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为探究淹水胁迫对杭菊黄酮合成途径中的关键基因类黄酮3’羟化酶基因(F3’H)的表达和活性成分的影响,该实验克隆了杭菊F3’H基因(暂命名Cm F3’H),并进行生物信息学分析;同时在杭菊花芽分化期进行淹水胁迫,以β-actin为内参基因,使用Real-time PCR检测Cm F3’H的相对表达量;然后使用HPLC测定杭菊CmF3’H下游产物及其他指标成分含量。通过研究得到一条总长1 562 bp的Cm F3’H基因,其中开放阅读框长1 527 bp,编码508个氨基酸,构建进化树发现CmF3’H与菊科其他种植物同源性很高;Real-time PCR结果表明Cm F3’H对淹水胁迫有明显响应,在淹水24 h后表达量最高,约为对照组的9倍,解除胁迫12天后恢复正常水平;HPLC结果显示淹水处理后的杭菊F3’H所催化形成的下游产物木犀草素和木犀草苷含量均显著高于对照组,同时发现其他2种指标成分绿原酸和3,5-O-二咖啡酰基奎宁酸含量也显著高于对照组。因此,杭菊在淹水胁迫下可以通过调节Cm F3’H的表达来调控下游产物的合成,从而对淹水胁迫做出应答,而且花芽分化期进行淹水胁迫可以显著增强杭菊活性成分的积累。
In order to explore the effect of flooding stress on the expression and activity of the key gene flavonoid 3’-hydroxylase gene (F3’H) in the pathway of flavonoids synthesis, we cloned the genes of Hangju F3’H (tentative name Cm F3’H), and analyzed by bioinformatics. At the same time, during the flower-bud differentiation of Hangzhou and Daqing, the expression of Cm F3’H was detected by using real-time PCR with β-actin as internal reference. The relative expression of Cm F3’H was detected by HPLC Determination of Chrysanthemum CmF3’H downstream products and other indicators of content. The Cm3’H gene with a total length of 1 562 bp was obtained. The open reading frame (ORF) was 1 527 bp, encoding 508 amino acids. Constructing the phylogenetic tree showed that CmF3’H shared high homology with other Compositae plants. Real- The results of time PCR showed that Cm F3’H had a significant response to waterlogging stress, and reached the highest level at 24 h after flooding, about 9 times of that of the control group, and resumed normal after 12 days of stress release. The results of HPLC showed that after flooding Hang chrysanthemum F3’H catalyzed the formation of the downstream products luteolin and luteolin glycosides were significantly higher than the control group, and found that the other two indicators of chlorogenic acid content and 3,5-O-dicofectionylquinic acid content Also significantly higher than the control group. Therefore, under the waterlogging stress, Hang Ju can regulate the synthesis of downstream products by regulating the expression of Cm F3’H, thus responding to waterlogging stress. Moreover, the flooding stress during flower bud differentiation can significantly enhance the activity of accumulation.