构建铁皮石斛多维指纹图谱方法的研究
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摘要
铁皮石斛为兰科石斛属植物，是一种名贵的药用植物。由于铁皮石斛特殊的生长环境和人们的过度采挖，目前铁皮石斛的野生资源已日趋枯竭，市场资源十分混乱。为促进该产业的健康发展，本文构建了铁皮石斛毛细管电泳指纹图谱、SRAP分子指纹图谱和多糖指纹图谱。
(1)毛细管电泳指纹图谱的构建：通过对提取条件和检测条件的优化，发现以40mM硼砂(pH9.5，含5％甲醇)为运行缓冲液，以20％乙醇为提取溶剂，能从10种产地铁皮石斛中得到15个特征指纹峰，并以第9号峰为内参比峰，计算各峰的相对保留时间和相对峰面积可作为指纹信息。在此基础上，对不同产地铁皮石斛药材进行了相似度分析、主成分分析、系统聚类分析，10种产地铁皮石斛可以被分为三类：浙江温州、浙江雁荡山、浙江富阳、江西、云南、广西、广东产地铁皮石斛聚为第一类，福建、安徽产地铁皮石斛聚为第二类，湖南产地铁皮石斛聚为第三类。此外，采用逐步判别法选取四个特征指纹峰面积(X_2、X7、X_(11)、X_(13))为原始数据，构建其典型判别函数，将待分类样品6个特征指纹峰的相对峰面积代入上述方程即可算出其所属类别。
(2)DNA分子指纹图谱的构建：通过对SRAP-PCR反应体系的优化，确立了铁皮石斛SRAP-PCR最佳反应体系：模板DNA30 ng，dNTP 0.25 mM，引物浓度0.1μM，Mg~(2+)浓度2.0 mM，Taq DNA聚合酶1.5 U，10×PCR Buffer 2.5μL，总反应体积为25μL。用四对引物组合对七种产地铁皮石斛进行扩增，共得到77条带，其中特异性分子标记39个，多态率达51％。四对引物中，Me5—Em6组合具有较多的多态性位点，Rp值也较高。遗传距离和系统聚类分析结果表明：七种产地铁皮石斛被分为四类，浙江温州、广东、广西为第一类；湖南为第二类；浙江雁荡山，江西为第三类；云南为第四类。
(3)多糖指纹图谱的构建：采用荧光辅助糖电泳技术构建了铁皮石斛多糖的酶解指纹图谱。多糖经β-甘露聚糖酶和纤维素酶分别水解，产物经聚丙烯酰胺凝胶电泳分离，发现不同产地铁皮石斛中聚合度较高寡糖片段位置和荧光强度有一定的差别，且β-甘露聚糖酶产生的多态性高于纤维素酶。按上述方法，对七种不同品种石斛多糖进行分析，发现高聚合度的片段与低聚合度的片段之间差异比较明显，且纤维素酶所产生的多态性要高于β-甘露聚糖酶。
研究结果不仅为铁皮石斛种质资源鉴定提供理论依据，而且能为进行中药材质量评价的研究提供方法学参考。
Dendrobium officinale is a precious traditional Chinese herbal medicine which belongs to Dendrobium species, Orchidaceae. Due to its unique growth condition and excessive utilization, the wild resources of Dendrobium officinale are nearly exhausted, resulting in market confusion. Multi-dimensional fingerprints of Dendrobium officinale including high performance capillary electrophoresis fingerprints, SRAP molecular fingerprints and polysaccharide fingerprints were constructed in this paper in order to promote the healthy development of this industry
(1) Construction of the fingerprints by high performance capillary electrophoresis:Though optimization of extraction and the separation conditions, 15 characteristic fingerprint peaks were identified from the ethanol extracts of Dendrobium officinales from 10 different producing areas by high performance capillary electrophoresis with 40 mM borax (pH 9.5, contains 5% methanol, v/v) as the background electrolyte and 20% ethanol as extraction solvent. Among these peaks, the ninth peak was chosen as reference peak for calculating the relative retention time and the relative peak area of the other peak. The quantitative data of the fingerprints were analyzed by similiarity analysis, principal component analysis and systematically cluster analysis. The results showed that the samples from 10 different producing areas could be clustered into three groups. The first consisted of samples from Yandangshan in Zhejiang province, Fuyang in Zhejiang province, Jiangxi province, Yunnan province, Guangxi province and Guangdo province. The second was composed of the samples from Fujian province and Anhui province. The third group contained only one sample from Hunan province. In addition, four characteristic fingerprint peaks (X_2, X_7, X_(11), X_(13)) were selected to construct discriminant analysis model, unclassified samples could be easily classified by calculating the relative peak areas of the four characteristic fingerprint peaks.
(2) Construction of DNA fingerprints by SRAP technology: SRAP reaction system was optimized and the optimal reaction system contained 30 ng DNA template, 0.25 mM dNTT, 0.1 mM primer, 2.0 mM Mg~(2+), 1.5 U Taq DNA polymerase and 2.5μL 10×PCR Buffer where the total volume was 25μL. The total of 77 bands was amplified by 4 pairs of primer and 39 of them were polymorphic accounting for 51%. Among all primers, the combination of Me5—Em6 showed the highest level of polymorphism and Rp value. The dendrogram constructed with SRAP molecular makers revealed that Dendrobium officinale from seven different producing areas could be divided into 4 groups: Dendrobium officinales from Wenzhou in Zhejiang province, Guangxi province, Guangdong province were the first cluster; Dendrobium officinale from Hunan province was the second; Dendrobium officinale from Yan dangshan in Zhejiang province and Jiangxi province were clustered in the third. The last contained only the sampe from Yunnan province.
(3) Construction of fingerprints of polysaccharides: Enzymatic fingerprint of Dendrobium officinale polysaccharides were investigated by fluorescence-assisted carbohydrate electrophoresis. Polysaccharides of Dendrobium officinale from different producing areas were hydrolyzed byβ-mannase and cellulose, respectively, and then separated by polyacrylamide gel electrophoresis. Some differences were observed in band location and fluorescence intensity of oligosaccharides with high polymerization degree. The polymorphism level produced byβ-mannase was higher than that by cellulase. Enzymatic fingerprints of polysaccharides from seven different Dendrobium species were also analyzed with this method and results indicated that there were significant differences not only in oligosaccharides with high polymerization degree but also in oligosaccharides with low polymerization degree. The polymorphism level produced by cellulose was higher than byβ-mannase.
The above results will not only provide the theoretical bases to identify the species of Dendrobium officinale, but also supply the methods for the quality evaluation of traditional Chinese medicine (TCM).
(1)毛细管电泳指纹图谱的构建：通过对提取条件和检测条件的优化，发现以40mM硼砂(pH9.5，含5％甲醇)为运行缓冲液，以20％乙醇为提取溶剂，能从10种产地铁皮石斛中得到15个特征指纹峰，并以第9号峰为内参比峰，计算各峰的相对保留时间和相对峰面积可作为指纹信息。在此基础上，对不同产地铁皮石斛药材进行了相似度分析、主成分分析、系统聚类分析，10种产地铁皮石斛可以被分为三类：浙江温州、浙江雁荡山、浙江富阳、江西、云南、广西、广东产地铁皮石斛聚为第一类，福建、安徽产地铁皮石斛聚为第二类，湖南产地铁皮石斛聚为第三类。此外，采用逐步判别法选取四个特征指纹峰面积(X_2、X7、X_(11)、X_(13))为原始数据，构建其典型判别函数，将待分类样品6个特征指纹峰的相对峰面积代入上述方程即可算出其所属类别。
(2)DNA分子指纹图谱的构建：通过对SRAP-PCR反应体系的优化，确立了铁皮石斛SRAP-PCR最佳反应体系：模板DNA30 ng，dNTP 0.25 mM，引物浓度0.1μM，Mg~(2+)浓度2.0 mM，Taq DNA聚合酶1.5 U，10×PCR Buffer 2.5μL，总反应体积为25μL。用四对引物组合对七种产地铁皮石斛进行扩增，共得到77条带，其中特异性分子标记39个，多态率达51％。四对引物中，Me5—Em6组合具有较多的多态性位点，Rp值也较高。遗传距离和系统聚类分析结果表明：七种产地铁皮石斛被分为四类，浙江温州、广东、广西为第一类；湖南为第二类；浙江雁荡山，江西为第三类；云南为第四类。
(3)多糖指纹图谱的构建：采用荧光辅助糖电泳技术构建了铁皮石斛多糖的酶解指纹图谱。多糖经β-甘露聚糖酶和纤维素酶分别水解，产物经聚丙烯酰胺凝胶电泳分离，发现不同产地铁皮石斛中聚合度较高寡糖片段位置和荧光强度有一定的差别，且β-甘露聚糖酶产生的多态性高于纤维素酶。按上述方法，对七种不同品种石斛多糖进行分析，发现高聚合度的片段与低聚合度的片段之间差异比较明显，且纤维素酶所产生的多态性要高于β-甘露聚糖酶。
研究结果不仅为铁皮石斛种质资源鉴定提供理论依据，而且能为进行中药材质量评价的研究提供方法学参考。
Dendrobium officinale is a precious traditional Chinese herbal medicine which belongs to Dendrobium species, Orchidaceae. Due to its unique growth condition and excessive utilization, the wild resources of Dendrobium officinale are nearly exhausted, resulting in market confusion. Multi-dimensional fingerprints of Dendrobium officinale including high performance capillary electrophoresis fingerprints, SRAP molecular fingerprints and polysaccharide fingerprints were constructed in this paper in order to promote the healthy development of this industry
(1) Construction of the fingerprints by high performance capillary electrophoresis:Though optimization of extraction and the separation conditions, 15 characteristic fingerprint peaks were identified from the ethanol extracts of Dendrobium officinales from 10 different producing areas by high performance capillary electrophoresis with 40 mM borax (pH 9.5, contains 5% methanol, v/v) as the background electrolyte and 20% ethanol as extraction solvent. Among these peaks, the ninth peak was chosen as reference peak for calculating the relative retention time and the relative peak area of the other peak. The quantitative data of the fingerprints were analyzed by similiarity analysis, principal component analysis and systematically cluster analysis. The results showed that the samples from 10 different producing areas could be clustered into three groups. The first consisted of samples from Yandangshan in Zhejiang province, Fuyang in Zhejiang province, Jiangxi province, Yunnan province, Guangxi province and Guangdo province. The second was composed of the samples from Fujian province and Anhui province. The third group contained only one sample from Hunan province. In addition, four characteristic fingerprint peaks (X_2, X_7, X_(11), X_(13)) were selected to construct discriminant analysis model, unclassified samples could be easily classified by calculating the relative peak areas of the four characteristic fingerprint peaks.
(2) Construction of DNA fingerprints by SRAP technology: SRAP reaction system was optimized and the optimal reaction system contained 30 ng DNA template, 0.25 mM dNTT, 0.1 mM primer, 2.0 mM Mg~(2+), 1.5 U Taq DNA polymerase and 2.5μL 10×PCR Buffer where the total volume was 25μL. The total of 77 bands was amplified by 4 pairs of primer and 39 of them were polymorphic accounting for 51%. Among all primers, the combination of Me5—Em6 showed the highest level of polymorphism and Rp value. The dendrogram constructed with SRAP molecular makers revealed that Dendrobium officinale from seven different producing areas could be divided into 4 groups: Dendrobium officinales from Wenzhou in Zhejiang province, Guangxi province, Guangdong province were the first cluster; Dendrobium officinale from Hunan province was the second; Dendrobium officinale from Yan dangshan in Zhejiang province and Jiangxi province were clustered in the third. The last contained only the sampe from Yunnan province.
(3) Construction of fingerprints of polysaccharides: Enzymatic fingerprint of Dendrobium officinale polysaccharides were investigated by fluorescence-assisted carbohydrate electrophoresis. Polysaccharides of Dendrobium officinale from different producing areas were hydrolyzed byβ-mannase and cellulose, respectively, and then separated by polyacrylamide gel electrophoresis. Some differences were observed in band location and fluorescence intensity of oligosaccharides with high polymerization degree. The polymorphism level produced byβ-mannase was higher than that by cellulase. Enzymatic fingerprints of polysaccharides from seven different Dendrobium species were also analyzed with this method and results indicated that there were significant differences not only in oligosaccharides with high polymerization degree but also in oligosaccharides with low polymerization degree. The polymorphism level produced by cellulose was higher than byβ-mannase.
The above results will not only provide the theoretical bases to identify the species of Dendrobium officinale, but also supply the methods for the quality evaluation of traditional Chinese medicine (TCM).
引文
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