狭叶松果菊组织培养、毛状根诱导及其松果菊苷和绿原酸积累的研究
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摘要
狭叶松果菊(Echinacea．angustifolia DC，也称狭叶紫锥菊，松果菊)，是菊科(Compositae)紫菀族(Aster family)，松果菊属(Echinacea)植物，原产美洲，是印地安人的传统草药。主要活性成分为多糖、多酚、烷基酰胺及咖啡酸衍生物等。具有增强人体免疫力的功效，为近年来欧洲和北美最流行的免疫制剂。松果菊苷是狭叶松果菊中咖啡酸衍生物的代表，是国际市场上狭叶松果菊相关制剂的质量控制标准，研究表明它具有抗衰老、调控细胞凋亡等功效。目前我国北京、沈阳、山东等地已成功引种松果菊，但由于松果菊种子的深度休眠性，严重制约了种苗的快速繁育。并且气候、土壤等条件直接影响引种松果菊活性成分的含量，为此利用生物技术解决无性系的快速繁育和提高松果菊中有用次生代谢产物的积累是发展松果菊产业的重要环节。
本文以狭叶松果菊种子萌发获得的无菌苗为材料通过正交试验的方法建立了狭叶松果菊离体快繁体系；采用发根农杆菌诱导出松果菊毛状根，并采用高效液相色谱(HPLC)对狭叶松果菊组织培养物和毛状根中次生代谢产物绿原酸及松果菊苷进行了含量分析。本文的主要研究内容及结果如下：
1．狭叶松果菊的组织培养和植株再生：以无菌苗叶片及叶柄为材料通过L9(3~4)正交试验筛选出最佳的愈伤组织诱导培养基：MS+6BA0.5mg／L+NAA1.0mg／L；最佳的丛芽诱导培养基：MS+6BA1.0mg／L+NAA0.5mg／L。再生芽经附加IBA0.5mg／L的1／2MS培养基生根后移栽成活获得再生植株。采用HPLC方法分析了狭叶松果菊组培苗各部位中次生代谢产物松果菊苷和绿原酸的含量，结果表明组培苗根中松果菊苷和绿原酸的含量高于地上部分，愈伤组织中含量最低，几乎检测不到松果菊苷的存在。
2．狭叶松果菊的毛状根诱导：利用发根农杆菌A4、R1000诱导得到狭叶松果菊毛状根，建立狭叶松果菊毛状根诱导体系。狭叶松果菊外植体被A4、R1000两种发根农杆菌感染后，20d后，外植体伤口处陆续分化生长出白色的毛状根。PCR实验结果表明，Ri质粒的T-DNA已整合在狭叶松果菊毛状根的基因组中。利用HPLC分别检测了A4-1、A4-2、R1000-1、R1000-2 4个单克隆毛状根培养系中松果菊苷和绿原酸的含量，结果表明不同株系次生代谢物含量差异较大。其中A4-2毛状根培养系中松果菊苷和绿原酸的含量分别达到0.16mg／gDW和0.82mg／gDW，均超过了原植物的0.15mg／gDW和0.68mg／gDW。R1000-2毛状根培养系的绿原酸含量0.94mg／gDW，为原植物的1.4倍。
3．狭叶松果菊根悬浮培养体系的建立：以1／2MS为基本培养液，通过在培养液中添加不同的生长激素，试验结果表明添加了0.5mg／L的IBA根长势最好，优于添加NAA及2，4-D的培养液，后两者培养过程中愈伤化严重。通过在根悬浮培养液中添加不同浓度苯丙氨酸(0，5，10，15，20，25mg／L)的实验表明苯丙氨酸有促进组培苗根生长的作用，其中5mg／L苯丙氨酸促进生根效果最明显。经HPLC检测添加20mg／L的苯丙氨酸最有利于松果菊苷的积累。
Echinacea angustifolia DC (Asteraceae) ,also named narrow-leafed purple coneflower, is native to North American and it is a traditional American indian's herb. The main active compounds of E. spp. are alkamides, polysaccharides,polyphenolic , caffeic acid derivatives and so on. Since they have the effect of immunostimulation, echinacea preparations have been widely used in Europe and North America. Echinacoside is the main caffeic acid derivatives of E. angustifolia. According to the results of recent pharmacopoeial articles, it has many effects such as antimicrobial, cell apoptosis regulation and so on. In recent years this species have been successfully introduced into some areas of China such as Beijing ,Shenyang and Shangdong. Because of dormancy, the seed germination percentage of E.angustifolia is very low, which highly limited the fast regenaretion of this plant. Influenced by climate and soil factors, the content of the active components fluctuates and it is difficult to control the quality of the related medicine. So, biotechniques is an essential way to solve these problems especially to improve the production of bioactive secondary metabolites.
In this research , a technical system for rapid propagation of E. angustifolia was established by Orthogonal Test , using mature seeds of the wild Canada E.angustifolia. The hairy roots inducing system of E.angustifolia was also established by two agropine strains. A method of High Performance Liquid Chromatography(HPLC) was developed for simultaneous determination of chlorogenic acid and echinacoside in the tissue cultures and hairy roots of E.angustifolia. The main research aspects are as follows:
1. Tissue culture and plants regenaretion of E. angustifolia: Efficient plant regeneration was achieved via organogenesis from leaf and stem explant of E. angustifolia. According to the results of Orthogonal Test L9(3~4) Murashige and Skoog(MS)medium supplemented with 6-benzylaminopurine (6-BA)0.5 mg/L and naphthaleneacetic acid (NAA)1.0 mg/L was most effective for callus inducing, while MS medium supplemented with 6-BA 1.0 mg/L and NAA 0.5 mg/L was best for shoots genisis. Plantlets were rooted on 1 / 2MS medium supplemented with different concentrations of indole- 3-butyric acid(IBA) high rooting and survival was achieved when the IBA concentration was 0.5 mg/L. The contents of chlorogenic acid and echinacoside in the callus, roots and upper ground parts of E. angustfolia were analyzed by HPLC respectively .The contents of the two secondary metabolites were both high in the roots ,while in the callus both of them were relatively low, especially echinacoside was hardly detectable .
2. Establishment of hairy root inducing system of E.angustifolia: Axenically grown E.angustifolia plantlets were inoculated with two Agrobacterium rhizogenes strains A4 and R1000. Hairy root lines were established 20 days after inoculation with the two agropine strains. Polymerase chain reaction with primers for the gene rolC confirmed the integration of the T-DNA fragment of Ri plasmid of A.rhizogenes to the genome of hairy roots. The content of echinacoside and chlorogenic acid in four hairy root strains ,A4-1,A4-2,R1000-1and R1000-2 were analyzed by HPLC.The results indicated there are differences in the content of secondary metabolites among the four strains.The content of echinacoside (0.16 mg/g DW)and chlorogenic acid(0.82mg/g DW) in A4-2 are more than the amount in nature roots (echinacoside 0.15 mg/g DW, chlorogenic acid 0.68mg/g DW). The amount of chlorogenic acid in R1000-2 was 0.94 mg/g DW 1.4 times of the content in nature roots(0.68mg/g DW).
3. Establishment of root suspension system for E.angustifolia: The root suspension system was established by supplementing 1 / 2MS liqid medium with different plant hormone. According to the results , adding IBA 0.5 mg/L in the medium was more profitable for root growth than adding 2,-4-D and NAA ,because 2,-4-D and NAA will lead to callus genesis of the suspension root.By adding phenylalanine to the medium for root suspension, we found that 5mg/L phenylalanine benefit for the growing of roots and when the phenylalanine concentration was 20mg/L the highest content of secondary metabolites were detected.
本文以狭叶松果菊种子萌发获得的无菌苗为材料通过正交试验的方法建立了狭叶松果菊离体快繁体系；采用发根农杆菌诱导出松果菊毛状根，并采用高效液相色谱(HPLC)对狭叶松果菊组织培养物和毛状根中次生代谢产物绿原酸及松果菊苷进行了含量分析。本文的主要研究内容及结果如下：
1．狭叶松果菊的组织培养和植株再生：以无菌苗叶片及叶柄为材料通过L9(3~4)正交试验筛选出最佳的愈伤组织诱导培养基：MS+6BA0.5mg／L+NAA1.0mg／L；最佳的丛芽诱导培养基：MS+6BA1.0mg／L+NAA0.5mg／L。再生芽经附加IBA0.5mg／L的1／2MS培养基生根后移栽成活获得再生植株。采用HPLC方法分析了狭叶松果菊组培苗各部位中次生代谢产物松果菊苷和绿原酸的含量，结果表明组培苗根中松果菊苷和绿原酸的含量高于地上部分，愈伤组织中含量最低，几乎检测不到松果菊苷的存在。
2．狭叶松果菊的毛状根诱导：利用发根农杆菌A4、R1000诱导得到狭叶松果菊毛状根，建立狭叶松果菊毛状根诱导体系。狭叶松果菊外植体被A4、R1000两种发根农杆菌感染后，20d后，外植体伤口处陆续分化生长出白色的毛状根。PCR实验结果表明，Ri质粒的T-DNA已整合在狭叶松果菊毛状根的基因组中。利用HPLC分别检测了A4-1、A4-2、R1000-1、R1000-2 4个单克隆毛状根培养系中松果菊苷和绿原酸的含量，结果表明不同株系次生代谢物含量差异较大。其中A4-2毛状根培养系中松果菊苷和绿原酸的含量分别达到0.16mg／gDW和0.82mg／gDW，均超过了原植物的0.15mg／gDW和0.68mg／gDW。R1000-2毛状根培养系的绿原酸含量0.94mg／gDW，为原植物的1.4倍。
3．狭叶松果菊根悬浮培养体系的建立：以1／2MS为基本培养液，通过在培养液中添加不同的生长激素，试验结果表明添加了0.5mg／L的IBA根长势最好，优于添加NAA及2，4-D的培养液，后两者培养过程中愈伤化严重。通过在根悬浮培养液中添加不同浓度苯丙氨酸(0，5，10，15，20，25mg／L)的实验表明苯丙氨酸有促进组培苗根生长的作用，其中5mg／L苯丙氨酸促进生根效果最明显。经HPLC检测添加20mg／L的苯丙氨酸最有利于松果菊苷的积累。
Echinacea angustifolia DC (Asteraceae) ,also named narrow-leafed purple coneflower, is native to North American and it is a traditional American indian's herb. The main active compounds of E. spp. are alkamides, polysaccharides,polyphenolic , caffeic acid derivatives and so on. Since they have the effect of immunostimulation, echinacea preparations have been widely used in Europe and North America. Echinacoside is the main caffeic acid derivatives of E. angustifolia. According to the results of recent pharmacopoeial articles, it has many effects such as antimicrobial, cell apoptosis regulation and so on. In recent years this species have been successfully introduced into some areas of China such as Beijing ,Shenyang and Shangdong. Because of dormancy, the seed germination percentage of E.angustifolia is very low, which highly limited the fast regenaretion of this plant. Influenced by climate and soil factors, the content of the active components fluctuates and it is difficult to control the quality of the related medicine. So, biotechniques is an essential way to solve these problems especially to improve the production of bioactive secondary metabolites.
In this research , a technical system for rapid propagation of E. angustifolia was established by Orthogonal Test , using mature seeds of the wild Canada E.angustifolia. The hairy roots inducing system of E.angustifolia was also established by two agropine strains. A method of High Performance Liquid Chromatography(HPLC) was developed for simultaneous determination of chlorogenic acid and echinacoside in the tissue cultures and hairy roots of E.angustifolia. The main research aspects are as follows:
1. Tissue culture and plants regenaretion of E. angustifolia: Efficient plant regeneration was achieved via organogenesis from leaf and stem explant of E. angustifolia. According to the results of Orthogonal Test L9(3~4) Murashige and Skoog(MS)medium supplemented with 6-benzylaminopurine (6-BA)0.5 mg/L and naphthaleneacetic acid (NAA)1.0 mg/L was most effective for callus inducing, while MS medium supplemented with 6-BA 1.0 mg/L and NAA 0.5 mg/L was best for shoots genisis. Plantlets were rooted on 1 / 2MS medium supplemented with different concentrations of indole- 3-butyric acid(IBA) high rooting and survival was achieved when the IBA concentration was 0.5 mg/L. The contents of chlorogenic acid and echinacoside in the callus, roots and upper ground parts of E. angustfolia were analyzed by HPLC respectively .The contents of the two secondary metabolites were both high in the roots ,while in the callus both of them were relatively low, especially echinacoside was hardly detectable .
2. Establishment of hairy root inducing system of E.angustifolia: Axenically grown E.angustifolia plantlets were inoculated with two Agrobacterium rhizogenes strains A4 and R1000. Hairy root lines were established 20 days after inoculation with the two agropine strains. Polymerase chain reaction with primers for the gene rolC confirmed the integration of the T-DNA fragment of Ri plasmid of A.rhizogenes to the genome of hairy roots. The content of echinacoside and chlorogenic acid in four hairy root strains ,A4-1,A4-2,R1000-1and R1000-2 were analyzed by HPLC.The results indicated there are differences in the content of secondary metabolites among the four strains.The content of echinacoside (0.16 mg/g DW)and chlorogenic acid(0.82mg/g DW) in A4-2 are more than the amount in nature roots (echinacoside 0.15 mg/g DW, chlorogenic acid 0.68mg/g DW). The amount of chlorogenic acid in R1000-2 was 0.94 mg/g DW 1.4 times of the content in nature roots(0.68mg/g DW).
3. Establishment of root suspension system for E.angustifolia: The root suspension system was established by supplementing 1 / 2MS liqid medium with different plant hormone. According to the results , adding IBA 0.5 mg/L in the medium was more profitable for root growth than adding 2,-4-D and NAA ,because 2,-4-D and NAA will lead to callus genesis of the suspension root.By adding phenylalanine to the medium for root suspension, we found that 5mg/L phenylalanine benefit for the growing of roots and when the phenylalanine concentration was 20mg/L the highest content of secondary metabolites were detected.
引文
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