高良姜1-脱氧-D-木酮糖5-磷酸还原异构酶cDNA克隆与表达调控

Cloning and expression regulation of 1-deoxy-D-xylulose-5-phosphate reductoisomerase cDNA from Alpinia officinarum

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。

The rhizome of Alpinia officinarum is a widely used Chinese herbal medicine. The essential oil in A. officinarum rhizome is mainly composed of 1, 8-cineole and other monoterpenes, as the major bioactive ingredients. In plants, monoterpenes are synthesized through the methylerythritol phosphate （MEP） pathway in the plastids, and 1-deoxy-D-xylulose 5-phosphate reductoisomerase （DXR） is an enzyme catalyzing a commined step of the MEP pathway. In the present study, the full-length eDNA encoding DXR was cloned from the rhizome of A. officinarum, using homology-based RT-PCR and rapid amplification of cDNA ends （RACE） techniques. The new eDNA was designated as AoDXR and submitted to GenBank to be assigned with an accession number HQ874658. The full-length cDNA of AoDXR was 1 670 bp containing a 1 419 bp open reading franle encoding a polypeptide of 472 amino acids with a calculated molecular mass of fi 1.48 kDa and an isoelectric point of 6.15. Bioinfornmtie analyses revealed that AoDXR showed extensive homology with DXRs from other plant species and contained a conserved plastids transit peptide, a Pro-rich region and two highly conserved NADPH-binding motifs in its N-terminal region characterized by all plant DXRs. The phylogenetic analysis revealed that AoDXR belonged to angiosperm DXRs. The structural modeling of AoDXR showed that AoDXR had the typical V-shaped strncture of DXR pro- teins. The tissue expression pattern analysis indicated that AoDXR expressed strongly in leaves, weak in rhizomes of A. officinarum. Exogenous methyl jasmonate （MeJA） could enhance the expression of AoDXR and the production of 1, 8-cineole in A. officinarum rhizomes. The cloning and characterization of AoDXR will be helpful to reveal the molecular regulation mechanism of monoterpene biosyn- thesis in A. offieinarum and provides a candidate gene for metabolic engineering in improving the medicinal quality of A. officinarum rhizome.