北柴胡和狭叶柴胡中黄酮类成分及其关键酶基因表达的组织差异分析
|
摘要
目的研究北柴胡Bupleurum chinense、狭叶柴胡B. scorzonerifolium不同组织中黄酮类成分含量及其关键酶基因表达量的关系。方法以北柴胡及狭叶柴胡的根、茎、叶、果实为实验材料,采用HPLC法测定不同组织中黄酮类成分(芦丁、槲皮素、山柰素、异鼠李素)的含量,用紫外分光光度法测定总黄酮含量,用实时荧光定量PCR(q RT-PCR)法检测黄酮类成分关键酶基因IFS、F3H、DFR表达量,用SPSS软件进行相关性分析。结果北柴胡、狭叶柴胡地上部分(茎、叶、果实)黄酮类成分含量显著高于根,其中黄酮类成分主要为芦丁,芦丁在北柴胡叶中含量最高可达106.961 mg/g;北柴胡、狭叶柴胡中总黄酮分布有明显的组织差异,含量由高到低为叶≥果实>茎>根;北柴胡IFS、F3H、DFR基因地上部分表达量远高于根,其中IFS基因表达量与芦丁含量呈显著正相关(P<0.05),F3H基因和DFR基因的组织表达量呈显著正相关(P<0.05),而狭叶柴胡各组织IFS、F3H、DFR基因表达均处于较低水平。结论北柴胡、狭叶柴胡黄酮类成分组织含量与关键酶基因的组织表达有一定的一致性,关键酶基因的组织差异表达调控了黄酮类成分在不同组织中的合成与积累。
Objective To investigate the relationship between the content of flavonoids and the key enzyme genes expression in different tissues of Bupleurum chinense and B. scorzonerifolium. Methods The roots, stems, leaves, and fruits of B. chinense and B. scorzonerifolium were used as test materials, determination of flavonoids(rutin, quercetin, kaempferol, and isorhamnetin) in different tissues by HPLC, determination of total flavonoids by UV spectrophotometry, the tissues expression of key enzyme genes(IFS, F3 H, and DFR) in flavonoids synthesis was determined by real-time quantitative PCR, correlation analysis was performed with SPSS. Results The content of flavonoids in the aerial parts(stems, leaves, and fruits) of B. chinense and B. scorzonerifolium was significantly higher than that in roots, the content of flavonoids was mainly rutin, and the content of rutin in the leaves of B. chinense leaves was up to 106.961 mg/g; The distribution of total flavonoids in B. chinense and B. scorzonerifolium was obviously different, the content was from high to low: leaves ≥ fruit > stem > root; The expression of B. chinense IFS, F3 H, and DFR gene in the aerial parts was much higher than that in roots, IFS gene was significantly positive correlated with rutin(P < 0.05), F3 H gene was significantly positive correlated with DFR gene(P < 0.05), but the expression of IFS, F3 H, and DFR gene in each tissues of B. scorzonerifolium was at lower level. Conclusion The content of flavonoids in different parts of B. chinense and B. scorzonerifolium was consistent with the expression of flavonoids synthesis key enzyme genes, the differential expression of key enzyme genes regulates the synthesis and accumulation of flavonoids in different tissues.
Objective To investigate the relationship between the content of flavonoids and the key enzyme genes expression in different tissues of Bupleurum chinense and B. scorzonerifolium. Methods The roots, stems, leaves, and fruits of B. chinense and B. scorzonerifolium were used as test materials, determination of flavonoids(rutin, quercetin, kaempferol, and isorhamnetin) in different tissues by HPLC, determination of total flavonoids by UV spectrophotometry, the tissues expression of key enzyme genes(IFS, F3 H, and DFR) in flavonoids synthesis was determined by real-time quantitative PCR, correlation analysis was performed with SPSS. Results The content of flavonoids in the aerial parts(stems, leaves, and fruits) of B. chinense and B. scorzonerifolium was significantly higher than that in roots, the content of flavonoids was mainly rutin, and the content of rutin in the leaves of B. chinense leaves was up to 106.961 mg/g; The distribution of total flavonoids in B. chinense and B. scorzonerifolium was obviously different, the content was from high to low: leaves ≥ fruit > stem > root; The expression of B. chinense IFS, F3 H, and DFR gene in the aerial parts was much higher than that in roots, IFS gene was significantly positive correlated with rutin(P < 0.05), F3 H gene was significantly positive correlated with DFR gene(P < 0.05), but the expression of IFS, F3 H, and DFR gene in each tissues of B. scorzonerifolium was at lower level. Conclusion The content of flavonoids in different parts of B. chinense and B. scorzonerifolium was consistent with the expression of flavonoids synthesis key enzyme genes, the differential expression of key enzyme genes regulates the synthesis and accumulation of flavonoids in different tissues.
引文
[1]Kuang H,Sun S,Yang B,et al.New megastigmane sesquiterpene and indole alkaloid glucosides from the aerial parts of Bupleurum chinense DC[J].Fitoterapia,2009,80(1):35-38.
[2]黄涵签,王潇晗,付航,等.柴胡属药用植物资源研究进展[J].中草药,2017,48(14):2989-2996.
[3]中国药典[S].一部.2015.
[4]Yang Z Y,Chao Z,Huo K K,et al.ITS sequence analysis used for molecular identification of the Bupleurum species from northwestern China[J].Phytomedicine,2007,14(6):416-423.
[5]韦英杰,王茉,宁青,等.HPLC法同时测定柴胡与春柴胡中皂苷类及黄酮类成分的含量[J].药物分析杂志,2011,31(5):879-883.
[6]谭玲玲,侯晓敏,胡正海.狭叶柴胡营养器官中柴胡皂苷和黄酮类化合物的积累部位及含量比较[J].西北植物学报,2014,34(2):276-281.
[7]潘俊倩,佟曦然,郭宝林.光对植物黄酮类化合物的影响研究进展[J].中国中药杂志,2016,41(21):3897-3903.
[8]冯欢,易姝利,左佳琦,等.基因工程及花色素合成途径在花色改良中的研究进展[J].基因组学与应用生物学,2014,33(2):445-451.
[9]汤立建,赵良才,李庆林,等.黄芩黄酮类成分抗肿瘤作用及机制研究进展[J].中国中药杂志,2007,32(1):21-23.
[10]Sui C,Chen M,Xu J,et al.Comparison of root transcriptomes and expressions of genes involved in main medicinal secondary metabolites from Bupleurum chinense and Bupleurum scorzonerifolium,the two Chinese official Radix Bupleuri source species[J].Physiol Plant,2015,153(2):230-242.
[11]Misra P,Pandey A,Tewari S K,et al.Characterization of isoflavone synthase gene from Psoralea corylifolia:Amedicinal plant[J].Plant Cell Rep,2010,29(7):747-755.
[12]Xiong S,Tian N,Long J,et al.Molecular cloning and characterization of a flavanone 3-Hydroxylase gene from Artemisia annua L[J].Plant Physiol Biochem,2016,105:29-36.
[13]Schijlen E G,Ric D V C,van Tunen A J,et al.Modification of flavonoid biosynthesis in crop plants[J].Phytochemistry,2004,65(19):2631-2648.
[14]Dick C A,Buenrostro J,Butler T,et al.Arctic mustard flower color polymorphism controlled by petal-specific downregulation at the threshold of the anthocyanin biosynthetic pathway[J].PLoS One,2011,6(4):e18230.
[15]周亚福,毛少利,李思锋,等.柴胡属5种植物总皂苷和总黄酮含量的测定[J].时珍国医国药,2013,24(5):1241-1243.
[16]Dong L,Sui C,Liu Y,et al.Validation and application of reference genes for quantitative gene expression analyses in various tissues of Bupleurum chinense[J].Mol Biol Rep,2011,38(8):5017-5023.
[17]梅青刚,袁伟成,王淳.黄酮醇类化合物的合成研究进展[J].有机化学,2015,35(1):70-84.
[18]谭玲玲,蔡霞,胡正海.北柴胡各器官中总黄酮动态变化研究[J].中草药,2008,39(2):286-287.
[19]谭玲玲,蔡霞,胡正海.狭叶柴胡中黄酮类化合物含量及其动态变化研究[J].南方农业学报,2012,43(2):223-226.
[20]梅赞,杨杰,范慧佳,等.4种柴胡地上部分黄酮类成分的含量测定[J].中国新药杂志,2011,20(10):932-935.
[21]段英姿,客绍英.不同引种柴胡不同生长期地上部总黄酮含量比较研究[J].中国药学杂志,2015,50(5):394-398.
[22]杨利民,张永刚,林红梅,等.中药材质量形成理论与控制技术研究进展[J].吉林农业大学学报,2012,34(2):119-124.
[23]孔垂华,徐涛,胡飞,等.环境胁迫下植物的化感作用及其诱导机制[J].生态学报,2000,20(5):849-854.
[2]黄涵签,王潇晗,付航,等.柴胡属药用植物资源研究进展[J].中草药,2017,48(14):2989-2996.
[3]中国药典[S].一部.2015.
[4]Yang Z Y,Chao Z,Huo K K,et al.ITS sequence analysis used for molecular identification of the Bupleurum species from northwestern China[J].Phytomedicine,2007,14(6):416-423.
[5]韦英杰,王茉,宁青,等.HPLC法同时测定柴胡与春柴胡中皂苷类及黄酮类成分的含量[J].药物分析杂志,2011,31(5):879-883.
[6]谭玲玲,侯晓敏,胡正海.狭叶柴胡营养器官中柴胡皂苷和黄酮类化合物的积累部位及含量比较[J].西北植物学报,2014,34(2):276-281.
[7]潘俊倩,佟曦然,郭宝林.光对植物黄酮类化合物的影响研究进展[J].中国中药杂志,2016,41(21):3897-3903.
[8]冯欢,易姝利,左佳琦,等.基因工程及花色素合成途径在花色改良中的研究进展[J].基因组学与应用生物学,2014,33(2):445-451.
[9]汤立建,赵良才,李庆林,等.黄芩黄酮类成分抗肿瘤作用及机制研究进展[J].中国中药杂志,2007,32(1):21-23.
[10]Sui C,Chen M,Xu J,et al.Comparison of root transcriptomes and expressions of genes involved in main medicinal secondary metabolites from Bupleurum chinense and Bupleurum scorzonerifolium,the two Chinese official Radix Bupleuri source species[J].Physiol Plant,2015,153(2):230-242.
[11]Misra P,Pandey A,Tewari S K,et al.Characterization of isoflavone synthase gene from Psoralea corylifolia:Amedicinal plant[J].Plant Cell Rep,2010,29(7):747-755.
[12]Xiong S,Tian N,Long J,et al.Molecular cloning and characterization of a flavanone 3-Hydroxylase gene from Artemisia annua L[J].Plant Physiol Biochem,2016,105:29-36.
[13]Schijlen E G,Ric D V C,van Tunen A J,et al.Modification of flavonoid biosynthesis in crop plants[J].Phytochemistry,2004,65(19):2631-2648.
[14]Dick C A,Buenrostro J,Butler T,et al.Arctic mustard flower color polymorphism controlled by petal-specific downregulation at the threshold of the anthocyanin biosynthetic pathway[J].PLoS One,2011,6(4):e18230.
[15]周亚福,毛少利,李思锋,等.柴胡属5种植物总皂苷和总黄酮含量的测定[J].时珍国医国药,2013,24(5):1241-1243.
[16]Dong L,Sui C,Liu Y,et al.Validation and application of reference genes for quantitative gene expression analyses in various tissues of Bupleurum chinense[J].Mol Biol Rep,2011,38(8):5017-5023.
[17]梅青刚,袁伟成,王淳.黄酮醇类化合物的合成研究进展[J].有机化学,2015,35(1):70-84.
[18]谭玲玲,蔡霞,胡正海.北柴胡各器官中总黄酮动态变化研究[J].中草药,2008,39(2):286-287.
[19]谭玲玲,蔡霞,胡正海.狭叶柴胡中黄酮类化合物含量及其动态变化研究[J].南方农业学报,2012,43(2):223-226.
[20]梅赞,杨杰,范慧佳,等.4种柴胡地上部分黄酮类成分的含量测定[J].中国新药杂志,2011,20(10):932-935.
[21]段英姿,客绍英.不同引种柴胡不同生长期地上部总黄酮含量比较研究[J].中国药学杂志,2015,50(5):394-398.
[22]杨利民,张永刚,林红梅,等.中药材质量形成理论与控制技术研究进展[J].吉林农业大学学报,2012,34(2):119-124.
[23]孔垂华,徐涛,胡飞,等.环境胁迫下植物的化感作用及其诱导机制[J].生态学报,2000,20(5):849-854.