蒺藜苜蓿叶绿素酸酯a加氧酶(MtCAO)基因的克隆与功能分析

Cloning and functional analysis of chlorophyllide a oxygenase encoding gene MtCAO in Medicago truncatula

叶绿素含量直接影响植物光合效率,从而决定其生物量。叶绿素酸酯a加氧酶(CAO)催化叶绿素酸酯a转化成叶绿素酸酯b,是叶绿素合成的限速酶之一。本研究克隆了蒺藜苜蓿MtCAO,该基因编码531个氨基酸。进化分析表明,MtCAO与双子叶植物的CAO亲缘关系较近。高等植物CAO基因的结构保守,均含有9个外显子。MtCAO主要在绿色组织尤其是叶片中高丰度表达。瞬时表达拟南芥原生质体试验显示,MtCAO-GFP融合蛋白定位于叶绿体。表达模式分析表明,茉莉酸甲酯(MeJA,100μmol·L^-1)及黑暗处理抑制MtCAO的表达,而24 h内NaCl(100 mmol·L^-1)及聚乙二醇(PEG,5%)处理先诱导后抑制其表达,且该基因受昼夜节律调控。这与对MtCAO启动子区顺式作用元件的预测结果一致。类似于超表达AtCAO的拟南芥,超表达MtCAO的拟南芥株系叶色正常,叶绿素含量及鲜重与野生型差异不显著,推测是由于MtCAO高度保守的A结构域中的降解子导致的。

Plant biomass is determined by photosynthesis efficiency,which in turn is affected directly by the chlorophyll concentration.Chlorophyllide a oxygenase(CAO)is one of the three speed-limiting enzymes in the chlorophyll synthesis pathway,and catalyzes chlorophyllide a into chlorophyllide b.In this study,the open reading frame of Medicago truncatula CAO(MtCAO),which encodes a protein of 531 amino acid residues,was cloned.Phylogenetic analysis demonstrated that MtCAO was closer to its dicot homologs than to those in more distantly related plant species.Different from CAOs in lower plants,CAOs in higher plants contain nine exons.MtCAO was preferentially expressed in green tissues,especially leaves.When transiently expressed,the MtCAO-GFP recombinant protein was localized in the chloroplast of Arabidopsis protoplast cells.The transcript of MtCAO was repressed by the treatment with methyl jasmonate(100μmol·L^-1)or darkness,but induced by NaCl(100 mmol·L^-1)or PEG(5%)within 4 h then repressed until 12 h from application.Also,the expression of MtCAO fluctuated with the circadian clock.These results are consistent with the cis-element analysis of the MtCAO promoter.MtCAO was ecotopically expressed in Arabidopsis.The analysis of two independent transgenic lines revealed no significant difference in the content of chlorophyll,the chlorophyll a∶b ratio or shoot weight from the wild type.The results are similar to those from AtCAO overexpression Arabidopsis.Therefore,we postulate that the non-abnormal phenotype of the transgenic Arabidopsis constitutively expressing MtCAO probably results from the fact that the conserved A domain contains a“degron”motif,which was proven to hamper the elevation of AtCAO in Arabidopsis overexpressing AtCAO.