野葛异黄酮类化合物生物合成新途径酶学证据的初步研究
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摘要
阐明植物的次级(生)代谢过程,是利用植物次生代谢产物进行医药工程及天然产物开发研究的前提和基础。与初级代谢相比,次级代谢是一个公认研究难度大,但又是潜力极大的研究领域。而在植物次生代谢的研究中,生物合成途径的研究属于机理研究范畴,是次级代谢研究的核心问题。因此,进行植物黄酮类化合物生物合成途径的研究,不仅可以揭示不同化学成分在植物体中的形成、转化及其相互关系的本质规律;而且对进一步利用现代生物技术方法进行黄酮化合物的生产也有着实际的意义。
野葛[Pueraria lobata(Willd)Ohwi.]分类学上属于豆科植物,是原产于我国的一种重要的野生植物资源,其主要生物活性成分为葛根素、大豆甙和大豆甙元等异黄酮化合物,含量(干重)高达7-8%以上,是大豆异黄酮含量(0.1-0.2%干重)的几十倍。野葛中异黄酮含量高的特点,易于次级代谢研究中产物的分离和检测;野葛悬浮细胞和野葛愈伤组织的成功培养,为开展的次级代谢研究建立了适宜的研究体系并提供了良好的研究基础,是进行植物黄酮类化合物生物合成途径研究的良好试材。
本论文实验以野葛培养细胞和野葛愈伤组织为研究体系,通过对预苯酸脱氢酶和对羟基苯丙酮酸还原酶活性与野葛离体培养体系中异黄酮化合物的生物合成的相关性研究,以及通过苯丙氨酸解氨酶(PAL)和对香豆酸连接酶(4CL)的专一性抑制剂处理实验,研究野葛离体培养体系中异黄酮化合物的生物合成以及异黄酮生物合成中可能的前体化合物预苯酸、对羟基苯丙酮酸和对羟基苯丙乳酸的积累情况,为以预苯酸为前体化合物,经对羟基苯丙酮酸、对羟基苯丙乳酸和香豆酸的中间代谢过程的一条异黄酮化合物生物合成新途径的证实提供酶学证据。实验取得以下主要结果:
在野葛细胞培养周期中,随着预苯酸脱氢酶和对羟基苯丙酮酸还原酶活性的提高,细胞培养体系中异黄酮的积累逐步增加,但异黄酮积累滞后于酶活的提高。通过对预苯酸脱氢酶和对羟基苯丙酮酸还原酶活性变化与野葛异黄酮化合物生物合成的相关性分析,结果表明两者具有较高的相关性,相关系数分别为0.8812和0.8046。
以野葛愈伤组织为试验材料,分别对PAL酶活性测定条件以及TTC法测定细胞活力条件进行了研究,结果表明,PAL的最适底物浓度为2mM,最适pH值为8.8,最适反应温度为60℃,酶促反应产物可在2.5h内保持稳定。细胞活力测定的最适TTC浓度为0.6%(w/v),最适pH值为7.5,TTC溶液处理的适宜时间范围为12～16h。
通过不同浓度的PAL专一性抑制剂(AOA)和4CL专一性抑制剂(MDCA)的处理实验,研究野葛愈伤组织的鲜重、干重、酶活、细胞活力和异黄酮含量的变化情况,得出AOA处理的适宜浓度为10μM,MDCA处理的适宜浓度为25μM。
选取10μM为AOA处理的适宜浓度,设计了三种不同的AOA处理,即①培养0天的野葛愈伤组织、②培养24天(无AOA处理)以及③培养24天(10μM AOA处理),分别收集样品,进行异黄酮含量的分析测定,结果显示,处理③较处理①异黄酮含量高出6.141mg.g~(-1),且有显著差异;而处理②较处理③高出18.197mg.g~(-1),两者间具有极显著差异;研究表明野葛愈伤组织中可能存在其他异黄酮合成途径。同时,异黄酮含量(处理③-处理①)与异黄酮含量(处理②-处理③)的差异,则反映了两条不同途径在野葛异黄酮生物合成中的贡献大小,即通过苯丙氨酸-肉桂酸-香豆酸-香豆酰辅酶A-查耳酮的异黄酮生物合成途径为野葛愈伤黄酮生物合成的主要途径,而本项目研究的可能存在的异黄酮生物合成途径为野葛愈伤黄酮合成的辅助途径。其外,10μMAOA处理条件下,野葛愈伤组织的预苯酸、对羟基苯丙酮酸和对羟基苯丙乳酸的含量都较对照的含量高,且差异极显著;表明AOA处理抑制了PAL酶活性以及异黄酮的生成,但具体机理不详。
培养第24天(25μM MDCA处理)野葛愈伤组织样品的异黄酮含量较培养0天的野葛愈伤组织样品中异黄酮含量高出2.217mg.g~(-1),但两者差异不显著。同时,培养第24天的野葛愈伤组织(无MDCA处理)样品较培养第24天的野葛愈伤组织(25μM MDCA处理)样品高出22.251mg.g~(-1),且两者具有极显著差异。表明4CL酶是异黄酮合成的关键酶,专一性抑制剂MDCA处理严重阻断了野葛愈伤体内异黄酮的生物合成。其外,25μM MDCA处理条件下,野葛愈伤组织的预苯酸含量都较对照的含量高,但差异不显著;而对羟基苯丙酮酸和对羟基苯丙乳酸含量都较对照的含量高,且差异极显著。同样表明MDCA阻断了香豆酸-香豆酰辅酶A的反应,进而阻断了黄酮化合物生物合成的途径,降低了异黄酮化合物的积累;并进一步通过反馈抑制作用,使得预苯酸、对羟基苯丙酮酸和对羟基苯丙乳酸含量都较对照有所增加。该研究结果也进一步证实了预苯酸、对羟基苯丙酮酸和对羟基苯丙乳酸为异黄酮生物合成的可能前体化合物。
To illustrate the plant secondary metabolism is a principal and basic work which is helpful for utilizing plant secondary metabolites to improve medicine production and push the research work about natural products.Compared with the primary metabolism,the secondary metabolism is very difficult to be recognized,but at the same time it is also an important research area with great potential.The research work about biosynthesis pathway is regarded as that in the area of mechanisms in the study of plant secondary metabolism,and it is also the core of the secondary metabolism.Thus,it is of great significance to study the biosynthesis pathway of plant flavonoids,not only for revealing the chemical composition of different plant in the formation,transformation and the relationship of different chemicals,but also in the production of flavonoids using modern biotechnique methods.
Kudzu[Pueraria lobata(Willd) Ohwi.]originating in China is a perennial leguminous plant and is also an important wild plant resource.The main components of biological activity are puerarin,daidzin and daidzein.Isoflavonoid Content(dry weight) is high up to 7-8 percent,is several times that of soybean isoflavonoids(0.1 to 0.2 percent dry weight),which is helpful for studying on the separation and detection of secondary metabolism.The establishment of kudzu cell suspension culture and kudzu callus provides a good research basis and the research system for secondary metabolism,and can be used as the good materials for illustrating the flavonoid biosynthesis pathway in plants.
In this paper,kudzu cell suspension cultures and callus cultures are selected as the materials,the correlation was examined between the activities of prephenate dehydrogenase and p-hydroxyphenylpyruvate reductase and the accumulation of isoflavonoids in kudzu in vitro,as well as the correlation of the isoflavone biosynthesis and the accumulation of the possible precursor compounds such as prephenate, p-hydroxyphenylpyruvate and p-hydroxyphenyllactate in kudzu in vitro is examined in the experiment about the PAL specific inhibitor and the 4CL specific inhibitor,which provide the enzymological evidences for verifying the novel possible pathway of isoflavone biosynthesis,and confirming the existence of the precursor compounds such as prephenate, p-hydroxyphenylpyruvate,p-hydroxyphenyllactate and p-coumaric acid during the production of kudzu isoflavonoids.The main results are as following:
With the increase of the activities of prephenate dehydrogenase and p-hydroxyphenylpyruvate reductase in the life cycle of kudzu cell culture,the accumulation of isoflavonoids increased gradually,and was examined several days later of the increase of enzyme activities.The correlations between the activities of prephenate dehydrogenase and p-hydroxyphenylpyruvate reductase and the accumulation of kudzu isoflavonoids are high,and the coefficients are 0.8812 and 0.8046,respectively.
The assay for the PAL activity and the TTC viability in kudzu callus were examined, the results showed that the optimum concentration of L-Phe is 2mM,the optimum pH value is 8.8,the optimum temperature is 60℃,and the reaction products of PAL could maintain stability within 2.5h.The optimum TTC concentration for kudzu viability is 0.6% (w/v),the optimum pH value is 7.5,and the appropriate time range is12～16h.
In the experiments using different concentrations of PAL-specific inhibitor(AOA) and 4CL-specific inhibitor(MDCA),the changes of the fresh weight,dry weight,the PAL activity,kudzu viability and the isoflavonoid content in kudzu callus were examined,the results showed that the optimal concentrations of AOA and MDCA were 10μM and 25μM,respectively.
Three different treatments were performed with the AOA concentration of 10μM,and were as followings:①kudzu callus sample after 0 day without AOA treatment,②kudzu callus sample after 24 day without AOA treatment,and③kudzu callus sample after 24 day with 10μM AOA treatment.Samples were collected for the analysis of the isoflavonoid contents.The results showed that the isoflavonoid content in the third sample was 6.141 mg.g~(-1) higher than that in the first sample,and showed the significant difference (P=0.05).The isoflavonoid content in the second sample was 18.197 mg.g~(-1) higher than that in the third sample,and also showed the significant difference(P=0.01).The above data suggested that the other pathway of isoflavone biosynthesis might exist in kudzu callus.At the same time,the difference between the isoflavonoid content(③-①) and the isoflavonoid content(②-③) reflected the contributions of two different pathways to the isoflavone biosynthesis in kudzu callus.The main pathway of flavonoids biosynthesis in kudzu callus was from phenylalanine,through cinnamate,p-coumaric acid and p-coumarate:coenzyme A to chalcone,and the novel possible pathway of isoflavone biosynthesis may be the auxiliary pathway in kudzu callus.Under the condition of 10μM AOA treatment,the contents of prephenate,p-hydroxyphenylpyruvate and p-hydroxyphenyllactate in kudzu callus were higher than the control,and showed the significant difference(P=0.01).The results show that AOA treatment inhibited the PAL activity,but the specific mechanism is unknown.
The isoflavonoid content in kudzu callus sample with 25μM MDCA treatment after 24 days was 2.217 mg.g~(-1) higher than that in the sample after 0 day,but showed no significant difference.At the same time,the isoflavonoid content in kudzu callus sample after 24 days without MDCA treatment was 22.251mg.g~(-1) higher than that after 24 days with 25μM MDCA treatment,and showed the significant difference(P=0.01).From which 4CL was the key enzyme of isoflavonoid biosynthesis,and specific inhibitor MDCA seriously blocked the isoflavone biosynthesis in kudzu Callus.Furthermore,the content of prephenate in kudzu callus with 25μM MDCA treatment was much higher than that in control,but showed no significant difference,and the contents of p-hydroxyphenylpyruvate and p-hydroxyphenyllactate were higher than control,and showed the significant difference(P=0.01).The results also showed that MDCA blocked the reaction from p-coumaric acid to p-coumarate:coenzyme A,and blocked the pathway of isoflavonoid biosynthesi,and reduced the accumulation of isoflavonoids,which further increased the content of prephenate,p-hydroxyphenylpyruvate and p-hydroxyphenyllactate in kudzu callus through feedback inhibition.The comparing results also further confirmed prephenate,p-hydroxyphenylpyruvate and p-hydroxyphenyllactate might be the precursors of isoflavone biosynthesis.
野葛[Pueraria lobata(Willd)Ohwi.]分类学上属于豆科植物,是原产于我国的一种重要的野生植物资源,其主要生物活性成分为葛根素、大豆甙和大豆甙元等异黄酮化合物,含量(干重)高达7-8%以上,是大豆异黄酮含量(0.1-0.2%干重)的几十倍。野葛中异黄酮含量高的特点,易于次级代谢研究中产物的分离和检测;野葛悬浮细胞和野葛愈伤组织的成功培养,为开展的次级代谢研究建立了适宜的研究体系并提供了良好的研究基础,是进行植物黄酮类化合物生物合成途径研究的良好试材。
本论文实验以野葛培养细胞和野葛愈伤组织为研究体系,通过对预苯酸脱氢酶和对羟基苯丙酮酸还原酶活性与野葛离体培养体系中异黄酮化合物的生物合成的相关性研究,以及通过苯丙氨酸解氨酶(PAL)和对香豆酸连接酶(4CL)的专一性抑制剂处理实验,研究野葛离体培养体系中异黄酮化合物的生物合成以及异黄酮生物合成中可能的前体化合物预苯酸、对羟基苯丙酮酸和对羟基苯丙乳酸的积累情况,为以预苯酸为前体化合物,经对羟基苯丙酮酸、对羟基苯丙乳酸和香豆酸的中间代谢过程的一条异黄酮化合物生物合成新途径的证实提供酶学证据。实验取得以下主要结果:
在野葛细胞培养周期中,随着预苯酸脱氢酶和对羟基苯丙酮酸还原酶活性的提高,细胞培养体系中异黄酮的积累逐步增加,但异黄酮积累滞后于酶活的提高。通过对预苯酸脱氢酶和对羟基苯丙酮酸还原酶活性变化与野葛异黄酮化合物生物合成的相关性分析,结果表明两者具有较高的相关性,相关系数分别为0.8812和0.8046。
以野葛愈伤组织为试验材料,分别对PAL酶活性测定条件以及TTC法测定细胞活力条件进行了研究,结果表明,PAL的最适底物浓度为2mM,最适pH值为8.8,最适反应温度为60℃,酶促反应产物可在2.5h内保持稳定。细胞活力测定的最适TTC浓度为0.6%(w/v),最适pH值为7.5,TTC溶液处理的适宜时间范围为12～16h。
通过不同浓度的PAL专一性抑制剂(AOA)和4CL专一性抑制剂(MDCA)的处理实验,研究野葛愈伤组织的鲜重、干重、酶活、细胞活力和异黄酮含量的变化情况,得出AOA处理的适宜浓度为10μM,MDCA处理的适宜浓度为25μM。
选取10μM为AOA处理的适宜浓度,设计了三种不同的AOA处理,即①培养0天的野葛愈伤组织、②培养24天(无AOA处理)以及③培养24天(10μM AOA处理),分别收集样品,进行异黄酮含量的分析测定,结果显示,处理③较处理①异黄酮含量高出6.141mg.g~(-1),且有显著差异;而处理②较处理③高出18.197mg.g~(-1),两者间具有极显著差异;研究表明野葛愈伤组织中可能存在其他异黄酮合成途径。同时,异黄酮含量(处理③-处理①)与异黄酮含量(处理②-处理③)的差异,则反映了两条不同途径在野葛异黄酮生物合成中的贡献大小,即通过苯丙氨酸-肉桂酸-香豆酸-香豆酰辅酶A-查耳酮的异黄酮生物合成途径为野葛愈伤黄酮生物合成的主要途径,而本项目研究的可能存在的异黄酮生物合成途径为野葛愈伤黄酮合成的辅助途径。其外,10μMAOA处理条件下,野葛愈伤组织的预苯酸、对羟基苯丙酮酸和对羟基苯丙乳酸的含量都较对照的含量高,且差异极显著;表明AOA处理抑制了PAL酶活性以及异黄酮的生成,但具体机理不详。
培养第24天(25μM MDCA处理)野葛愈伤组织样品的异黄酮含量较培养0天的野葛愈伤组织样品中异黄酮含量高出2.217mg.g~(-1),但两者差异不显著。同时,培养第24天的野葛愈伤组织(无MDCA处理)样品较培养第24天的野葛愈伤组织(25μM MDCA处理)样品高出22.251mg.g~(-1),且两者具有极显著差异。表明4CL酶是异黄酮合成的关键酶,专一性抑制剂MDCA处理严重阻断了野葛愈伤体内异黄酮的生物合成。其外,25μM MDCA处理条件下,野葛愈伤组织的预苯酸含量都较对照的含量高,但差异不显著;而对羟基苯丙酮酸和对羟基苯丙乳酸含量都较对照的含量高,且差异极显著。同样表明MDCA阻断了香豆酸-香豆酰辅酶A的反应,进而阻断了黄酮化合物生物合成的途径,降低了异黄酮化合物的积累;并进一步通过反馈抑制作用,使得预苯酸、对羟基苯丙酮酸和对羟基苯丙乳酸含量都较对照有所增加。该研究结果也进一步证实了预苯酸、对羟基苯丙酮酸和对羟基苯丙乳酸为异黄酮生物合成的可能前体化合物。
To illustrate the plant secondary metabolism is a principal and basic work which is helpful for utilizing plant secondary metabolites to improve medicine production and push the research work about natural products.Compared with the primary metabolism,the secondary metabolism is very difficult to be recognized,but at the same time it is also an important research area with great potential.The research work about biosynthesis pathway is regarded as that in the area of mechanisms in the study of plant secondary metabolism,and it is also the core of the secondary metabolism.Thus,it is of great significance to study the biosynthesis pathway of plant flavonoids,not only for revealing the chemical composition of different plant in the formation,transformation and the relationship of different chemicals,but also in the production of flavonoids using modern biotechnique methods.
Kudzu[Pueraria lobata(Willd) Ohwi.]originating in China is a perennial leguminous plant and is also an important wild plant resource.The main components of biological activity are puerarin,daidzin and daidzein.Isoflavonoid Content(dry weight) is high up to 7-8 percent,is several times that of soybean isoflavonoids(0.1 to 0.2 percent dry weight),which is helpful for studying on the separation and detection of secondary metabolism.The establishment of kudzu cell suspension culture and kudzu callus provides a good research basis and the research system for secondary metabolism,and can be used as the good materials for illustrating the flavonoid biosynthesis pathway in plants.
In this paper,kudzu cell suspension cultures and callus cultures are selected as the materials,the correlation was examined between the activities of prephenate dehydrogenase and p-hydroxyphenylpyruvate reductase and the accumulation of isoflavonoids in kudzu in vitro,as well as the correlation of the isoflavone biosynthesis and the accumulation of the possible precursor compounds such as prephenate, p-hydroxyphenylpyruvate and p-hydroxyphenyllactate in kudzu in vitro is examined in the experiment about the PAL specific inhibitor and the 4CL specific inhibitor,which provide the enzymological evidences for verifying the novel possible pathway of isoflavone biosynthesis,and confirming the existence of the precursor compounds such as prephenate, p-hydroxyphenylpyruvate,p-hydroxyphenyllactate and p-coumaric acid during the production of kudzu isoflavonoids.The main results are as following:
With the increase of the activities of prephenate dehydrogenase and p-hydroxyphenylpyruvate reductase in the life cycle of kudzu cell culture,the accumulation of isoflavonoids increased gradually,and was examined several days later of the increase of enzyme activities.The correlations between the activities of prephenate dehydrogenase and p-hydroxyphenylpyruvate reductase and the accumulation of kudzu isoflavonoids are high,and the coefficients are 0.8812 and 0.8046,respectively.
The assay for the PAL activity and the TTC viability in kudzu callus were examined, the results showed that the optimum concentration of L-Phe is 2mM,the optimum pH value is 8.8,the optimum temperature is 60℃,and the reaction products of PAL could maintain stability within 2.5h.The optimum TTC concentration for kudzu viability is 0.6% (w/v),the optimum pH value is 7.5,and the appropriate time range is12～16h.
In the experiments using different concentrations of PAL-specific inhibitor(AOA) and 4CL-specific inhibitor(MDCA),the changes of the fresh weight,dry weight,the PAL activity,kudzu viability and the isoflavonoid content in kudzu callus were examined,the results showed that the optimal concentrations of AOA and MDCA were 10μM and 25μM,respectively.
Three different treatments were performed with the AOA concentration of 10μM,and were as followings:①kudzu callus sample after 0 day without AOA treatment,②kudzu callus sample after 24 day without AOA treatment,and③kudzu callus sample after 24 day with 10μM AOA treatment.Samples were collected for the analysis of the isoflavonoid contents.The results showed that the isoflavonoid content in the third sample was 6.141 mg.g~(-1) higher than that in the first sample,and showed the significant difference (P=0.05).The isoflavonoid content in the second sample was 18.197 mg.g~(-1) higher than that in the third sample,and also showed the significant difference(P=0.01).The above data suggested that the other pathway of isoflavone biosynthesis might exist in kudzu callus.At the same time,the difference between the isoflavonoid content(③-①) and the isoflavonoid content(②-③) reflected the contributions of two different pathways to the isoflavone biosynthesis in kudzu callus.The main pathway of flavonoids biosynthesis in kudzu callus was from phenylalanine,through cinnamate,p-coumaric acid and p-coumarate:coenzyme A to chalcone,and the novel possible pathway of isoflavone biosynthesis may be the auxiliary pathway in kudzu callus.Under the condition of 10μM AOA treatment,the contents of prephenate,p-hydroxyphenylpyruvate and p-hydroxyphenyllactate in kudzu callus were higher than the control,and showed the significant difference(P=0.01).The results show that AOA treatment inhibited the PAL activity,but the specific mechanism is unknown.
The isoflavonoid content in kudzu callus sample with 25μM MDCA treatment after 24 days was 2.217 mg.g~(-1) higher than that in the sample after 0 day,but showed no significant difference.At the same time,the isoflavonoid content in kudzu callus sample after 24 days without MDCA treatment was 22.251mg.g~(-1) higher than that after 24 days with 25μM MDCA treatment,and showed the significant difference(P=0.01).From which 4CL was the key enzyme of isoflavonoid biosynthesis,and specific inhibitor MDCA seriously blocked the isoflavone biosynthesis in kudzu Callus.Furthermore,the content of prephenate in kudzu callus with 25μM MDCA treatment was much higher than that in control,but showed no significant difference,and the contents of p-hydroxyphenylpyruvate and p-hydroxyphenyllactate were higher than control,and showed the significant difference(P=0.01).The results also showed that MDCA blocked the reaction from p-coumaric acid to p-coumarate:coenzyme A,and blocked the pathway of isoflavonoid biosynthesi,and reduced the accumulation of isoflavonoids,which further increased the content of prephenate,p-hydroxyphenylpyruvate and p-hydroxyphenyllactate in kudzu callus through feedback inhibition.The comparing results also further confirmed prephenate,p-hydroxyphenylpyruvate and p-hydroxyphenyllactate might be the precursors of isoflavone biosynthesis.
引文
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