【作者】 黄伟；

【导师】 卢孟柱；

【作者基本信息】 中国林业科学研究院 ， 林木遗传育种， 2014， 博士

【摘要】 杜仲（Eucommia ulmoides Oliv），为杜仲科Eucommiaceae杜仲属Eucommia Oliver落叶乔木，是第四纪冰川侵袭后残留下来的孑遗古老树种，其近缘种类都已绝灭，有“活化石植物”的美称，现主要以栽培为主，被列为国家二类重点保护树种，我国是现存杜仲资源的惟一保存地。本研究以杜仲不同组织器官（根、叶、树皮）为实验材料，根据2010年版《中国药典》规定的松脂醇二葡萄糖苷、绿原酸的含量测定方法对不同产地杜仲进行了评测，并对不同产地杜仲不同组织器官化学组分进行了全面分析，同时采用ISSR（inter-simple sequence repeat）分子标记技术对不同产地杜仲的遗传差异进行分析评价。得到如下主要结论：（1）HPLC分析结果表明，湖北宜昌、贵州遵义的杜仲皮样品中松脂醇二葡萄糖苷的含量不符合药典标准；河南、陕西产杜仲皮中松脂醇二葡萄糖苷的含量最高，是药典标准的两倍还多；湖北、贵州、重庆等地的杜仲皮样品的均一性较好，而四川旺苍产杜仲皮中松脂醇二葡萄糖苷的含量要远低于省内其余地方的含量。此外，河南、陕西产杜仲叶中所含绿原酸含量最高，而整个四川产杜仲叶中绿原酸含量接近，湖北、贵州产杜仲叶中的绿原酸含量为最低，因此，区分不同产地来源对于杜仲的质量控制具有重要意义。（2）用8对ISSR引物对15个不同产地的杜仲进行遗传标记，共扩增出102个DNA片段，多态性比率为88.2%；其有效等位基因数平均为1.781，基因多样度平均为0.443，Shannon信息指数平均为0.758，表明杜仲的不同产地的存在着较大的遗传差异，利用这些差异的分子标记可以鉴别不同产地来源。15个不同产地杜仲植物的遗传基因的相似度有65%，可大致分为三大类；从15个产地杜仲的遗传距离来看，杜仲产地间的遗传差异与其所在的地理位置远近分布相关性明显。（3）从杜仲叶中提取了酸性多糖，通过对该酸性多糖进行部分酸水解、甲基化分析等研究证明EOP-1为杜仲叶中首次发现的一种多分支的酸性杂多糖，分子量约为6.0×105D，它由半乳糖醛酸、半乳糖、鼠李糖、甘露糖、阿拉伯糖及葡萄糖组成，其中半乳糖含量较高。EOP-1主链结构可能为1，4连接的半乳糖醛酸聚糖，分支由多分支的1,3连接及1,6连接的半乳糖及1,5连接的阿拉伯糖组成。（4）对我国由北到南的五个不同产区的杜仲不同组织器官（叶、根、皮）细胞壁木质素、纤维素、果胶和半纤维素四个组分进行含量分析，并进一步测定了果胶和半纤维素中的中性单糖。研究结果表明，总中性糖在叶组织中含量最高，在根、皮中呈现逐渐递减的趋势；糖醛酸组分在叶组织中含量也是最高，树皮中含量次之，根中最低；纤维素组分在根、皮和叶中呈现逐渐递减的趋势，木质素含量在叶、根和皮中呈现递增的趋势。杜仲叶中总中性糖及糖醛酸含量均较皮和根含量高，以糖类含量角度分析，叶片能取代杜仲皮。杜仲不同产地、不同组织器官的有效成分和化学组分以及中性单糖的差异，说明可以进一步高效利用和开发杜仲的药用价值。有效成分与遗传差异聚类的相一致性，表明可以采用分子标记进行产地的分析，有利于药材的质量控制。遗传与环境的相互作用决定了有效成分的差异，因此还需要通过解析遗传与环境因子的关系，进一步解析遗传差异和环境对于有效成分的影响。更多还原

【Abstract】 Eucommia ulmoides Olive., a deciduous tree species classified into Eucommia Oliver ofEucommiaceae, is the relict ancient tree species left after the invasion of Quaternary glacier. As"living fossil plant", E. ulmoide is a type of second-class national key protected species, whoseclosely related species have been extinct and the existing E. ulmoide resources are only savedin China. In this study the barks and leaves are used as experimental materials to analyze thecontent of the pinoresinol diglucoside and chlorogenic acid of E. ulmoide. Additionally, theISSR (inter-simple sequence repeat) technology is applied to explore the genetic diversity ofsamples collected from different origins of E. ulmoide. The main results are as follows：1. HPLC analysis results show that the pinoresinol glucoside in E. ulmoide barks fromYichang, Hubei, Guizhou Zunyi do not meet Pharmacopoeia standards. The samples of Henanand Shaanxi yield the highest level of the pinoresinol glucoside. and the content of plantsamples in Henan and Shaanxi ranks top is more than twice of the standard. Samples of E.ulmoide from Hubei, Guizhou, Chongqing and other places have great homogeneity, while thecontent of pinoresinol glucoside in E. ulmoide barks from Wangcang Sichuan is much lowerthan that from other place in that province. Additionally, the chlorogenic acid contained inleaves of E. ulmoide from Henan and Shaanixi is the highest, while the content in leaves of E.ulmoide within the whole Sichuan is intermediate and stable, and that in leaves from Hubei andGuizhou is the minimum. Therefore, it is important to identify the origions of the samples.2. Eight pairs of ISSR primers were used to amplify E. ulmoide DNAs from15differentorigins, and102DNA fragments were amplified with polymorphic rate at88.2%. The E.ulmoide plants genes within species had a large diversity. The effective number of alleles hit1.781on average and the average gene diversity was0.443. The information index of Shannonwas0.758on average. Genetic similarity at65%. The E. ulmoide in15different habitats can bebroadly divided into three categories, and the genetic distances showed similar with theirgeographical distances. 3. Another finding of this study is the identification of an acidic heteropolysaccharide,EOP-1extracted from E. ulmoide leaves by partial acid hydrolysis, methylation analysis et al.The molecular weight is about6×105, and consists of galacturonic acid, galactose, rhamnose,mannose, glucose and arabinose. In this polysaccharide, the content of glucose, galactose wasrelatively high, which included main part of homogalacturonan fragments and residues of sugarconstituents were mainly D-GalpA, the backbone was composed of1,4-linked-D-GalpA and1,3-D-Galp、1,4-D-Galp、1,5-L-Araf were the mainly consititution of the branch. Thebioactivity of this acidic heteropolysaccharide deserves a further anlaysis.4. Based on the pharmacological components of E. ulmoide, polysaccharide was one ofimportant effective components. The compositions of cell wall of E. ulmoide from differentorgans and different areas of production are compared in this paper. Four components of lignin,cellulose, pectin and hemicellulose of cell wall of E. ulmoide in different organs (leaf, root,bark) in five different areas from the North to the South China, and the neutral monosaccharidein pectin and hemicellulose was further analyzed. The results showed that the total content ofneutral sugar was the highest in leaf tissue and showed gradual decline in root and bark; thecontent of uronic was is also the highest in leaf tissue, followed by the root, that it was thelowest in the bark. The results showed the fraction of cellulose indicated gradual decline in root,bark and leaf. The content of lignin showed an increasing trend in leaf, root and bark. totalneutral sugar and uronic acid content of the leaf was higher than that of the bark and root whichindicated that the bark could be replaced by the leaf.The varied contents and ratios of pharmacological components including neutralmonosaccharides in the leaves and barks of E. ulmoide from different organs and from samplesin different origins demonstrate the needs to develop cut-edge technology to fully use E.ulmoide resources. The similarity of the contents of pinoresinol glucoside and chlorogenic acidof the E. ulmoide samples with their genetic distances provides the opportunity to developmoleculat markers to identify the origins thus to control the quanlity of medicines. The geneticvaribility and enviromental factors influence the comttent of the pharmacological components thus it is required to disseminate the effects of genetic and enviromental determination on thefinal output of the medical herb.更多还原