淹水胁迫对杭菊F3'H基因表达及其下游产物含量的影响
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摘要
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.
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.
引文
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[23]黄璐琦.分子生药学[M].2版.北京:北京大学医学出版社,2006.
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[25]Liu L,Zhu Z B,Guo Q S,et al.Variation in contents of major bioactive compounds in Glechoma longituba related to harvesting time and geographic distribution[J].J Med Plant Res,2012,6(1):122.
[2]邵清松,郭巧生,李育川,等.药用菊花HPLC图谱分析及其模式识别研究[J].中草药,2011,42(11):2330.
[3]王亮,汪涛,郭巧生,等.昆仑雪菊与杭菊、贡菊主要活性成分比较[J].中国中药杂志,2013,38(20):3442.
[4]Wang T,Xi M Q,Guo Q S,et al.Chemical components and antioxidant activity of volatile oil of a Compositae tea(Coreopsis tinctoria Nutt.)from Mt.Kunlun[J].Ind Crop Prod,2015,67(4):318.
[5]汪涛,郭巧生,沈学根,等.杭菊不同栽培类型内在质量比较研究[J].中国中药杂志,2007,32(9):783.
[6]Lin L Z,James M H.Identification of the phenolic components of chrysanthemum flower(Chrysanthemum morifolium Ramat.)[J].Food Chem,2010,120(1):319.
[7]华波,吕圭源.杭白菊黄酮类化合物的心血管药理实验研究进展[J].青岛医药卫生,2006,38(4):282.
[8]Lai J P,Lim Y H,Su J,et al.Identification and characterization of major flavonoids and caffeoylquinic acids in three Compositae plants by LC/DAD-APCI/MS.[J].J Chromatogr B:Analyt Technol Biomed Life Sci,2007,848(2):215.
[9]Chongkuei L,Lei Y P,Yao H T,et al.Chrysanthemum morifolium Ramat.reduces the oxidized LDL-induced expression of intercellular adhesion molecule-1 and E-selectin in human umbilical vein endothelial cells[J].J Ethnopharmacol,2010,128(1):213.
[10]Luo H J,Wang J Z,Chen J F,et al.Docking study on chlorogenic acid as a potential H5N1 influenza A virus neuraminidase inhibitor[J].Med Chem Res,2011,20(5):554.
[11]Wang T,Zhu Z B,Guo Q S,et al.Variation in major flavonoids glycosides and caffeoylquinic acids during florescence of three Chrysanthemum morifolium Ramat cv.'Hangju'genotypes[J].Biochem Syst Ecol,2013,47:74.
[12]石万里,姚毓璆.菊花花芽分化初步研究[J].园艺学报,1990(4):309.
[13]倪月荷,汪觉先.菊花栽培与鉴赏[M].上海:上海科学技术出版社,2000:11.
[14]Feldmann K A.Cytochrome P450s as genes for crop improvement[J].Curr Opin Plant Bio,2001,4(2):162.
[15]Seitz C,Ameres S,Forkmann G.Identification of the molecular basis for the functional difference between flavonoid 3'-hydroxylase and flavonoid 3',5'-hydroxylase[J].Febs Lett,2007,581(18):3429.
[16]乔小燕,马春雷,陈亮.植物类黄酮生物合成途径及重要基因的调控[J].天然产物研究与开发,2009,21(2):354.
[17]Wang T,Guo Q S,Mao P F.Flavonoid accumulation during florescence in three Chrysanthemum morifolium Ramat cv.'Hangju'genotypes[J].Biochem Syst Ecol,2014,55:79.
[18]郭巧生,汪涛,程俐陶,等.药用菊花不同栽培类型总黄酮动态积累研究[J].中国中药杂志,2008,33(11):1237.
[19]谭勇,梁宗锁,董娟娥,等.水分胁迫对菘蓝生长发育和有效成分积累的影响[J].中国中药杂志,2008,33(1):19.
[20]黄璐琦,郭兰萍.环境胁迫下次生代谢产物的积累及道地药材的形成[J].中国中药杂志,2007,32(4):277.
[21]Yang D F,Ma P D,Liang X,et al.PEG and ABA trigger methyl jasmonate accumulation to induce the MEP pathway and increase tanshinone production in Salvia miltiorrhiza hairy roots[J].Physiol Plantarum,2012,146(2):173.
[22]张松焕,郭惠明,裴熙祥,等.紫茎泽兰类黄酮3'-羟化酶在烟草中的表达[J].中国农业科学,2009,42(12):4182.
[23]黄璐琦.分子生药学[M].2版.北京:北京大学医学出版社,2006.
[24]Huang L Q,Guo L P,Ma C Y,et al.Top-geoherbs of traditional Chinese medicine:common traits,quality characteristics and formation[J].Front Med,2011,5(2):185.
[25]Liu L,Zhu Z B,Guo Q S,et al.Variation in contents of major bioactive compounds in Glechoma longituba related to harvesting time and geographic distribution[J].J Med Plant Res,2012,6(1):122.