Gene expression analysis of key enzymes and MYB transcription factors in flavonoid biosynthesis pathway during germination of Fagopyrum tataricum | 苦荞芽期黄酮合成关键酶和MYB转录因子基因的表达分析

Chinese. 苦荞(Fagopyrum tataricum)作为一种药食两用植物，富含以芦丁为主的黄酮类化合物。苦荞芽期芦丁含量较高，其分子机制尚不清楚。本研究选用西荞2号，采用AlCl3法测定了苦荞芽期6~10 d胚轴和子叶中的总黄酮，采用半定量RT-PCR分析其黄酮合成途径中主要关键酶基因苯丙氨酸氨裂解酶基因(Pal)、查尔酮异构酶基因(Chi)和黄酮醇合酶基因(Fls)，以及MYB转录因子基因FtMyb1、FtMyb2和FtMyb3的相对表达水平，并对三者之间的相关性进行了统计学分析。结果表明，以相关系数绝对值大于0.75为阈值，子叶中，总黄酮的积累与FtMyb3表达显著正相关(0.9625)，与FtMyb2表达显著负相关(－0.8572)；Chi与FtMyb2表达显著正相关(0.8468)，与FtMyb3表达显著负相关(－0.8010)；Pal、Chi和Fls表达彼此显著正相关，相关系数分别为0.9119、0.8920和0.7584。子叶中总黄酮含量在4.58%~5.54%之间，且随芽期递增。Pal、Chi和Fls整体表达趋势相似，均呈现先升高后降低的趋势，且Fls的表达水平明显高于前二者。FtMyb2 和FtMyb3整体表达趋势相反，FtMyb2呈下降趋势，FtMyb3呈上升趋势。胚轴中，总黄酮含量与Chi显著负相关(－0.8989)；Fls与Chi显著负相关(－0.7498)。结果提示，苦荞芽期黄酮合成的分子机制较为复杂，但部分基因表达仍存在显著相关性联系，为进一步选择苦荞分子操作靶位点提供参考。

English. As a medicinal and edible plant, tartary buckwheat (Fagopyrum tataricum) is rich in flavonoids especially rutin. During germination, it has a higher concentration of ruin, but the molecular mechanism is still unclear. In this study, the total flavonoids in cotyledon and hypocotyl of F. tataricum during 6~10 days after germination were extracted by the methanol extraction method, and were determined by aluminum chloride spectrophotometry. Semi-quantitative RT-PCR was used to determine the expression level of the phenylalnine ammonialyase gene (Pal), chalcone isomeerase gene (Chi), flavonol synthase gene (Fls) and MYB transcription factor genes (FtMyb1, FtMyb2 and FtMyb3) in the cotyledon and hypocotyl during the seed germination, the relation among them analysed by statistics method. The result showed that: in cotyledons, the total flavonoids content was significantly and positively associated with FtMyb3 (0.9625), and negatively with FtMyb2 (－0.8572); Chi was significantly and positively associated with FtMyb2 (0.8468), and negatively with FtMyb3 (－0.8010); the three key enzyme genes were significantly and positively correlated to each other with 0.9119, 0.8920 and 0.7584, respectively. In hypocotyls, the total flavonoids content was significantly and negatively associated with Chi (－0.8989), while Fls was significantly negative associated with Chi (－0.7498). Visiblely, the regulation of flavonoid biosynthesis is rather complex during germination of F. tataricum, but some gene's expression is still significantly associated with each other.