遗传与环境因子对甘草生长及药材质量的影响研究
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摘要
中药资源的生态学研究是中药资源保护和可持续利用的基础。生态学与中药资源的关系虽然很早就已被关注,但一直以来没有采用定量化的技术方法,导致了中药资源生态学长期停留在定性描述阶段,阻碍了学科的发展。本文在充分分析生态学科发展方向的基础上,抓住现代生态学研究有越来越向宏观发展,更加重视地下生物系统以及地下生物系统与地上生物系统相互关系的趋势,以甘草Glycryrrhizauralensis Fish.为研究对象,采用近地摄影技术对甘草的野外群落进行了调查,采用Biolog方法对甘草生长环境进行了评价,从外观形态、无机元素、有机物质三个层面开展了甘草药材质量方面的研究,开展了两种源两产地的甘草生态学种植实验,探讨影响甘草生长及药材质量的因素。综合以上研究,得出如下结论：
(1)近地摄影技术可以应用于甘草植物的野外调查。调查结果表明了其可以很好的弥补传统样方调查对盖度估计不够准确的缺陷,大大提高了野外调查方法数据的定量水平,保证了数据的可靠性。
(2)Biolog方法应用于根际土壤微生物多样性分析能够反映周围环境影响,可以用于评价甘草的生态环境变化。结果表明不同环境生长的甘草根际微生物对不同碳源的利用差异较大,选择合适的多样性指数对于实现研究目的具有很重要的意义,利用该方法还发现了一些对多种碳源利用效率均较高的微生物群落,为进一步开发微生物肥料提供了基础,有望进一步推动本学科对地下生态系统的关注。
(3)甘草药材质量的全面评价有赖于多种方法的联合使用。结果表明,采用多种方法结合才能更好的全面反映药材质量信息,单纯从形态学、有效成分都不足以保证全面反映药材的质量,本研究中野生甘草出现了形态学、有效成分比较都无统计学显著差异的现象,但是通过红外光谱技术可以很好的将两个产地的样品区分开,而采用无机元素分析方法可以很好的区别野生样品和栽培样品,某些微量元素如Mo还体现出产地间的差异,也可以用于区分产地。
(4)出于生态学研究目的的甘草药材质量评价应该选择适宜的生长时期。结果表明,甘草生长的第二年是植物的快速生长期,在植株外在形态和有效成分含量上都发生很大变化,该阶段是生态学研究质量差异比较的首选阶段,而不是以往认为的药材收获期。
(5)植物的遗传学背景分析是中药资源生态学栽培实验的首要条件。分析结果表明,调查的两个产区遗传距离较远,产地间的差异远远大于产地内部的遗传差异,可以考虑作为实验种植的材料来源,同时比较了采集的种子和从市场上不同产地收集的种苗,结果显示,种苗间存在种源混乱现象,据深入调查发现该现象应该来自种子的大规模跨地区调用,今后工作中应大力开展优良品种的选育,并加大对优质种源的保护力度,开展生态学栽培实验在目前阶段还是应选用自己亲自采集的种子。
(6)遗传因素和环境因素对不同质量指标的影响程度有所差异。两种源两产地实验结果表明：测量4个形态学指标中,株高、根重统计学上差异不显著,遗传因素与环境因素,及两者间的交互作用均会对植株根长、根粗两个指标产生影响,具体而言,根长受环境、遗传单独影响因素影响较大,根粗主要受遗传因素影响较大。甘草两种有效成分受遗传因素和环境因素双重控制,但两种因素间互作效应不明显,甘草酸受两种因素影响,遗传因素影响更大,甘草苷主要受到环境因素的影响；红外实验结果表明许多内在成分都是受遗传与环境的双重影响,并且环境因素可能影响更大。
本论文的研究特色和创新点为：(1)评价了近地摄影技术和Biolog技术在本学科中的应用可行性,完善了中药资源调查技术体系,有望推动本学科对地下生态系统的关注。(2)建立了较为全面的基于生态学角度的甘草质量评价技术体系,为全面评价甘草药材质量提供了技术支持。(3)将遗传多样性分析方法用于评价种植实验材料选择的可行性,有利于克服目前药用植物中遗传稳定性材料缺乏的弊病。(4)揭示了遗传因素和环境因素对甘草质量的影响程度,为进一步遗传育种工作和生态学研究奠定了基础。
Ecology study of Traditional Chinese Medicine (TCM) is the basis of resource protection and sustainable utilization. Ecological influence on TCM have been observed ages ago, while few application of modern technology hindered the development of ecological study of TCM resource. In this article, the author introduced some technology based on analysis of the trends of ecological and TCM resource, such as ground digital photography in resource survey, community-level physiological profiling based on Biolog ECO MicroPlates in distinguish spatial difference in microbial communities of Glycyrrhiza uralensis plant rhizosphere, and the author evaluate the quality of Glycyrrhiza uralensis based on multilevel information such as exterior morphology data, intrinsic contents of inorganic and organic substance, cultivation experiment of different genotype seed in different environment were done to evaluate the effects of genotype and environment on morphology and intrinsic contents. Some conclusions were achieved from the work.
1. Ground digital photography is suitable for the resource survey of Glycyrrhiza uralensis, which can supply much help for coverage measure in traditional sample investigation by statistical analysis of GIS of photo, avoiding the error caused by subjective observation, the data reliability was improved much.
2. It was demonstrated in our research that Biolog is an appropriate method to evaluate the environment, which the microbial community spatial change of Glycyrrhiza uralensis plant rhizosphere is correlation with environment change the plant exist. And the diversity index must be chose for realization of certain purpose. Some microbial community was found that the utilization effective is many high for many carbon sources, which can be taken as a substance basis for further cultivation of microbial fertilizer.
3. The quality evaluation must consider multi-level methods. The study showed only with one or two method, we may lose some useful information to evaluate the quality. In the study morphology data, intrinsic contents of effective substance between samples come from different ecological zone showed few difference statistics, but with infrared spectroscopy, the difference was disclosed and the different group samples can be distinguished by PCA. Inorganic substance analysis differentiated wild and cultivation type sample easily, and some microelement such as Mo can also differentiated the producing area.
4. The choice of appropriate growth periods is needed for ecological purpose, and the time maybe different from traditional plant harvest time, which showed in our research Glycyrrhiza uralensis plant grow much faster in the second year, the morphology intrinsic contents of effective substance both increased greatly, so this period is the optimal choice for quality difference evaluation especially for ecological purpose,while harvest time maybe not suitable for that.
5. The application of ISSR showed that the methods can disclose the genetics difference between groups and in group, and can provide support for ecological cultivation experiment, it also showed the disorder existed in medicinal plant cultivation, further investigation showed which comes from seed transfer among different area, so the breeding work should be done and excellent genetic substance should be protected.
6. The cultivation experiment results suggest that morphology data, intrinsic contents may influenced by genotype, environment and the interaction of two factors, though the degree is different.e.g. the genotype, environment and the interaction of two factors have much influence on the root length and the diameter of root, the root length influenced by genotype, environment respectively, while the diameter of root influenced more by genotype. Two effective substance, glycyrrhizin and liquiritin was influenced by genotype, environment but not the interaction of two factors, and glycyrrhizin influenced more by genotype, while liquiritin was influenced more by environment. Infrared spectroscopy analysis also showed that many chemical substance influenced by genotype and environment, but not one factor, and the environment is more important in the two factors.
The innovative points and the special feature of this research are:(1) The feasibility of methods of ground digital photography and Biolog in TCM ecology study was evaluated. (2) Technology system of quality evaluation with multi-level methods was established. (3) Gene background of materials was analyzed before field cultivation. (4)The effects of genotype, environment and the interaction of two factors were disclosed, which may be helpful for breeding and further ecological study.
(1)近地摄影技术可以应用于甘草植物的野外调查。调查结果表明了其可以很好的弥补传统样方调查对盖度估计不够准确的缺陷,大大提高了野外调查方法数据的定量水平,保证了数据的可靠性。
(2)Biolog方法应用于根际土壤微生物多样性分析能够反映周围环境影响,可以用于评价甘草的生态环境变化。结果表明不同环境生长的甘草根际微生物对不同碳源的利用差异较大,选择合适的多样性指数对于实现研究目的具有很重要的意义,利用该方法还发现了一些对多种碳源利用效率均较高的微生物群落,为进一步开发微生物肥料提供了基础,有望进一步推动本学科对地下生态系统的关注。
(3)甘草药材质量的全面评价有赖于多种方法的联合使用。结果表明,采用多种方法结合才能更好的全面反映药材质量信息,单纯从形态学、有效成分都不足以保证全面反映药材的质量,本研究中野生甘草出现了形态学、有效成分比较都无统计学显著差异的现象,但是通过红外光谱技术可以很好的将两个产地的样品区分开,而采用无机元素分析方法可以很好的区别野生样品和栽培样品,某些微量元素如Mo还体现出产地间的差异,也可以用于区分产地。
(4)出于生态学研究目的的甘草药材质量评价应该选择适宜的生长时期。结果表明,甘草生长的第二年是植物的快速生长期,在植株外在形态和有效成分含量上都发生很大变化,该阶段是生态学研究质量差异比较的首选阶段,而不是以往认为的药材收获期。
(5)植物的遗传学背景分析是中药资源生态学栽培实验的首要条件。分析结果表明,调查的两个产区遗传距离较远,产地间的差异远远大于产地内部的遗传差异,可以考虑作为实验种植的材料来源,同时比较了采集的种子和从市场上不同产地收集的种苗,结果显示,种苗间存在种源混乱现象,据深入调查发现该现象应该来自种子的大规模跨地区调用,今后工作中应大力开展优良品种的选育,并加大对优质种源的保护力度,开展生态学栽培实验在目前阶段还是应选用自己亲自采集的种子。
(6)遗传因素和环境因素对不同质量指标的影响程度有所差异。两种源两产地实验结果表明：测量4个形态学指标中,株高、根重统计学上差异不显著,遗传因素与环境因素,及两者间的交互作用均会对植株根长、根粗两个指标产生影响,具体而言,根长受环境、遗传单独影响因素影响较大,根粗主要受遗传因素影响较大。甘草两种有效成分受遗传因素和环境因素双重控制,但两种因素间互作效应不明显,甘草酸受两种因素影响,遗传因素影响更大,甘草苷主要受到环境因素的影响；红外实验结果表明许多内在成分都是受遗传与环境的双重影响,并且环境因素可能影响更大。
本论文的研究特色和创新点为：(1)评价了近地摄影技术和Biolog技术在本学科中的应用可行性,完善了中药资源调查技术体系,有望推动本学科对地下生态系统的关注。(2)建立了较为全面的基于生态学角度的甘草质量评价技术体系,为全面评价甘草药材质量提供了技术支持。(3)将遗传多样性分析方法用于评价种植实验材料选择的可行性,有利于克服目前药用植物中遗传稳定性材料缺乏的弊病。(4)揭示了遗传因素和环境因素对甘草质量的影响程度,为进一步遗传育种工作和生态学研究奠定了基础。
Ecology study of Traditional Chinese Medicine (TCM) is the basis of resource protection and sustainable utilization. Ecological influence on TCM have been observed ages ago, while few application of modern technology hindered the development of ecological study of TCM resource. In this article, the author introduced some technology based on analysis of the trends of ecological and TCM resource, such as ground digital photography in resource survey, community-level physiological profiling based on Biolog ECO MicroPlates in distinguish spatial difference in microbial communities of Glycyrrhiza uralensis plant rhizosphere, and the author evaluate the quality of Glycyrrhiza uralensis based on multilevel information such as exterior morphology data, intrinsic contents of inorganic and organic substance, cultivation experiment of different genotype seed in different environment were done to evaluate the effects of genotype and environment on morphology and intrinsic contents. Some conclusions were achieved from the work.
1. Ground digital photography is suitable for the resource survey of Glycyrrhiza uralensis, which can supply much help for coverage measure in traditional sample investigation by statistical analysis of GIS of photo, avoiding the error caused by subjective observation, the data reliability was improved much.
2. It was demonstrated in our research that Biolog is an appropriate method to evaluate the environment, which the microbial community spatial change of Glycyrrhiza uralensis plant rhizosphere is correlation with environment change the plant exist. And the diversity index must be chose for realization of certain purpose. Some microbial community was found that the utilization effective is many high for many carbon sources, which can be taken as a substance basis for further cultivation of microbial fertilizer.
3. The quality evaluation must consider multi-level methods. The study showed only with one or two method, we may lose some useful information to evaluate the quality. In the study morphology data, intrinsic contents of effective substance between samples come from different ecological zone showed few difference statistics, but with infrared spectroscopy, the difference was disclosed and the different group samples can be distinguished by PCA. Inorganic substance analysis differentiated wild and cultivation type sample easily, and some microelement such as Mo can also differentiated the producing area.
4. The choice of appropriate growth periods is needed for ecological purpose, and the time maybe different from traditional plant harvest time, which showed in our research Glycyrrhiza uralensis plant grow much faster in the second year, the morphology intrinsic contents of effective substance both increased greatly, so this period is the optimal choice for quality difference evaluation especially for ecological purpose,while harvest time maybe not suitable for that.
5. The application of ISSR showed that the methods can disclose the genetics difference between groups and in group, and can provide support for ecological cultivation experiment, it also showed the disorder existed in medicinal plant cultivation, further investigation showed which comes from seed transfer among different area, so the breeding work should be done and excellent genetic substance should be protected.
6. The cultivation experiment results suggest that morphology data, intrinsic contents may influenced by genotype, environment and the interaction of two factors, though the degree is different.e.g. the genotype, environment and the interaction of two factors have much influence on the root length and the diameter of root, the root length influenced by genotype, environment respectively, while the diameter of root influenced more by genotype. Two effective substance, glycyrrhizin and liquiritin was influenced by genotype, environment but not the interaction of two factors, and glycyrrhizin influenced more by genotype, while liquiritin was influenced more by environment. Infrared spectroscopy analysis also showed that many chemical substance influenced by genotype and environment, but not one factor, and the environment is more important in the two factors.
The innovative points and the special feature of this research are:(1) The feasibility of methods of ground digital photography and Biolog in TCM ecology study was evaluated. (2) Technology system of quality evaluation with multi-level methods was established. (3) Gene background of materials was analyzed before field cultivation. (4)The effects of genotype, environment and the interaction of two factors were disclosed, which may be helpful for breeding and further ecological study.
引文
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