RACE技术从高良姜根茎中克隆DXR全长cDNA,运用生物信息学解析其编码的蛋白质结构,实时荧光定量PCR法分析其组织表达模式和MeJA的调控模式。结果:克隆了高良姜DXR全长cDNA序列(AoDXR),开放读码框长1 419 bp,编码的蛋白质含472个氨基酸残基、相对分子质量约51.48 kDa。推导的AoDXR氨基酸序列与其他高等植物的DXR具有高度的序列一致性(73%～99%)。AoDXR在高良姜叶片中表达量最强,而在根茎中表达量较弱。外源茉莉酸甲酯(MeJA)处理提高了根茎AoDXR的转录水平和1,8-桉油精含量。结论:AoDXR在高良姜根茎中的表达水平与1,8-桉油精的积累不一致,反应了AoDXR催化的终产物的多样性和表达调控的复杂性。外源MeJA可促进根茎AoDXR的表达和1,8-桉油精的积累,对提高药材品质有应用价值。
OBJECTIVE: To clone the full-length cDNA of galactose-1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) from Alpinia officinarum and analyze its tissue expression pattern and regulation mode of MeJA. Composition of gene regulation and genetic engineering laid the foundation for breeding. METHODS: DXR full-length cDNA was cloned from roots of Alpinia officinalis using degenerate primers RT-PCR and RACE. The protein structure of the full-length DXR cDNA was analyzed by bioinformatics analysis. The expression patterns of MeJA and the regulation of MeJA were analyzed by real-time fluorescence quantitative PCR. Results: The full length cDNA of Aloxacum curcas DXR was cloned and the open reading frame was 1 419 bp in length. The encoded protein contained 472 amino acid residues with a relative molecular mass of 51.48 kDa. The deduced AoDXR amino acid sequence has a high degree of sequence identity (73% -99%) with that of other higher plants. AoDXR expression in the galangal leaves the strongest, while the expression in the rhizome weaker. Exogenous methyl jasmonate (MeJA) treatment increased rhizome AoDXR transcription and 1,8-eucalyptol content. CONCLUSION: The expression level of AoDXR in Alpinia officinarum roots is inconsistent with the accumulation of 1,8-eucalyptol, reflecting the diversity of AoDXR-catalyzed end products and the complexity of expression regulation. Exogenous MeJA can promote the expression of AoDXR in rhizome and the accumulation of 1,8-eucalyptol, which has application value to improve the quality of medicinal materials.