红花檵木LcFLS1基因的克隆及其表达与转化研究

Cloning,Expression and Transformation of LcFLS1 Gene fromLoropetalum chinense var.rubrum

该研究以红花檵木(Loropetalum chinense var.rubrum)为材料,根据转录组测序结果和PCR方法克隆到1个黄酮醇合成酶(FLS)同源基因,命名为LcFLS1。生物信息学分析显示,LcFLS1的开放阅读框为996bp,编码331个氨基酸。氨基酸序列分析显示,LcFLS1具有典型的2-酮戊二酸和铁依赖性双加氧酶结构域;蛋白结构预测表明,球形蛋白结构的核心区域存在10个与2-酮戊二酸配体互作的位点。进化树分析结果表明,LcFLS1与茶树(Camellia sinensis)等木本植物的亲缘关系较近,而与拟南芥(Arabidopsis thaliana)等草本植物的亲缘关系较远。荧光定量PCR检测显示,LcFLS1在红花檵木的花中相对表达量最高,而在茎中最少。成功构建了LcFLS1基因的过表达载体pLcFLS1-SUPER1300,经农杆菌侵染花序法将pLcFLS1-SUPER1300质粒转入拟南芥中获得转基因植株,PCR鉴定表明获得了转LcFLS1基因拟南芥阳性植株。该研究结果为红花檵木黄酮醇的生物合成机制研究,以及药用价值的开发利用奠定了基础。

In this study,Loropetalum chinense var.rubrum was used as tested material.A flavonol synthase(FLS)homology gene named LcFLS1 was cloned by PCR based on the transcriptome sequencing results.The bioinformatic analysis shows that the open reading frame of LcFLS1 gene contains 996 base pairs encoding 331 amino acids.Amino acid alignment results indicate that LcFLS1 may encodes a typical2-oxoglutarate and Fe(II)-dependent oxygenase.Protein structure prediction indicates there are ten loci at the core domain of globular protein interacting with 2-oxoglutarate ligand in LcFLS1.The phylogenetic tree analysis illustrates that LcFLS1 is closely related to Camellia sinensis and other woody plants but is far related to herbaceous plants like Arabidopsis thaliana.The express pattern of different tissues by qPCR shows that LcFLS1 expressed highest in L.chinense var.rubrumflower while lowest in stem.The pLcFLS1-SUPER1300 overexpression vector was successfully constructed and transferred into Arabidopsis thaliana by Agrobacterium infection inflorescence method to obtain transgenic plants.PCR identificationshows that positive transgenic plants of LcFLS1 were obtained.This research laid important foundations for studying the mechanism of flavonol biosynthesis as well as development and utilization of medicine value in L.chinense var.rubrum.