药用菊花中绿原酸合成途径关键酶基因的克隆及表达分析
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摘要
目的探究淹水胁迫对杭菊绿原酸合成途径中的关键酶香豆酰基喹酸酯3’-单加氧酶(C3’H)和莽草酸羟基肉桂酰基转移酶(HCT)基因的表达和活性成分的影响。方法使用实时荧光定量PCR(qRT-PCR)方法基于杭菊转录组数据库克隆HCT和C3’H基因各2个,其中C3’H酶的2个基因CmC3’H1、CmC3’H2,HCT酶的2个基因CmHCT1、CmHCT2,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qRT-PCR检测以上4个基因的相对表达量;然后使用HPLC法测定杭菊这4个基因的下游产物及其他指标成分含量。结果通过研究获得了4条基因的完整开放阅读框,并且预测其氨基酸序列的相对分子质量和理论等电点(pI),同时构建蛋白质三级结构模型。qRT-PCR结果显示在花芽分化期对杭菊进行持续3 d的淹水处理会导致以上4个基因在不同生长时期内的表达显著变化。HPLC结果显示淹水处理后的杭菊C3’H和HCT所催化形成的下游产物绿原酸含量显著高于对照组,同时发现其他2种指标成分木犀草苷和3,5-O-二咖啡酰基奎宁酸含量也显著高于对照组。结论杭菊在淹水胁迫下可以通过调节4种基因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 coumaroylquinate 3'-monooxygenase(C3'H) and shikimate O-hydroxycinnamoyltransferase(HCT) in the chlorogenic acid-producing pathway and active ingredients in Chrysanthemum morifolium under flooding stress, we cloned two C3'H genes which were CmC3'H1 and CmC3'H2 and two HCT genes which were CmHCT1 and Cm HCT2 by the RT-PCR from Hangju and conducted bioinformatics analysis. During the flower bud differentiation stage, we flooded the C. morifolium and then used β-actin as the reference gene to detect the relative expression of the four genes by the qRT-PCR. Finally,the content of downstream products and other indicators of these four genes in C. morifolium were measured by HPLC. We obtained the four genes' complete open reading frame and predicted the relative molecular mass of the amino acid sequence and the theoretical isoelectric point(pI). And the protein tertiary structure models were constructed. The q RT-PCR results showed that flooding the C.morifolium for 3 days during the flower bud differentiation stage resulted in significant expression changes of the four genes at different growth stages. The results of HPLC showed that chlorogenic acid, the downstream product catalyzed by the C3'H and the HCT, was significantly higher than that in the control group. It was also found that the content of luteoloside and 3,5-O-di-caffeoylquinic acid was also significantly higher than those of the control group. Therefore, C. morifolium regulates the synthesis of downstream products by regulating the expression of the four genes under flooding stress, thereby responding to flooding stress. And the flooding stress during flower bud differentiation can significantly enhance the accumulation of active ingredients of C. morifolium.
To investigate the effects of the expression of coumaroylquinate 3'-monooxygenase(C3'H) and shikimate O-hydroxycinnamoyltransferase(HCT) in the chlorogenic acid-producing pathway and active ingredients in Chrysanthemum morifolium under flooding stress, we cloned two C3'H genes which were CmC3'H1 and CmC3'H2 and two HCT genes which were CmHCT1 and Cm HCT2 by the RT-PCR from Hangju and conducted bioinformatics analysis. During the flower bud differentiation stage, we flooded the C. morifolium and then used β-actin as the reference gene to detect the relative expression of the four genes by the qRT-PCR. Finally,the content of downstream products and other indicators of these four genes in C. morifolium were measured by HPLC. We obtained the four genes' complete open reading frame and predicted the relative molecular mass of the amino acid sequence and the theoretical isoelectric point(pI). And the protein tertiary structure models were constructed. The q RT-PCR results showed that flooding the C.morifolium for 3 days during the flower bud differentiation stage resulted in significant expression changes of the four genes at different growth stages. The results of HPLC showed that chlorogenic acid, the downstream product catalyzed by the C3'H and the HCT, was significantly higher than that in the control group. It was also found that the content of luteoloside and 3,5-O-di-caffeoylquinic acid was also significantly higher than those of the control group. Therefore, C. morifolium regulates the synthesis of downstream products by regulating the expression of the four genes under flooding stress, thereby responding to flooding stress. And the flooding stress during flower bud differentiation can significantly enhance the accumulation of active ingredients of C. morifolium.
引文
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[17]林郑和,钟秋生,陈常颂,等.茶树叶片GDH、GS、GOGAT基因的克隆及荧光定量PCR分析[J].茶叶科学,2012,32(6):523-529.
[18]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):19-22.
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[20]Yang D F,Ma P D,Liang X,et al.PEG and ABA trigger methyl jasmonate accumulation to induce the MEPpathway and increase tanshinone production in Salvia miltiorrhiza hairy roots[J].Physiol Planta,2012,146(2):173-183.
[21]黄璐琦.分子生药学[M].第2版.北京:北京大学医学出版社,2006.
[22]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):185-194.
[23]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):122-128.
[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):2330-2334.
[3]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,2013,47:74-79.
[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):318-323.
[5]刘波.杏香兔耳风活性成分研究[D].北京:首都师范大学,2005.
[6]张鞍灵,马琼,高锦明,等.绿原酸及其类似物与生物活性[J].中草药,2001,32(2):173-174.
[7]石万里,姚毓璆.菊花花芽分化初步研究[J].园艺学报,1990,17(4):309-312.
[8]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.
[9]Hoffmann L,Maury S,Martz F,et al.Purification,cloning,and properties of an acyltransferase controlling shikimate and quinate ester intermediates in phenylpropanoid metabolism[J].J Biol Chem,2003,278(1):95-103.
[10]Hoffmann L,Besseau S,Geoffroy P,et al.Silencing of hydroxycinnamoy-coenzymeashikimate/quinate hydroxyl cinnamoyltransferase affects phenylpropanoid biosynthesis[J].Plant Cell,2004,16(6):1446-1465.
[11]Schoch G,Goepfert S,Morant M,et al.CYP98A3 from Arabidopsis thaliana is a 3’hydroxylase of phenolic esters,a missing link in the phenylpropanoid pathway[J].J Biol Chem,2001,276(39):36566-36574.
[12]Franke R,Humphreys J M,Hemm M R,et al.The Arabidopsis REF8 gene encodes the 3-hydroxylase of phenylpropanoid metabolism[J].Plant J,2002,30(1):33-45.
[13]Moglia A,Comino C,Portis E,et al.Isolation and mapping of a C3’H gene(CYP98A49)from globe artichoke,and its expression upon UV-C stress[J].Plant Cell Rep,2009,28(6):963-974.
[14]何柳,陈士林.植物中绿原酸合成途径研究进展[J].药物生物技术,2013,20(5):463-466.
[15]Rudkin G O,Nelson J M.Chlorogenic acid and respiration of sweet potatoes[J].J Am Chem Soc,1947,69(6):1470-1475.
[16]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):1237-1239.
[17]林郑和,钟秋生,陈常颂,等.茶树叶片GDH、GS、GOGAT基因的克隆及荧光定量PCR分析[J].茶叶科学,2012,32(6):523-529.
[18]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):19-22.
[19]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):277-280.
[20]Yang D F,Ma P D,Liang X,et al.PEG and ABA trigger methyl jasmonate accumulation to induce the MEPpathway and increase tanshinone production in Salvia miltiorrhiza hairy roots[J].Physiol Planta,2012,146(2):173-183.
[21]黄璐琦.分子生药学[M].第2版.北京:北京大学医学出版社,2006.
[22]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):185-194.
[23]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):122-128.