青蒿素生物合成途径基因组织表达分析与青蒿素积累研究

Relative expression of genes involved in artemisinin biosynthesis and artemisinin accumulation in different tissues of Artemisia annua

目的:研究青蒿根、茎、叶和花中与青蒿素合成相关基因的相对表达量,建立相关基因表达量与青蒿素积累的关系,为发现青蒿素生物合成中起主要作用的基因奠定基础。方法:采用qRT-PCR技术对不同组织中青蒿素合成途径涉及到的7个功能基因(HMGR,DXR,FPS,ADS,CYP71AV1,CPR,AAR)的表达水平进行分析,同时测定对应组织中青蒿素含量。结果:青蒿素生物合成上游途径涉及到的3个关键酶基因HMGR,DXR,FPS在花中的表达量最高;青蒿素生物合成特有途径涉及到的4个基因功能在根、茎、叶和花中均有表达;ADS表达量在叶中最高,其次为花,在茎中表达量最低;CYP71AV1表达量在花中最高,在叶中最低;CPR的最高表达量也出现在叶中;而AAR在各个组织中表达量相对而言都较低。青蒿素质量分数在叶中最高(0.343 mg.g-1),花中次之(0.152 mg.g-1),在根(0.062 mg.g-1)和茎(0.060 mg.g-1)中含量很低。结论:在青蒿素生物合成中,上游途径包括MVA和MEP途径在花中的代谢更加活跃,花可能是青蒿素前体合成的主要部位,其中来自于MEP途径的DXR对于青蒿素积累具有较大贡献;在青蒿素生物合成下游途径的4个功能基因中,ADS在各组织中的表达量与青蒿素含量完全一致,表现为正相关,表明ADS在青蒿素合成中起到重要作用,是该途径遗传改造的重要靶点。青蒿中各基因在不同组织中不是均一表达,而是有选择性的协同表达。

Objective： To study the relative expression of the genes involved in artemisinin biosynthesis in different tissues including roots,stems,leaves and flowers of Artemisia annua,and establish the relationship between gene expression and artemisinin accumulation,eventually leading to discover the mainly effective genes involved in artemisinin biosynthesis.Method： The 7 functional genes involved in artemisinin biosynthesis were detected at the level of expression by using qRT-PCR,and simultaneously the content of artemisinin in the 4 investigated tissues was detected in parallel.Result： The 3 genes including HMGR,DXR and FPS which were involved in the upstream pathway of artemisinin biosynthesis showed the highest expression levels in flowers,and the 4 functional genes including ADS,CYP71AV1,CPR and AAR which were involved in the artemisinin-specific biosynthetic pathway were found to be expressed in all the 4 detected tissues.The highest expression level of ADS was found in leaves,then followed by flowers,and the lowest expression level of ADS was found in roots and stems.CYP71AV1 had highest expression level in flowers and lowest in leaves.CPR showed highest expression level in flowers,and AAR had lower expression levels in the other 3 artemisinin-specific pathway genes in all the tissues.The highest content of artemisinin was found in leaves（0.343 mg·g^-1）,then followed by flowers（0.152 mg·g^-1,roots（0.062 mg·g^-1 and stems（0.060 mg·g^-1.Conclusion： In the biosynthesis of artemisinin,the upstream genes including HMGR from the MVA pathway,DXR from the MEP pathway and the checkpoint gene FPS were much more active in flowers,and this suggested that flowers might be the tissues of artemisinin precursor biosynthesis,and further DXR contributed more to artemisinin biosynthesis.The positive correlation of ADS expression and artemisinin content in tissues demonstrated that ADS played a very important role in artemisinin biosynthesis,which was the ideal target for engineering the artemisinin biosynthetic pathway.In summary,the functional genes involved in artemisinin biosynthesis do not express at the same level but synergistically.