苦荞黄酮醇合酶FLS同源基因的分子克隆、催化特征及其对芦丁合成的影响

The tartary buckwheat (Fagopyrum tataricum Garetn.) is an excellent medicinal and nutrient-rich crop containing aboudance of rutin (a flavonol glycoside), which is the main quality character and economic indicator for this crop. In plant, flavonol synthase (FLS) and its isoforms (homologous genes) are the key enzymes and limiting factors in rutin biosynthesis. An important cause of plant species accumulate different flavonols, including the types and contents, mainly contribute to the isoform of FLSs, which have distinguishing expression pattern, catalytic activity, and substrate preference. However, the study on isoform of FLSs for tartary buckwheat have not systematically reported yet. In our previous studies, the full-length cDNA of FtFLS1 and FtFLS3 were isolated from tartary buckwheat, and the FtFLS1 was charactered and identificated. Thus, this project focuses on molecular cloning homologous gene of FLSs from tartary buckwheat, and characterization and functional identification the isoform of FLS proteins. Via homologous cloning and RACE techniques, other cDNA of FLSs will be cloned, and their molecular features will be characterized by bioinformatics. Through quantitative PCR and HPLC, the expression level of FLSs and the flavonoids contents in florescence of tartary buckwheat and transgenic tobaccos will be tested, and these will provide the basic data for understanding the accumulation of flavonoids caused by different FLSs. Also, the biological activity of different isoform of FLS proteins and their effect on flavonol biosynthesis pathway will be detected. Then, prokaryotic expression and affinity chromatography will be performed to obtain the isoform of FLS proteins in vitro, and their enzymatic kinetic parameters will be analyzed by assaying. Totally, the aim of this project will reveal the molecular and enzymatic features of FLSs from tartary buckwheat, and it also provide the scientific theories and materials for improving the flavonols or rutin biosynthesis pathway in tartary buckwheat and molecular breeding.

苦荞作为富含芦丁(黄酮醇)而兼具药用和保健功能的小杂粮，芦丁含量是其主要质量性状和经济指标。黄酮醇合酶(FLS)及其同源基因是芦丁合成和积累的关键酶和限制因素，FLS同源蛋白的催化活性和底物偏爱性是植物黄酮醇种类和含量差异的重要原因。本项目拟在获得苦荞FtFLS1和FtFLS3的基础上，进一步利用RACE技术克隆2-4条FLS同源基因并进行分子鉴定；采用荧光定量PCR和HPLC技术，分析苦荞花期FLS同源基因的表达对黄酮醇代谢支路的影响，检测转基因烟草中FLS同源基因的生物活性与产物的关系；采用原核表达和亲和层析技术，分离纯化FLS同源蛋白，分析其酶促动力学参数。本项目可望初步阐明苦荞FLS同源基因的分子特征、表达产物的活性和主效基因，明晰FLS对苦荞黄酮醇合成支路的代谢调控，为未来进行高芦丁苦荞的分子育种提供理论依据和基础材料。

苦荞作为富含芦丁(黄酮醇)而兼具药用和保健功能的小杂粮，芦丁含量是其主要质量性状和经济指标。黄酮醇合酶(FLS)及其同源基因是芦丁合成和积累的关键酶和限制因素，FLS同源蛋白的催化活性和底物偏爱性是植物黄酮醇种类和含量差异的重要原因。本研究成功获得3条新的FLS同源基因，命名为FtFLS3、FtFLS4和FtFLS5，分子特征鉴定结果表明FtFLS1-5在分子特征上FtFLS1和FtFLS3较为类似，FtFLS2、FtFLS4和FtFLS5较为接近；花期苦荞FtFLS1有最高的表达量且与黄酮含量强烈正相关可能为影响苦荞黄酮醇及芦丁合成和积累的主要因素。转FtFLS1-5的拟南芥转基因阳性植株中FtFLS1-5的超表达均引起拟南芥总黄酮含量的提高，但含不同基因的植株中黄酮组分具有显著差异。重组FtFLS1-5蛋白显示出催化能力的强弱和酶催特征的差异，FtFLS1具有最高的比活力和最强的底物竞争力。研究结果提示，苦荞黄酮醇合成代谢支路的主效基因为FtFLS1，FtFLS3与之具有类似的催化功能但其转录水平受到FtFLS1的抑制；FtFLS2、FtFLS4和FtFLS5的催化能力较弱，可能参与苦荞其他黄酮的合成，其表达可能受到其他内外环境的影响。