中国中药杂志

2018, v.43(01) 52-57

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淹水胁迫对杭菊F3'H基因表达及其下游产物含量的影响
Cloning and expression analysis of F3'H and quantification of downstream products in Chrysanthemum morifolium under flooding stress

邹庆军;汪涛;郭巧生;肖有梅;武立伟;
ZOU Qing-jun;WANG Tao;GUO Qiao-sheng;XIAO You-mei;WU Li-wei;Institute of Chinese Medicinal Materials,Nanjing Agricultural University;

摘要(Abstract):

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CmF3'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CmF3'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1条总长1 562 bp的CmF3'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CmF3'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 d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CmF3'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。
To investigate the effects of the expression of flavonoid 3' hydroxylase gene(F3'H) and active ingredients in Chrysanthemum morifolium under flooding stress,we cloned F3'H from Hangju(temporarily named CmF3'H) and conducted bioinformatics analysis.During the flower bud differentiation stage,we flooded the Ch.morifolium and then used the Real-time PCR to detect the relative expression of CmF3'H;Finally,active ingredients of the inflorescence were measured by HPLC.The sequencing results showed that 1 562 bp sequence was acquired with the largest open reading frame of 1 527 bp,which encoded 508 amino acids.The phylogenetic tree found that CmF3'H was highly homologous to other species of Compositae.Real-time PCR results showed that CmF3'H had a significant response to flooding stress and had the highest expression level after flooding for 24 h,which was about 9 times as that of the control group.The results of HPLC showed that luteolin and luteoloside,the downstream products catalyzed by the F3'H,were significantly higher than those in the control group.It was also found that the contents of chlorogenic acid and 3,5-O-di-caffeoylquinic acid were also significantly higher than those of the control group.Therefore,Ch.morifolium regulates the synthesis of downstream products by regulating the expression of CmF3'H in the flavonoid synthesis pathway under flooding stress,thereby responding to flooding stress.The flooding stress during flower bud differentiation can significantly enhance the accumulation of active ingredients.

关键词(KeyWords): 杭菊;F3'H;基因克隆;淹水胁迫;黄酮;活性成分
Chrysanthemum morifolium;F3'H;clone;flooding stress;flavonoids;active ingredients

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金青年基金项目(81503180);; 中央高校基本科研业务费专项(KJQN201643,KYZ201608)

作者(Author): 邹庆军;汪涛;郭巧生;肖有梅;武立伟;
ZOU Qing-jun;WANG Tao;GUO Qiao-sheng;XIAO You-mei;WU Li-wei;Institute of Chinese Medicinal Materials,Nanjing Agricultural University;

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DOI: 10.19540/j.cnki.cjcmm.20171030.008

参考文献(References):

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