苹果MdAFS叶绿体定位表达对拟南芥萜类挥发物的影响

Effects of Chloroplast-targeted MdAFS on Terpene Volatiles of Transgenic Arabidopsis

以‘青香蕉’苹果(Malus×domestica‘White Winter Pearmain’)α–法尼烯合酶基因(MdAFS)为目标基因,构建叶绿体定位的植物过表达载体,并通过农杆菌介导遗传转化拟南芥,获得使MdAFS在叶绿体中过表达的拟南芥转基因株系。将pBI122-rbcS1-pla-MdAFS-GFP表达载体瞬时表达本氏烟,分析表明叶绿体定位肽能成功将MdAFS蛋白定位到叶绿体中。定量测定莲座期和盛花期拟南芥光合色素,结果显示,转基因拟南芥叶绿素和类胡萝卜素含量较野生型显著增加。qRT-PCR分析萜类代谢相关基因转录表达水平表明,尤其在盛花期,过表达MdAFS能显著影响萜类代谢基因表达上调。应用静态顶空固相微萃取法收集拟南芥挥发物并结合GC–MS分析鉴定萜类挥发物成分,转基因拟南芥中萜类挥发物含量和种类明显增加。这些结果表明,过表达MdAFS能显著影响萜类代谢流的重新定向,增加萜类代谢挥发物。叶绿体是萜类代谢工程一个理想的亚细胞空间。

The apple α-farnesene synthase gene（MdAFS）was cloned by combination of the homologous clone and PCR technology from apple（Malus × domestica‘White Winter Pearmain＇）. By using chloroplast transit peptide（pla）of the At Cor15 A gene from Arabidopsis thaliana,we targeted MdAFS to the chloroplast of Arabidopsis to generate pla ＋ MdAFS transgenic lines. Transgenic Arabidopsis was generated by Agrobacterium-mediated genetic transformation. The subcellular localization of the pla-MdAFS-GFP fusion fluorescent protein revealed that the MdAFS protein was localized in chloroplast. The quantification of photosynthesis-related isoprenoid metabolites in all plants at the rosette and flowering stages showed that chlorophyll a,chlorophyll b,total chlorophyll,and carotenoid were higher in the transgenic lines as compared to wild-type control. The expressions of terpenoid biosynthetic genes were investigated in different development stages by qRT-PCR. Especially,at the flowering stage,the expression of several terpenoid biosynthetic genes significantly increased in the transgenic lines. Gas chromatography mass spectrometry（GC–MS）analysis of volatile extracts from the transgenic plants revealed the biosynthesis of several novel sesquiterpenes,including（E,E）-α-farnesene. These results indicated that overexpressing MdAFS in chloroplast of Arabidopsis could significantly affect the reorientation of terpene metabolic pathway,and increase the transgenic plant emitting terpene volatiles.