三种药用甘草遗传多样性和次生代谢物含量研究
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摘要
甘草是我国西北部荒漠半荒漠生态环境的重要固沙植物，为豆科（Fabaceae）甘草属多年生植物。《中国药典》（2010版）规定的药用甘草为甘草(GlycyrrhizauralensisFisch.)、胀果甘草（GlycyrrhizainflateBat.）和光果甘草（GlycyrrhizaglabraL.），这3种甘草是我国药用甘草的主要来源，除药用外，还广泛应用于食品、烟草、化妆品等行业。近年来，由于国内外甘草市场需求量猛增，野生资源的过度采挖不仅导致甘草药材产量和质量的急剧下降，同时也加速了我国北方荒漠化的扩展，造成了严重生态灾难。另一方面，甘草属植物的地理分布跨度大，形态变异十分显著，药材质量差异也很大。迄今为止，有关栽培甘草属植物甘草、胀果甘草、光果甘草在驯化过程中的植物外形变异、遗传多样性、居群结构与药材质量的相关性未见系统研究报道。开展本课题的研究具有重要的科学意义和应用价值。
本文对2007年引种到甘肃民勤4年生的宁夏盐池的甘草、新疆喀什的胀果甘草和光果甘草分别进行了植物外形鉴别、有效成分次生代谢物含量测定、遗传多样性差异的系统研究，建立了化学指纹图谱与分子指纹图谱，用两个相关联的图谱比较在同一生境同一生长年限不同种源的三种药用甘草的种源内和种源间的差异，主要研究结果如下：
12010年5月份到10月份，对不同生长时期三种药用甘草植物的外形从植株的株高、茎粗、叶片形态、根及根茎、果穗、果荚、种子等形态指标进行测定，结果显示：此三种药用甘草与《中国植物志》及《中国药典》描述相比变异程度较小，除胀果甘草叶子边缘产生了大幅度波浪式的蜷曲，光果甘草株高大幅度增高，平均高度在160cm以上，成“巨株”，而甘草果荚的腺毛变得更为粗大。通过上述研究结果显示：三种药用甘草在驯化过程中为适应生存环境导致某些表型发生变异。
2通过对三种药用药用部位每月进行有效成分次生代谢物甘草苷、甘草酸含量的测定，结果显示：5、6月份3种甘草的甘草苷和甘草酸含量均不达国家药典规定标准，但是从7月份开始含量一直上升，到9、10月份时含量达标，可进入药材市场。
3利用ISSR分子标记技术对三种药用甘草遗传多样性进行了分析，并利用筛选出的5条ISSR引物进行扩增，从三种药用甘草60份样品中共扩增出886条条带，其中多态带数498条，多态带百分率为56.19%。遗传多样性分析结果显示，同种胀果甘草的20份样品遗传多样性指数（h）和Shannon’sInformation指数（I）分别为0.25和0.37；而光果甘草分别为0.23和0.34；甘草最低为0.19和0.28。AMOVA分析结果显示：同种甘草不同植株间有37%的变异，而种间变异为63%。依据Nei遗传距离对三种药用甘草分别进行UPGMA聚类，结果显示甘草和光果甘草的遗传关系较近，聚为一组，而胀果甘草另成一组，与其它2种甘草遗传关系较远。4通过使用中药色谱指纹图谱相似度评价系统A版软件对三种药用甘草色谱含量
建立化学指纹图谱，通过图谱比较甘草苷和甘草酸两个有效特征峰，结果显示，甘草和光果甘草的甘草苷峰高相似，均是胀果甘草的1.1倍，具有明显差异，与上述遗传多样性研究结果相吻合。
本文的研究特色和创新点
（1）首次对不同种源同一生境同一生长年限引种4年的三种药用甘草进行植物形态学研究，从植物的株高、茎粗、叶片形态、根及根茎、果穗、果荚、种子这几方面进行比较，发现三种药用甘草植物形态变异与生境改变和自身表现性状改变均有影响。这一研究结果表明栽培甘草所选用的种子为优良种，含有丰富的杂合体基因可有效预防在栽培驯化过程中出现的同种种源内植物的退化现象。
（2）首次应用ISSR技术对不同种源同一生境同一生长年限引种4年的三种药用甘草的遗传多样性进行了系统研究，发现该实验材料的遗传多样性水平依然很高，提示尽管野生甘草采挖过度，但只要合理引种驯化栽培还不至于导致该种群遗传多样性的丧失而灭绝，这一研究结果为甘草资源的保护和抚育，以及我国北方荒漠地区的生态保护、恢复和重建，提供了科学依据。
（3）首次应用化学指纹图谱和分子指纹图谱相联系的方式对三种药用甘草进行遗传多样性差异与有效成分化学含量测定系统研究，发现胀果甘草与甘草具有较近的遗传关系，而与光果甘草遗传关系较远。揭示了遗传多样性差异与产地环境，以及药材质量有关，为甘草药材质量的评价及优良种的选育提供了科学依据。
Licorice belongs to the genus Glycyrrhiza of the family Leguminosae. Glycyrrhiza uralensis Fisch., Glycyrrhiza inflate Bat.and Glycyrrhiza glabra L.are three kinds Statutory Glycyrrhiza. They are perennial herbs and mainly distributed in the northern China. Their roots, have been used as a traditional drug for at least2000years in China. In addition, it contains glycyrrhizin, a non-sugar sweetener, and thus is widely used in the manufacture of food, tobacco, cosmetic, etc. For these reasons, the market demands for Glycyrrhiza production at home and abroad were expanded enormously in recent years. Thus, overcollection has resulted in a rapid decline of the wild resources of Glycyrrhiza and caused a severe ecological disaster in North China. Therefore, our research in this thesis is of great scientific and applied values.
In this paper, introduced in2007to four years in Minqin Gansu, Ningxia Yanchi of Glycyrrhiza uralensis, Glycyrrhiza inflate and Glycyrrhiza glabra of Kashi in Xinjiang were carried out to identify plant form, the active ingredient content of secondary metabolites determination, the systematic study of differences in genetic diversity, established a chemical fingerprint and molecular fingerprint patterns associated with the two systems compared in the same habitat the same period the growth of three different provenances of cultivated Glycyrrhiza populations within populations and the differences between the changes, The main findings are as follows:cultivated Glycyrrhiza Liquiritin and Glycyrrhizic acid
1Starts to the different vegetal period three kinds of cultivated Glycyrrhiza plant's contour from adult plant's high, the stem is thick, shape targets and so on leaf blade shape, root and rhizome, cob, fruit pod, seed carry on the determination, and "the Chinese Pharmacopoeia" describes with "the Chinese Flora" compares the variation degree to be small, the result is as follows, The Glycyrrhiza inflate leaf edge has produced large scale wave form curling up; Starts from the mature period, Glycyrrhiza glabra average height above160cm, becomes large scale "great"; The Glycyrrhiza uralensis strawberry pod's glandular hair becomes thicker. Through the above findings demonstrated that three kinds of cultivated Glycyrrhiza can have the compatible variation in the domesticated process to the external environment, three kinds of cultivated Glycyrrhiza seeds have the many heterozygous body genes, enables oneself to have the compatible change to the environment, observes from the phenotype to the varying degree variation situation. Simultaneously also explains this introduction the wild Glycyrrhiza for the fine mother generation, and the domesticated cultivation's habitat choice is also reasonable.
2This experiment to cultivated Glycyrrhiza raw material for medicine quality rating standard is according to2010editions "the Chinese Pharmacopoeia" stipulated that the Liquiritin, the glycyrrhizic acid content do not be lower than0.50%and2.0%, carries on the effective component secondary metabolite Liquiritin, the glycyrrhizic acid content determination every month to three kinds of cultivated Glycyrrhiza for medicinal purposes spot, finally demonstrated that in May and June determines the cultivated Glycyrrhiza Liquiritin and the glycyrrhizic acid content cannot achieve the standard, but starts the content from July to rise, to September and October when the content achieves the national pharmacopoeia to stipulate, may enter raw material for medicine market. The research indicated that the cultivated Glycyrrhiza is stable in four year domesticated process traditional Chinese medicine quality content, for next step promoted the large-scale domesticated cultivated Glycyrrhiza to provide the reasonable theory basis.
3Carries on the DNA genome team to three kinds of cultivated Glycyrrhiza young tender leaf blade the extraction, has carried on the analysis using the ISSR member mark method to its heredity multiplicity. Using5ISSR which screens directs the thing, occupies the group60sample Chinese Communist Party from3to increase886bandings, polymorphism belt several498, the polymorphism belt percentage is56.19%. Occupies the group heredity multiplicity analysis result to demonstrate, the Glycyrrhiza inflate in occupies in the group level, the total heredity multiple indices (h) and the Shannon index (I) respectively is0.25and0.37; But the Glycyrrhiza glabra respectively is0.23and0.34; The most Glycyrrhiza uralensis is low is0.19and0.28. The AMOVA analysis result showed:Three kinds of cultivated Glycyrrhiza have37%hereditary change to occur in occupy in the group,63%hereditary change occur in occupies the group. It's a way from based on the Nei heredity to three kinds of cultivated Glycyrrhiza carries on the UPGMA cluster separately, finally demonstrated that the Glycyrrhiza uralensis the heredity similarity is smallest, it has the rich genetic background, adaptation environment selection pressure ability some. Occupies three groups to carry on again the UPGMA cluster, the Glycyrrhiza inflate and the Glycyrrhiza glabra gathers a group to constitute separately with the two branches, the result demonstration Glycyrrhiza uralensisand the Glycyrrhiza glabra genetic affinity are near, it is far with the Glycyrrhiza inflate genetic affinity. This experimental result showed that three kinds of cultivated Glycyrrhiza are still retaining the high heredity multiple levels, was its idioplasm resources preservation, the protection and the nurture has provided the scientific basis.
4Through use the traditional Chinese medicine chromatograph fingerprint atlas similarity evaluation system. A version software to three kinds of cultivated Glycyrrhiza chromatograph content establishment chemistry fingerprint atlas, through the atlas comparison Liquiritin and the glycyrrhizic acid two effective characteristic peaks, demonstrated finally that Glycyrrhiza uralensis and Glycyrrhiza glabra Liquiritin peaks high similar, is Glycyrrhiza inflate1.1times, has the obvious difference, tallies with the above heredity multiplicity findings. Indicated that several testing methods unify and the verification experimental method mutually may to test a more comprehensive reasonable explanation, enables experiment's reliability, the feasibility to obtain the effective guarantee, also provides effectively, the reasonable theory basis for the cultivated Glycyrrhiza big area manual cultivation domestication.
This paper research characteristic and innovation spots:
(1) Introduces a fine variety to the different seedling source identical habitat identical growth age limit four year three kinds of cultivated Glycyrrhiza to conduct the plant-morphology research for the first time, from plant's high, the stem is thick, the leaf blade shape, the root and the rhizome, the cob, the fruit pod, seed these aspects carry on the comparison, discovered that three kinds of cultivated Glycyrrhiza plant morphological change and the habitat change and own performance character change is influential. This finding indicated the cultivated Glycyrrhiza selects the seed is the improved seed, includes the rich heterozygous body gene to be possible to prevent effectively in the cultivation domestication process appears occupies in the group plant's degenerated phenomenon.
(2) Introduced a fine variety using the ISSR technology to the different seedling source identical habitat identical growth age limit four year three kinds of cultivated Glycyrrhiza to occupy the group heredity multiplicity for the first time to conduct the system research, discovered that should occupy the group heredity multiple levels to be very still high, prompts the wild Glycyrrhiza to excavate freely excessively, so long as but introduced a fine variety the domesticated cultivation as for not to cause this population heredity reasonably multiply to lose exterminates, this findings for the Glycyrrhiza resources'protection and the nurture, as well as north part of our country wilderness area's ecological protection, restored and reconstruct, have provided the scientific basis.
(3) For the first time the applied chemistry fingerprint atlas and the molecular fingerprint atlas relate the way carries on the heredity multiple differences and the effective component chemistry content determination system research to three kinds of cultivated Glycyrrhiza, discovered that between Glycyrrhiza inflate and Glycyrrhiza uralensis Glycyrrhiza glabra the two difference is remarkable, and has promulgated the heredity multiple differences and the habitat environment, as well as raw material for medicine quality's relevance, has provided the scientific basis for the Glycyrrhiza raw material for medicine quality's appraisal and improved seed's selective breeding.
本文对2007年引种到甘肃民勤4年生的宁夏盐池的甘草、新疆喀什的胀果甘草和光果甘草分别进行了植物外形鉴别、有效成分次生代谢物含量测定、遗传多样性差异的系统研究，建立了化学指纹图谱与分子指纹图谱，用两个相关联的图谱比较在同一生境同一生长年限不同种源的三种药用甘草的种源内和种源间的差异，主要研究结果如下：
12010年5月份到10月份，对不同生长时期三种药用甘草植物的外形从植株的株高、茎粗、叶片形态、根及根茎、果穗、果荚、种子等形态指标进行测定，结果显示：此三种药用甘草与《中国植物志》及《中国药典》描述相比变异程度较小，除胀果甘草叶子边缘产生了大幅度波浪式的蜷曲，光果甘草株高大幅度增高，平均高度在160cm以上，成“巨株”，而甘草果荚的腺毛变得更为粗大。通过上述研究结果显示：三种药用甘草在驯化过程中为适应生存环境导致某些表型发生变异。
2通过对三种药用药用部位每月进行有效成分次生代谢物甘草苷、甘草酸含量的测定，结果显示：5、6月份3种甘草的甘草苷和甘草酸含量均不达国家药典规定标准，但是从7月份开始含量一直上升，到9、10月份时含量达标，可进入药材市场。
3利用ISSR分子标记技术对三种药用甘草遗传多样性进行了分析，并利用筛选出的5条ISSR引物进行扩增，从三种药用甘草60份样品中共扩增出886条条带，其中多态带数498条，多态带百分率为56.19%。遗传多样性分析结果显示，同种胀果甘草的20份样品遗传多样性指数（h）和Shannon’sInformation指数（I）分别为0.25和0.37；而光果甘草分别为0.23和0.34；甘草最低为0.19和0.28。AMOVA分析结果显示：同种甘草不同植株间有37%的变异，而种间变异为63%。依据Nei遗传距离对三种药用甘草分别进行UPGMA聚类，结果显示甘草和光果甘草的遗传关系较近，聚为一组，而胀果甘草另成一组，与其它2种甘草遗传关系较远。4通过使用中药色谱指纹图谱相似度评价系统A版软件对三种药用甘草色谱含量
建立化学指纹图谱，通过图谱比较甘草苷和甘草酸两个有效特征峰，结果显示，甘草和光果甘草的甘草苷峰高相似，均是胀果甘草的1.1倍，具有明显差异，与上述遗传多样性研究结果相吻合。
本文的研究特色和创新点
（1）首次对不同种源同一生境同一生长年限引种4年的三种药用甘草进行植物形态学研究，从植物的株高、茎粗、叶片形态、根及根茎、果穗、果荚、种子这几方面进行比较，发现三种药用甘草植物形态变异与生境改变和自身表现性状改变均有影响。这一研究结果表明栽培甘草所选用的种子为优良种，含有丰富的杂合体基因可有效预防在栽培驯化过程中出现的同种种源内植物的退化现象。
（2）首次应用ISSR技术对不同种源同一生境同一生长年限引种4年的三种药用甘草的遗传多样性进行了系统研究，发现该实验材料的遗传多样性水平依然很高，提示尽管野生甘草采挖过度，但只要合理引种驯化栽培还不至于导致该种群遗传多样性的丧失而灭绝，这一研究结果为甘草资源的保护和抚育，以及我国北方荒漠地区的生态保护、恢复和重建，提供了科学依据。
（3）首次应用化学指纹图谱和分子指纹图谱相联系的方式对三种药用甘草进行遗传多样性差异与有效成分化学含量测定系统研究，发现胀果甘草与甘草具有较近的遗传关系，而与光果甘草遗传关系较远。揭示了遗传多样性差异与产地环境，以及药材质量有关，为甘草药材质量的评价及优良种的选育提供了科学依据。
Licorice belongs to the genus Glycyrrhiza of the family Leguminosae. Glycyrrhiza uralensis Fisch., Glycyrrhiza inflate Bat.and Glycyrrhiza glabra L.are three kinds Statutory Glycyrrhiza. They are perennial herbs and mainly distributed in the northern China. Their roots, have been used as a traditional drug for at least2000years in China. In addition, it contains glycyrrhizin, a non-sugar sweetener, and thus is widely used in the manufacture of food, tobacco, cosmetic, etc. For these reasons, the market demands for Glycyrrhiza production at home and abroad were expanded enormously in recent years. Thus, overcollection has resulted in a rapid decline of the wild resources of Glycyrrhiza and caused a severe ecological disaster in North China. Therefore, our research in this thesis is of great scientific and applied values.
In this paper, introduced in2007to four years in Minqin Gansu, Ningxia Yanchi of Glycyrrhiza uralensis, Glycyrrhiza inflate and Glycyrrhiza glabra of Kashi in Xinjiang were carried out to identify plant form, the active ingredient content of secondary metabolites determination, the systematic study of differences in genetic diversity, established a chemical fingerprint and molecular fingerprint patterns associated with the two systems compared in the same habitat the same period the growth of three different provenances of cultivated Glycyrrhiza populations within populations and the differences between the changes, The main findings are as follows:cultivated Glycyrrhiza Liquiritin and Glycyrrhizic acid
1Starts to the different vegetal period three kinds of cultivated Glycyrrhiza plant's contour from adult plant's high, the stem is thick, shape targets and so on leaf blade shape, root and rhizome, cob, fruit pod, seed carry on the determination, and "the Chinese Pharmacopoeia" describes with "the Chinese Flora" compares the variation degree to be small, the result is as follows, The Glycyrrhiza inflate leaf edge has produced large scale wave form curling up; Starts from the mature period, Glycyrrhiza glabra average height above160cm, becomes large scale "great"; The Glycyrrhiza uralensis strawberry pod's glandular hair becomes thicker. Through the above findings demonstrated that three kinds of cultivated Glycyrrhiza can have the compatible variation in the domesticated process to the external environment, three kinds of cultivated Glycyrrhiza seeds have the many heterozygous body genes, enables oneself to have the compatible change to the environment, observes from the phenotype to the varying degree variation situation. Simultaneously also explains this introduction the wild Glycyrrhiza for the fine mother generation, and the domesticated cultivation's habitat choice is also reasonable.
2This experiment to cultivated Glycyrrhiza raw material for medicine quality rating standard is according to2010editions "the Chinese Pharmacopoeia" stipulated that the Liquiritin, the glycyrrhizic acid content do not be lower than0.50%and2.0%, carries on the effective component secondary metabolite Liquiritin, the glycyrrhizic acid content determination every month to three kinds of cultivated Glycyrrhiza for medicinal purposes spot, finally demonstrated that in May and June determines the cultivated Glycyrrhiza Liquiritin and the glycyrrhizic acid content cannot achieve the standard, but starts the content from July to rise, to September and October when the content achieves the national pharmacopoeia to stipulate, may enter raw material for medicine market. The research indicated that the cultivated Glycyrrhiza is stable in four year domesticated process traditional Chinese medicine quality content, for next step promoted the large-scale domesticated cultivated Glycyrrhiza to provide the reasonable theory basis.
3Carries on the DNA genome team to three kinds of cultivated Glycyrrhiza young tender leaf blade the extraction, has carried on the analysis using the ISSR member mark method to its heredity multiplicity. Using5ISSR which screens directs the thing, occupies the group60sample Chinese Communist Party from3to increase886bandings, polymorphism belt several498, the polymorphism belt percentage is56.19%. Occupies the group heredity multiplicity analysis result to demonstrate, the Glycyrrhiza inflate in occupies in the group level, the total heredity multiple indices (h) and the Shannon index (I) respectively is0.25and0.37; But the Glycyrrhiza glabra respectively is0.23and0.34; The most Glycyrrhiza uralensis is low is0.19and0.28. The AMOVA analysis result showed:Three kinds of cultivated Glycyrrhiza have37%hereditary change to occur in occupy in the group,63%hereditary change occur in occupies the group. It's a way from based on the Nei heredity to three kinds of cultivated Glycyrrhiza carries on the UPGMA cluster separately, finally demonstrated that the Glycyrrhiza uralensis the heredity similarity is smallest, it has the rich genetic background, adaptation environment selection pressure ability some. Occupies three groups to carry on again the UPGMA cluster, the Glycyrrhiza inflate and the Glycyrrhiza glabra gathers a group to constitute separately with the two branches, the result demonstration Glycyrrhiza uralensisand the Glycyrrhiza glabra genetic affinity are near, it is far with the Glycyrrhiza inflate genetic affinity. This experimental result showed that three kinds of cultivated Glycyrrhiza are still retaining the high heredity multiple levels, was its idioplasm resources preservation, the protection and the nurture has provided the scientific basis.
4Through use the traditional Chinese medicine chromatograph fingerprint atlas similarity evaluation system. A version software to three kinds of cultivated Glycyrrhiza chromatograph content establishment chemistry fingerprint atlas, through the atlas comparison Liquiritin and the glycyrrhizic acid two effective characteristic peaks, demonstrated finally that Glycyrrhiza uralensis and Glycyrrhiza glabra Liquiritin peaks high similar, is Glycyrrhiza inflate1.1times, has the obvious difference, tallies with the above heredity multiplicity findings. Indicated that several testing methods unify and the verification experimental method mutually may to test a more comprehensive reasonable explanation, enables experiment's reliability, the feasibility to obtain the effective guarantee, also provides effectively, the reasonable theory basis for the cultivated Glycyrrhiza big area manual cultivation domestication.
This paper research characteristic and innovation spots:
(1) Introduces a fine variety to the different seedling source identical habitat identical growth age limit four year three kinds of cultivated Glycyrrhiza to conduct the plant-morphology research for the first time, from plant's high, the stem is thick, the leaf blade shape, the root and the rhizome, the cob, the fruit pod, seed these aspects carry on the comparison, discovered that three kinds of cultivated Glycyrrhiza plant morphological change and the habitat change and own performance character change is influential. This finding indicated the cultivated Glycyrrhiza selects the seed is the improved seed, includes the rich heterozygous body gene to be possible to prevent effectively in the cultivation domestication process appears occupies in the group plant's degenerated phenomenon.
(2) Introduced a fine variety using the ISSR technology to the different seedling source identical habitat identical growth age limit four year three kinds of cultivated Glycyrrhiza to occupy the group heredity multiplicity for the first time to conduct the system research, discovered that should occupy the group heredity multiple levels to be very still high, prompts the wild Glycyrrhiza to excavate freely excessively, so long as but introduced a fine variety the domesticated cultivation as for not to cause this population heredity reasonably multiply to lose exterminates, this findings for the Glycyrrhiza resources'protection and the nurture, as well as north part of our country wilderness area's ecological protection, restored and reconstruct, have provided the scientific basis.
(3) For the first time the applied chemistry fingerprint atlas and the molecular fingerprint atlas relate the way carries on the heredity multiple differences and the effective component chemistry content determination system research to three kinds of cultivated Glycyrrhiza, discovered that between Glycyrrhiza inflate and Glycyrrhiza uralensis Glycyrrhiza glabra the two difference is remarkable, and has promulgated the heredity multiple differences and the habitat environment, as well as raw material for medicine quality's relevance, has provided the scientific basis for the Glycyrrhiza raw material for medicine quality's appraisal and improved seed's selective breeding.
引文
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