吴茱萸种质资源遗传多样性及抗旱生理研究
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摘要
中药吴茱萸来源于芸香科(Rutaceae)吴茱萸属(Evodia)植物吴茱萸Evodia rutaecarpa(Juss.) Benth.、疏毛吴茱萸Evoida rutaecarpa(Juss.) Benth.var.bodinieri(Dode) Huang和石虎Evodia rutaecarpa(Juss.)Benth.var.officinalis(Dode)Huang的干燥近成熟果实,是我国常用的大宗中药材。吴茱萸药材基原植物、自然资源丰富且分布较广,但由于各地吴茱萸生境差异很大,生态条件复杂,栽培广泛；数千年来各地间种质资源交流频繁；各地风俗迥异,用药习惯差别较大且吴茱萸属内种和变种较多等原因造成了吴茱萸遗传变异复杂,遗传背景不明确。至今人们对吴茱萸种内间的演化关系及栽培种间的亲缘关系以及不同基原的正品药材的质量评价尚不清晰。此外,吴茱萸生长过程中易受到干旱胁迫,导致植株体内活性氧累积过多,活性氧清除系统失调,对植物生长和药材品质带来严重的不良影响。所以,针对吴茱萸的种质资源多样性和抗旱生理进行基础研究,这对吴茱萸资源的保护和可持续利用、促进吴茱萸药材产业化生产的良好发展、保证吴茱萸药材质量以及临床用药的安全性等方面都具有重要的科学意义和现实意义。
目的：
尽管前人采用AFLP、RAPD等分子标记技术对吴茱萸种质资源遗传多样性进行了研究,但由于受到样本数量和样本来源地的限制,没有广泛收集其它吴茱萸主产区的种质,因此其遗传多样性分析具有一定的局限性。本研究广泛收集吴茱萸正品药材基源植物的栽培种和药材,分析了它们的种质资源遗传多样性,构建了HPLC化学指纹图谱,客观的评价了吴茱萸种质资源,进一步探讨了其遗传多样性,为阐明吴茱萸栽培种的亲缘关系提供了科学的实验依据。
吴茱萸在生长过程中易受干旱胁迫,严重影响药材质量和产量。目前,干旱胁迫的抵御机制研究主要集中在农作物上,药用植物相关研究较少,还未见吴茱萸抗旱生理研究的报道。本实验利用人为模拟的干旱胁迫环境,研究了吴茱萸扦插苗在受到干旱胁迫和复水过程各项生理生化指标的变化规律并克隆了抗逆关键基因—Cu/Zn-SOD和Mn/Fe-SOD基因的全长序列,为下一步深入研究奠定了坚实的基础。
方法：
1.文献研究
检索与本研究相关的古籍和国内外文献,并进行了总结和综述,概括了与本论文相关的研究动态。主要包括以下4个方面：
(1)吴茱萸药用历史、资源分布、性味功效与应用、吴茱萸植物特征与品种鉴别等吴茱萸本草考证概况；
(2)吴茱萸植物形态学标记、细胞学分析、DNA分子标记和DNA条形码等吴茱萸植物种质资源多样性研究进展；
(3)干旱胁迫环境下植物细胞渗透调节物质和活性氧(ROS)清除系统及植物SOD研究进展；
(4)吴茱萸化学成分、化学指纹图谱等吴茱萸化学质量评价研究进展。
2.实验研究
本文利用DNA分子标记技术、DNA条形码技术、分析化学方法对不同来源地和产地的吴茱萸种质资源及药材进行了多样性评价；并且,对吴茱萸扦插苗在人工模拟的干旱胁迫环境条件下,植株内多种生理生化指标变化规律及SOD基因的克隆进行了研究。本文研究内容包括：
(1)采用ISSR分子标记技术对来源于贵州省、江西省、湖南省、湖北省、广东省的71份栽培吴茱萸属植物种质资源进行了遗传多样性研究分析。
(2)采用ITS2DNA条形码技术分析了代表不同来源地、不同种的吴茱萸属植物样品18份。
(3)使用HPLC法测定了绿原酸、异鼠李素-3-O-半乳糖苷、槲皮素、吴茱萸碱、吴茱萸次碱和吴茱萸新碱等6种有效成分的含量并构建了37个批次的吴茱萸药材HPLC化学指纹图谱。
(4)使用不同浓度PEG—-6000溶液,经过不同时间处理吴茱萸扦插苗,测定可溶性蛋白、可溶性糖、游离脯氨酸、丙二醛(MDA)、过氧化氢酶(CAT)、过氧化物酶(POD)、超氧化物歧化酶(SOD)等生理生化指标。
(5)采用同源克隆法克隆了吴茱萸Cu/Zn-SOD和Mn/Fe-SOD基因的核心片段,根据片段序列设计特异性引物。使用3’,5’-末端快速扩增(RACE)技术克隆了上述2个基因的全长cDNA序列。
成果：
通过完成上述实验研究后,本论文取得了以下成果：
(1)吴茱萸栽培种种质资源遗传多样性ISSR分析实验获得了以下实验结果。贵州省吴茱萸居群的遗传多样性比江西省石虎居群的遗传多样性丰富；贵州省石虎居群的遗传多样性较高；不同产地居群遗传多样性从高到低依次为：7,10,1,11,9,3,6,2,4,吴茱萸药材3个基原品种群的遗传多样性从高到低依次为：吴茱萸,石虎和疏毛吴茱萸；不同产地及不同种群群体分化程度都较大,尤其是不同产地居群间,Gst达到了0.8164,不同品种群间的Gst为0.6898,而基因流的计算结果刚好相反,不同产地居群Nm小于不同种群,但是两者的基因流都较弱,可能与吴茱萸的繁殖方式多为扦插繁殖有关,同时生长环境的条件也有一定影响。UPGMA聚类表明,基于ISSR的遗传多样性分析不仅可以将正品吴茱萸基原植物与伪品—楝叶吴茱萸区分开,还能将不同吴茱萸药材不同基原种甚至是不同产地的样本进行有效分辨,显示了它们之间的亲缘关系,遗传距离和聚类分析表明,基于ISSR的遗传多样性分析是研究吴茱萸属植物栽培种亲缘关系和品种鉴定的可靠手段。
(2)吴茱萸种质资源ITS2DNA条形码实验获得了以下实验结果。ITS2序列长度均为220bp,共产生了17个单核苷酸(SNP)变异位点,占序列总长的7.73%,其中特异性位点7个。石虎与吴茱萸的种间遗传距离最近为0.036,疏毛吴茱萸与石虎的种间遗传距离最远为0.039。基原物种与楝叶吴茱萸的K2-P距离分布在0.042—0.067之间,疏毛吴茱萸与楝叶吴茱萸遗传距离最远,吴茱萸与楝叶吴茱萸遗传距离最近。邻接法(NJ)构建的吴茱萸属植物系统聚类树显示：18个样品主要分成3大支,楝叶吴茱萸单独形成一个分支,亲缘关系与吴茱萸3个基原种植物较远。
(3)药材含量测定实验获得了以下实验结果,吴茱萸和疏毛吴茱萸的吴茱萸碱、吴茱萸次碱、吴茱萸新碱等生物碱含量较石虎高,表现出与海拔高度呈正相关的趋势,而绿原酸、异鼠李素-3-O-半乳糖苷、槲皮素等非生物碱类物质则没有表现出上述的规律。HPLC化学指纹图谱实验获得了以下实验结果。10批次江西吴茱萸样品有19个共有峰,保留时间的RSD均小于0.4%,而峰面积的RSD比较大,说明江西省吴茱萸药材在化学成分的组成上基本一样,但是组分的量上存在差异。贵州省余庆县龙溪镇、贵州省石阡县白沙镇、贵州省石阡县白沙镇、贵州省松桃县正大乡、湖北、湖南样品指纹图谱与江西对照指纹图谱相似度为0.952、0.969、0.967、0.965,0.874,0.973,由此可知,吴茱萸和石虎两个不同种的相似度较高,说明两品种的吴茱萸药材品质较为一致。HPLC化学指纹图谱欧氏距离矩阵与ISSR分子标记遗传相似系数矩阵相关系数为0.469,相关性不显著。
(4)吴茱萸扦插苗干旱胁迫实验获得了以下实验结果。叶片可溶性蛋白含量变化总体趋势为：随胁迫强度和时间延长而呈先上升后下降,复水过程中,可溶性蛋白含量缓慢下降,但均高于胁迫状态时的含量。叶片可溶性糖含量变化总体趋势为：随胁迫强度和时间延长一直呈上升趋势,复水过程中,可溶性糖含量呈明显下降趋势,但均比CK高,不能恢复到CK水平。叶片游离脯氨酸含量变化总体趋势为：随胁迫强度和时间延长一直呈上升趋势,复水过程中,其含量较胁迫时均呈迅速下降趋势,但随着复水时间延长,其含量又缓慢上升。叶片MDA变化总体趋势为：随胁迫强度和时间延长一直呈上升趋势,复水过程中,除了在重度胁迫时,胁迫7d后复水条件下,MDA含量继续上升外,MDA含量较胁迫时均呈下降状态。叶片SOD活性变化总体趋势为：随胁迫强度和时间延长一直呈上升趋势,复水过程中,前期SOD活性继续升高,后期活性缓慢下降。叶片CAT活性变化总体趋势为：随胁迫强度呈先上升后下降的趋势,复水过程中,随时间延长而缓慢上升。叶片POD活性变化总体趋势为：随胁迫强度和时间延长一直呈上升趋势,复水过程中,短时间迅速下降,随时间延长降幅减缓,但均不能恢复到CK水平。
(5)吴茱萸Cu/Zn-SOD和Mn/Fe-SOD基因克隆实验获得了以下实验结果。吴茱萸Cu/Zn-SOD基因全长cDNA序列为717bp,开放阅读框长459bp,编码152个氨基酸,GenBank登录号为JQ285851。该基因编码的氨基酸分子量为15142.7Da,理论等电点pI为5.47,属亲水性酸性氨基酸,为稳定类蛋白。吴茱萸Mn/Fe-SOD基因全长cDNA序列为1048bp,开放阅读框长687bp,编码229个氨基酸,GenBank登录号为JQ285852。该基因编码的氨基酸分子量为25434.1Da,理论等电点pI为7.16,属亲水性碱性蛋白,为稳定类蛋白。
结论：
71份不同品种、不同来源地的吴茱萸属植物种质资源遗传多样性ISSR分析和DNA条形码ITS2区段分析的实验结果均证明了吴茱萸药材3个基原种中,石虎和疏毛吴茱萸的亲缘关系较为接近,但还是受到产地、环境和繁殖方式的影响而有所分化。ISSR标记和ITS2DNA条形码能作为吴茱萸及其种内变异种类有效鉴别的方法。吴茱萸药材HPLC化学指纹图谱证明了石虎药材和疏毛吴茱萸药材所含化学成分和含量相当,从化学成分方面证实了两者亲缘关系接近,与分子标记的实验结果一致。但HPLC指纹图谱聚类分析与ISSR聚类、ITS2聚类结果不完全一致,表明药用植物次生代谢产物积累可能受环境条件和种植水平等客观因素影响较大。HPLC含量测定结果表明,吴茱萸药材生物碱含量高低与栽培地海拔高度呈正相关趋势。
石虎扦插苗干旱胁迫和复水过程中植株体内细胞渗透调节物质和活性氧清除系统变化规律的实验证明了在逆境条件下细胞渗透调节物质和活性氧清除系统并不是同步运行的,变化规律复杂,其中可溶性蛋白、可溶性糖、游离脯氨酸含量及POD活性呈先上升后下降的变化趋势,MDA含量和SOD活性一直呈上升趋势,CAT活性呈现“N”型变曲线变化,即先上升后下降再上升的趋势。
吴茱萸SOD基因克隆实验证明了吴茱萸Cu/Zn-SOD和Mn/Fe-SOD基因与其他植物同类基因的相似度较高。该实验为下一步研究SOD基因调控对吴茱萸抗旱强弱的影响及抗旱优良品种选育打下基础。
Euodiae Fructus was a commonly used Chinese herbal medicine. Evoida rutaecarpas natural resources were rich and had a wide distribution but genetic variation were complex and genetic background was not clear because these reasons that the eclolgical environment very different,the ecological condition complicated,widely cultivated,for thousands of years all over the frequent exchanges between germplasm resources,drug habits vary greatly and had many species and varieties in Evodia.Now people about the phylogenetic evolution relationship between Evoida rutaecarpa of different species and cultivars and varieties and quality evaluation of genuine medicinal materials of different origin was not clear. In addition,drought stress susceptible to Evodia rutaecarpa during the growth of plants,resulting in excessive accumulation of active oxygen and active oxygen scavenging system disorders,caused serious adverse effects on the growth and quality of medicinal material plant.Therefore, studies on the diversity of germplasm resources of Evodia rutaecarpa and physiological drought has important scientific and practical significance.
Objective
Although molecular marker technique such as AFLP and RAPD on Evodia rutaecarpa germplasm genetic diversity study were used, but because of the number of samples and sample source, germplasm without extensive collection of other Evodia rutaecarpa advocate produce a division, so the genetic analysis had the certain limitation. In this study, Evodia rutaecarpa genuine medicine source plant cultivation and medicinal materials were collected extensively. The genetic germplasm resources and their diversity were analyzed and HPLC fingerprinting was construced. Germplasm resource of Evodia rutaecarpa was evaluated objectively. Its genetic diversity was discussed. These experiments could clarify the phylogenetic relationship of Evodia rutaecarpa cultivation. In addition, Evodia rutaecarpa in the growth process is vulnerable to drought stress, seriously affect the quality and yield of medicinal materials. At present, research on the mechanism of resistance to drought stress mainly concentrated on crops, and little study of medicinal plants, also study on drought resistance of Evodia rutaecarpa not been reported. This experiment using artificial simulated drought stress environment, study of Evodia rutaecarpa cuttings under variation of drought stress and rewatering during various physiological and biochemical indexes and cloned the full-length sequence of key resistance gene Cu/Zn-SOD and Mn/Fe-SOD genes, the next step for the research has laid a solid foundation.
Methods
1. Literature research
The ancient books related to this study and the literature home and abroad were retrivealed and summarized. The research trends related to the paper was reviewed. Mainly includes the following four aspects:
(1) Herbal research of Evodia rutaecarpa include Survey medical history, resource distribution, function and application, plant characteristics and variety identification.
(2) Evodia rutaecarpa germplasm genentic diversity's research progress about morphological markers, cytological analysis, DNA molecurlar markers and DNA barcoding.
(3) Evodia rutaecarpa s cell osmotic adjustment and Active Oxygen Scavenging system(ROS) and plant SOD progress in drought stress environment.
(4) Research progress of Evodia rutaecarpa on chemical quality evaluation include chemical fingerprint and chemical composition.
2. Experimenal research
DNA molecular marker technology, DNA barcoding and chemical analysis method for the evaluation of germplasm resources and diversity of Evodia rutaecarpa herbs of different source and origin in this paper. The several physiological and biochemical indexes of cutting seedling in artifical simulated drought stress conditions were studied. Finally, Cu/Zn-SOD and Mn/Fe-SOD gene of Evodia rutaecarpa were cloned. The research contents of this paper include:
(1) Seventy-one cultivars of Evodia on originates from Guizhou province, Jiangxi province, Hunan province, Hubei province and Guangdong province of plant germplasm resources were analyzed to study the genetic diversity by ISSR molecular markers.
(2) The Evoida genus18samples of different varieties and different sources were analyzed using ITS2DNA barcoding technology.
(3) The contents of six kinds of chlorogenic acid, isorhamnetin-3-O-galactoside,quercetin, evodiamine, rutaecarpine and evocarpine were determined in37batches of material medicines used HPLC method. HPLC chemical fingerpring on37batches of material medicines of Evodia rutaecarpa were constructed using HPLC method.
(4) The Evodia rutaecarpa cutting seedlings were treated using different concentration of PEG-6000solution and different time. The physiological and biochemical indexes such as soluble protein, soluble sugar, praline, malondialdehyde(MDA), catalase(CAT), peroxidase(POD) and superoxide dismutase(SOD) were determinated.
(5) By homology cloning method for cloning the core fragments of Evodia rutaecarpa's Cu/Zn-SOD and Mn/Fe-SOD genes, specific primers were designed according to the sequences.3,5-Rapid Amplification of cDNA Ends(RACE) technology was used to clone the full-length cDNA sequence of the two genes.
Results
Through the experimental study, this paper has made the following achievements:
(1)ISSR genetic diversity of germplasm resources of Evodia rutaecarpa experiment has obtained the following results. The genetic divertiy of Wuzhuyu populations in Guizhou province than Shihu groups of Jiangxi province. The diversity of Shihu populations was higher. The genetic diversity of the populations of different regions are arranged from high to low is:7,10,1,11,9,3,6,2and4. Three genetic base original species of Evodia rutaecarpa medicine diversity from high to low for:Wuzhuyu>Shihu>Shumaowuzhuyu. Different areas and different varieties of groups differentiation are larger, especially the Gst reached0.8164that from different pophlations and different varieties of group Gst was0.6898, but gene flow calculation result was just the opposite. Different populations of Nm less than cultivars in different groups, but the gene flow were weak. The reasons are which reproduction of Evodia rutaecarpa are cutting propagation and growth environments conditions also had certain influence. UPGMA clustering showed that ISSR genetic diversity analysis can not only separate the genuine Evodia rutaecarpa original plant with fake-Evodia glabrifolia and Evodia rutaecarpa also would be different samples of different varieties or even different areas of original samples are effective resolution and shows the relationship between the species, the genetic distance and cluster analysis. ISSR genetic diversity analysis is a reliable means to study the Evodia rutaecarpa genus relationship and cultivar identification.
(2) Evodia rutaecarpa germplasm resources ITS2DNA barcoding experiment has obtained the following experimental results. ITS2sequences length are220bp, yielding a total of17single nucleotide(SNP) sequence variation sites, accounting for7.73%of the total length, in which specific stie are seven. The interspecific genetic distance Shihu and Wuzhuyu recently0.036, interspecific genetic distance Shumaowuzhuyu and Shihu up to0.039. The K2-P distance distribution of base the original species and Evodia glabrifolia in the0.042to0.067. Evodia rutaecarpa and Evodia glabrifolia nearest genetic distance. Neighbor-joining(NJ) to construct that Evodia rutaecarpa genus system clustering tree display that18samples are divided into three branches which Evodia glabrifolia to form a single branch,phylogenetic relationships and Evodia rutaecarpa three base stock plant far.
(3) The following experimental results of drug content determination were obtained that wuzhuyu and shumaowuzhuyu's contents of evodiamine, rutaecarpine and evocarpine were higher than shihu's. These alkaloids contents had the tendency that correlated with altitude while the contents of chlorogenic acid, isorhamnetin-3-O-galactoside and quercetin did not show the above rule.HPLC fingerprint experiment has obtained the following experimental results. Ten batches samples of Evodia rutaecarpa in Jiangxi has19common peaks and the retention time of RSD is less than0.4%and the peak area of RSD is large, which show that Jiangxi province medicinal Evodia rutaecarpa is basically the same as in the chemical composition of the components but the amount of different Guizhou province Yuqing country dragon town, Guizhou Shiqian baisha town, Guizhou Shiqian baisha town, Guizhou province Songtao country zhengda town, Hubei, Hunan and Jiangxi control sample fingerprint similarity was0.952,0.969,0.965,0.874,0.973. Therefore, two different varieties of Evodia rutaecarpa and Shihu similarity, two species of medicinal Evodia rutaecarpa quality more consistent. Study on HPLC fingerprint of the Euclidean distance martrix and ISSR molecular marker genetic similarity coefficient matrix of related coefficient was0.469that the correlation was not significant.
(4) Drought stress experiment of Evoida rutaecarpa cutting seeding has obtained the following experimental results. Changes in the trend of the overall content of soluble protein in leaves is that increased first and then decreased with the increasing of stress intensity and time, the complex process of water, soluble protein content decreased slowly but higher than the content of stress state. Changes in the trend of overall soluble sugar content is that an upward trend with the stress intensity and time, the complex process of water, soluble sugar content was significantly decreased but higher than the CK can not be restored to the level of CK. Changes in the trend of overallproline content is that has been an upward trend with the stress intensity and time and at rehydration process that its content is stress showed the downtrend but with the rehydration time prolonging the content and slowly rising. Leaf blade MDA change the general trend is that has been an upward trend with the stress intensity and time and rehydration process, except in severe stress, seven days stress after rewatering conditions the content of MDA continues to rise the content of MDA is relatively stress showed decline. Change tendency of SOD activity in leaf is that has been an upward trend with the stress intensity and time and rewatering process its activity continues to rise and the late activity decreased slowly. Change tendency of CAT activity in leaf is that increased first and then decreased with increasing stress intensity and at rewatering process with the extension of time and slow rise. Change tendency of POD activity in leaf is that has been an upward trend with the stress intensity and time and at rewatering process decreased rapiadly in short time with the time prolonging fell down but could not recover to the level of CK.
(5) Evoida rutaecarpa' s Cu/Zn-SOD and Mn/Fe-SOD gene cloning experiment has obtained the following experimental results. The full-length cDNA sequence of Cu/Zn-SOD gene is717bp and the open reading frame is459bp that encoding152amino acids. Its Genbank accession number is JQ285851This amino acid molecular weight is15142.7Da and isoelectric point is5.47, belonging to the hydrophilic acidic amion acid and stable protein. The full-length cDNA sequence of Mn/Fe-SOD gene is1048bp and the open reading frame is687bp that endoding229amino acids. Its Genbank accession number is JQ285852. This amino acid molecular weight is25434.1Da and isoelectric point is7.16, belonging to the hydrophilic basic protein and stable protein.
Conslusion
Analysis of senenty-one samples of germplasm of genus Evoida rutaecarpa in different varieties and different source of the genetic diversity of ISSR analysis and DNA barcoding region ITS2experiments proved that the genetic relationship of the original plants is closer but still affected areas, environment and reproduction method but has the differentiation. HPLC chemical fingerprint proved that Shihu and Shumuwuzhuyu contained in the chemical composition and content. The chemical composition of both confirmed the close genetic relationship. This result is common that the experimental result of molecular marker.But HPLC fingerprinting culster analysis and ISSR clustering and ITS2culstering results were not entirely consistent that showed themedicinal plant's secondary metabolite accumualtion may be affected by the objective factors such as environmental conditions and culture level.HPLC determination results showed that Evodia rutaecarpa's alkaloid content had the tendency that related to altitude and cultivation.
The change of cell osmotic substances and ROS system in cutting seedling at drought stress and rewatering time proves that the permeability of the somotic and ROS system is not synchronous operation under adverse conditions and the changing law is complicated. Soluble protein, free proline content and POD activity were increased firstly and then decreased. MDA content and SOD activity were increased. CAT activity showed "N"type curve change, namely the first increase then decrease and increase trend.
SOD gene cloning experiments proved that Cu/Zn-SOD and Mn/Fe-SOD gene have high similarity with other plants of similar genes. This experiment lays a foundation for breeding of drought resistance and SOD gene regulation.
目的：
尽管前人采用AFLP、RAPD等分子标记技术对吴茱萸种质资源遗传多样性进行了研究,但由于受到样本数量和样本来源地的限制,没有广泛收集其它吴茱萸主产区的种质,因此其遗传多样性分析具有一定的局限性。本研究广泛收集吴茱萸正品药材基源植物的栽培种和药材,分析了它们的种质资源遗传多样性,构建了HPLC化学指纹图谱,客观的评价了吴茱萸种质资源,进一步探讨了其遗传多样性,为阐明吴茱萸栽培种的亲缘关系提供了科学的实验依据。
吴茱萸在生长过程中易受干旱胁迫,严重影响药材质量和产量。目前,干旱胁迫的抵御机制研究主要集中在农作物上,药用植物相关研究较少,还未见吴茱萸抗旱生理研究的报道。本实验利用人为模拟的干旱胁迫环境,研究了吴茱萸扦插苗在受到干旱胁迫和复水过程各项生理生化指标的变化规律并克隆了抗逆关键基因—Cu/Zn-SOD和Mn/Fe-SOD基因的全长序列,为下一步深入研究奠定了坚实的基础。
方法：
1.文献研究
检索与本研究相关的古籍和国内外文献,并进行了总结和综述,概括了与本论文相关的研究动态。主要包括以下4个方面：
(1)吴茱萸药用历史、资源分布、性味功效与应用、吴茱萸植物特征与品种鉴别等吴茱萸本草考证概况；
(2)吴茱萸植物形态学标记、细胞学分析、DNA分子标记和DNA条形码等吴茱萸植物种质资源多样性研究进展；
(3)干旱胁迫环境下植物细胞渗透调节物质和活性氧(ROS)清除系统及植物SOD研究进展；
(4)吴茱萸化学成分、化学指纹图谱等吴茱萸化学质量评价研究进展。
2.实验研究
本文利用DNA分子标记技术、DNA条形码技术、分析化学方法对不同来源地和产地的吴茱萸种质资源及药材进行了多样性评价；并且,对吴茱萸扦插苗在人工模拟的干旱胁迫环境条件下,植株内多种生理生化指标变化规律及SOD基因的克隆进行了研究。本文研究内容包括：
(1)采用ISSR分子标记技术对来源于贵州省、江西省、湖南省、湖北省、广东省的71份栽培吴茱萸属植物种质资源进行了遗传多样性研究分析。
(2)采用ITS2DNA条形码技术分析了代表不同来源地、不同种的吴茱萸属植物样品18份。
(3)使用HPLC法测定了绿原酸、异鼠李素-3-O-半乳糖苷、槲皮素、吴茱萸碱、吴茱萸次碱和吴茱萸新碱等6种有效成分的含量并构建了37个批次的吴茱萸药材HPLC化学指纹图谱。
(4)使用不同浓度PEG—-6000溶液,经过不同时间处理吴茱萸扦插苗,测定可溶性蛋白、可溶性糖、游离脯氨酸、丙二醛(MDA)、过氧化氢酶(CAT)、过氧化物酶(POD)、超氧化物歧化酶(SOD)等生理生化指标。
(5)采用同源克隆法克隆了吴茱萸Cu/Zn-SOD和Mn/Fe-SOD基因的核心片段,根据片段序列设计特异性引物。使用3’,5’-末端快速扩增(RACE)技术克隆了上述2个基因的全长cDNA序列。
成果：
通过完成上述实验研究后,本论文取得了以下成果：
(1)吴茱萸栽培种种质资源遗传多样性ISSR分析实验获得了以下实验结果。贵州省吴茱萸居群的遗传多样性比江西省石虎居群的遗传多样性丰富；贵州省石虎居群的遗传多样性较高；不同产地居群遗传多样性从高到低依次为：7,10,1,11,9,3,6,2,4,吴茱萸药材3个基原品种群的遗传多样性从高到低依次为：吴茱萸,石虎和疏毛吴茱萸；不同产地及不同种群群体分化程度都较大,尤其是不同产地居群间,Gst达到了0.8164,不同品种群间的Gst为0.6898,而基因流的计算结果刚好相反,不同产地居群Nm小于不同种群,但是两者的基因流都较弱,可能与吴茱萸的繁殖方式多为扦插繁殖有关,同时生长环境的条件也有一定影响。UPGMA聚类表明,基于ISSR的遗传多样性分析不仅可以将正品吴茱萸基原植物与伪品—楝叶吴茱萸区分开,还能将不同吴茱萸药材不同基原种甚至是不同产地的样本进行有效分辨,显示了它们之间的亲缘关系,遗传距离和聚类分析表明,基于ISSR的遗传多样性分析是研究吴茱萸属植物栽培种亲缘关系和品种鉴定的可靠手段。
(2)吴茱萸种质资源ITS2DNA条形码实验获得了以下实验结果。ITS2序列长度均为220bp,共产生了17个单核苷酸(SNP)变异位点,占序列总长的7.73%,其中特异性位点7个。石虎与吴茱萸的种间遗传距离最近为0.036,疏毛吴茱萸与石虎的种间遗传距离最远为0.039。基原物种与楝叶吴茱萸的K2-P距离分布在0.042—0.067之间,疏毛吴茱萸与楝叶吴茱萸遗传距离最远,吴茱萸与楝叶吴茱萸遗传距离最近。邻接法(NJ)构建的吴茱萸属植物系统聚类树显示：18个样品主要分成3大支,楝叶吴茱萸单独形成一个分支,亲缘关系与吴茱萸3个基原种植物较远。
(3)药材含量测定实验获得了以下实验结果,吴茱萸和疏毛吴茱萸的吴茱萸碱、吴茱萸次碱、吴茱萸新碱等生物碱含量较石虎高,表现出与海拔高度呈正相关的趋势,而绿原酸、异鼠李素-3-O-半乳糖苷、槲皮素等非生物碱类物质则没有表现出上述的规律。HPLC化学指纹图谱实验获得了以下实验结果。10批次江西吴茱萸样品有19个共有峰,保留时间的RSD均小于0.4%,而峰面积的RSD比较大,说明江西省吴茱萸药材在化学成分的组成上基本一样,但是组分的量上存在差异。贵州省余庆县龙溪镇、贵州省石阡县白沙镇、贵州省石阡县白沙镇、贵州省松桃县正大乡、湖北、湖南样品指纹图谱与江西对照指纹图谱相似度为0.952、0.969、0.967、0.965,0.874,0.973,由此可知,吴茱萸和石虎两个不同种的相似度较高,说明两品种的吴茱萸药材品质较为一致。HPLC化学指纹图谱欧氏距离矩阵与ISSR分子标记遗传相似系数矩阵相关系数为0.469,相关性不显著。
(4)吴茱萸扦插苗干旱胁迫实验获得了以下实验结果。叶片可溶性蛋白含量变化总体趋势为：随胁迫强度和时间延长而呈先上升后下降,复水过程中,可溶性蛋白含量缓慢下降,但均高于胁迫状态时的含量。叶片可溶性糖含量变化总体趋势为：随胁迫强度和时间延长一直呈上升趋势,复水过程中,可溶性糖含量呈明显下降趋势,但均比CK高,不能恢复到CK水平。叶片游离脯氨酸含量变化总体趋势为：随胁迫强度和时间延长一直呈上升趋势,复水过程中,其含量较胁迫时均呈迅速下降趋势,但随着复水时间延长,其含量又缓慢上升。叶片MDA变化总体趋势为：随胁迫强度和时间延长一直呈上升趋势,复水过程中,除了在重度胁迫时,胁迫7d后复水条件下,MDA含量继续上升外,MDA含量较胁迫时均呈下降状态。叶片SOD活性变化总体趋势为：随胁迫强度和时间延长一直呈上升趋势,复水过程中,前期SOD活性继续升高,后期活性缓慢下降。叶片CAT活性变化总体趋势为：随胁迫强度呈先上升后下降的趋势,复水过程中,随时间延长而缓慢上升。叶片POD活性变化总体趋势为：随胁迫强度和时间延长一直呈上升趋势,复水过程中,短时间迅速下降,随时间延长降幅减缓,但均不能恢复到CK水平。
(5)吴茱萸Cu/Zn-SOD和Mn/Fe-SOD基因克隆实验获得了以下实验结果。吴茱萸Cu/Zn-SOD基因全长cDNA序列为717bp,开放阅读框长459bp,编码152个氨基酸,GenBank登录号为JQ285851。该基因编码的氨基酸分子量为15142.7Da,理论等电点pI为5.47,属亲水性酸性氨基酸,为稳定类蛋白。吴茱萸Mn/Fe-SOD基因全长cDNA序列为1048bp,开放阅读框长687bp,编码229个氨基酸,GenBank登录号为JQ285852。该基因编码的氨基酸分子量为25434.1Da,理论等电点pI为7.16,属亲水性碱性蛋白,为稳定类蛋白。
结论：
71份不同品种、不同来源地的吴茱萸属植物种质资源遗传多样性ISSR分析和DNA条形码ITS2区段分析的实验结果均证明了吴茱萸药材3个基原种中,石虎和疏毛吴茱萸的亲缘关系较为接近,但还是受到产地、环境和繁殖方式的影响而有所分化。ISSR标记和ITS2DNA条形码能作为吴茱萸及其种内变异种类有效鉴别的方法。吴茱萸药材HPLC化学指纹图谱证明了石虎药材和疏毛吴茱萸药材所含化学成分和含量相当,从化学成分方面证实了两者亲缘关系接近,与分子标记的实验结果一致。但HPLC指纹图谱聚类分析与ISSR聚类、ITS2聚类结果不完全一致,表明药用植物次生代谢产物积累可能受环境条件和种植水平等客观因素影响较大。HPLC含量测定结果表明,吴茱萸药材生物碱含量高低与栽培地海拔高度呈正相关趋势。
石虎扦插苗干旱胁迫和复水过程中植株体内细胞渗透调节物质和活性氧清除系统变化规律的实验证明了在逆境条件下细胞渗透调节物质和活性氧清除系统并不是同步运行的,变化规律复杂,其中可溶性蛋白、可溶性糖、游离脯氨酸含量及POD活性呈先上升后下降的变化趋势,MDA含量和SOD活性一直呈上升趋势,CAT活性呈现“N”型变曲线变化,即先上升后下降再上升的趋势。
吴茱萸SOD基因克隆实验证明了吴茱萸Cu/Zn-SOD和Mn/Fe-SOD基因与其他植物同类基因的相似度较高。该实验为下一步研究SOD基因调控对吴茱萸抗旱强弱的影响及抗旱优良品种选育打下基础。
Euodiae Fructus was a commonly used Chinese herbal medicine. Evoida rutaecarpas natural resources were rich and had a wide distribution but genetic variation were complex and genetic background was not clear because these reasons that the eclolgical environment very different,the ecological condition complicated,widely cultivated,for thousands of years all over the frequent exchanges between germplasm resources,drug habits vary greatly and had many species and varieties in Evodia.Now people about the phylogenetic evolution relationship between Evoida rutaecarpa of different species and cultivars and varieties and quality evaluation of genuine medicinal materials of different origin was not clear. In addition,drought stress susceptible to Evodia rutaecarpa during the growth of plants,resulting in excessive accumulation of active oxygen and active oxygen scavenging system disorders,caused serious adverse effects on the growth and quality of medicinal material plant.Therefore, studies on the diversity of germplasm resources of Evodia rutaecarpa and physiological drought has important scientific and practical significance.
Objective
Although molecular marker technique such as AFLP and RAPD on Evodia rutaecarpa germplasm genetic diversity study were used, but because of the number of samples and sample source, germplasm without extensive collection of other Evodia rutaecarpa advocate produce a division, so the genetic analysis had the certain limitation. In this study, Evodia rutaecarpa genuine medicine source plant cultivation and medicinal materials were collected extensively. The genetic germplasm resources and their diversity were analyzed and HPLC fingerprinting was construced. Germplasm resource of Evodia rutaecarpa was evaluated objectively. Its genetic diversity was discussed. These experiments could clarify the phylogenetic relationship of Evodia rutaecarpa cultivation. In addition, Evodia rutaecarpa in the growth process is vulnerable to drought stress, seriously affect the quality and yield of medicinal materials. At present, research on the mechanism of resistance to drought stress mainly concentrated on crops, and little study of medicinal plants, also study on drought resistance of Evodia rutaecarpa not been reported. This experiment using artificial simulated drought stress environment, study of Evodia rutaecarpa cuttings under variation of drought stress and rewatering during various physiological and biochemical indexes and cloned the full-length sequence of key resistance gene Cu/Zn-SOD and Mn/Fe-SOD genes, the next step for the research has laid a solid foundation.
Methods
1. Literature research
The ancient books related to this study and the literature home and abroad were retrivealed and summarized. The research trends related to the paper was reviewed. Mainly includes the following four aspects:
(1) Herbal research of Evodia rutaecarpa include Survey medical history, resource distribution, function and application, plant characteristics and variety identification.
(2) Evodia rutaecarpa germplasm genentic diversity's research progress about morphological markers, cytological analysis, DNA molecurlar markers and DNA barcoding.
(3) Evodia rutaecarpa s cell osmotic adjustment and Active Oxygen Scavenging system(ROS) and plant SOD progress in drought stress environment.
(4) Research progress of Evodia rutaecarpa on chemical quality evaluation include chemical fingerprint and chemical composition.
2. Experimenal research
DNA molecular marker technology, DNA barcoding and chemical analysis method for the evaluation of germplasm resources and diversity of Evodia rutaecarpa herbs of different source and origin in this paper. The several physiological and biochemical indexes of cutting seedling in artifical simulated drought stress conditions were studied. Finally, Cu/Zn-SOD and Mn/Fe-SOD gene of Evodia rutaecarpa were cloned. The research contents of this paper include:
(1) Seventy-one cultivars of Evodia on originates from Guizhou province, Jiangxi province, Hunan province, Hubei province and Guangdong province of plant germplasm resources were analyzed to study the genetic diversity by ISSR molecular markers.
(2) The Evoida genus18samples of different varieties and different sources were analyzed using ITS2DNA barcoding technology.
(3) The contents of six kinds of chlorogenic acid, isorhamnetin-3-O-galactoside,quercetin, evodiamine, rutaecarpine and evocarpine were determined in37batches of material medicines used HPLC method. HPLC chemical fingerpring on37batches of material medicines of Evodia rutaecarpa were constructed using HPLC method.
(4) The Evodia rutaecarpa cutting seedlings were treated using different concentration of PEG-6000solution and different time. The physiological and biochemical indexes such as soluble protein, soluble sugar, praline, malondialdehyde(MDA), catalase(CAT), peroxidase(POD) and superoxide dismutase(SOD) were determinated.
(5) By homology cloning method for cloning the core fragments of Evodia rutaecarpa's Cu/Zn-SOD and Mn/Fe-SOD genes, specific primers were designed according to the sequences.3,5-Rapid Amplification of cDNA Ends(RACE) technology was used to clone the full-length cDNA sequence of the two genes.
Results
Through the experimental study, this paper has made the following achievements:
(1)ISSR genetic diversity of germplasm resources of Evodia rutaecarpa experiment has obtained the following results. The genetic divertiy of Wuzhuyu populations in Guizhou province than Shihu groups of Jiangxi province. The diversity of Shihu populations was higher. The genetic diversity of the populations of different regions are arranged from high to low is:7,10,1,11,9,3,6,2and4. Three genetic base original species of Evodia rutaecarpa medicine diversity from high to low for:Wuzhuyu>Shihu>Shumaowuzhuyu. Different areas and different varieties of groups differentiation are larger, especially the Gst reached0.8164that from different pophlations and different varieties of group Gst was0.6898, but gene flow calculation result was just the opposite. Different populations of Nm less than cultivars in different groups, but the gene flow were weak. The reasons are which reproduction of Evodia rutaecarpa are cutting propagation and growth environments conditions also had certain influence. UPGMA clustering showed that ISSR genetic diversity analysis can not only separate the genuine Evodia rutaecarpa original plant with fake-Evodia glabrifolia and Evodia rutaecarpa also would be different samples of different varieties or even different areas of original samples are effective resolution and shows the relationship between the species, the genetic distance and cluster analysis. ISSR genetic diversity analysis is a reliable means to study the Evodia rutaecarpa genus relationship and cultivar identification.
(2) Evodia rutaecarpa germplasm resources ITS2DNA barcoding experiment has obtained the following experimental results. ITS2sequences length are220bp, yielding a total of17single nucleotide(SNP) sequence variation sites, accounting for7.73%of the total length, in which specific stie are seven. The interspecific genetic distance Shihu and Wuzhuyu recently0.036, interspecific genetic distance Shumaowuzhuyu and Shihu up to0.039. The K2-P distance distribution of base the original species and Evodia glabrifolia in the0.042to0.067. Evodia rutaecarpa and Evodia glabrifolia nearest genetic distance. Neighbor-joining(NJ) to construct that Evodia rutaecarpa genus system clustering tree display that18samples are divided into three branches which Evodia glabrifolia to form a single branch,phylogenetic relationships and Evodia rutaecarpa three base stock plant far.
(3) The following experimental results of drug content determination were obtained that wuzhuyu and shumaowuzhuyu's contents of evodiamine, rutaecarpine and evocarpine were higher than shihu's. These alkaloids contents had the tendency that correlated with altitude while the contents of chlorogenic acid, isorhamnetin-3-O-galactoside and quercetin did not show the above rule.HPLC fingerprint experiment has obtained the following experimental results. Ten batches samples of Evodia rutaecarpa in Jiangxi has19common peaks and the retention time of RSD is less than0.4%and the peak area of RSD is large, which show that Jiangxi province medicinal Evodia rutaecarpa is basically the same as in the chemical composition of the components but the amount of different Guizhou province Yuqing country dragon town, Guizhou Shiqian baisha town, Guizhou Shiqian baisha town, Guizhou province Songtao country zhengda town, Hubei, Hunan and Jiangxi control sample fingerprint similarity was0.952,0.969,0.965,0.874,0.973. Therefore, two different varieties of Evodia rutaecarpa and Shihu similarity, two species of medicinal Evodia rutaecarpa quality more consistent. Study on HPLC fingerprint of the Euclidean distance martrix and ISSR molecular marker genetic similarity coefficient matrix of related coefficient was0.469that the correlation was not significant.
(4) Drought stress experiment of Evoida rutaecarpa cutting seeding has obtained the following experimental results. Changes in the trend of the overall content of soluble protein in leaves is that increased first and then decreased with the increasing of stress intensity and time, the complex process of water, soluble protein content decreased slowly but higher than the content of stress state. Changes in the trend of overall soluble sugar content is that an upward trend with the stress intensity and time, the complex process of water, soluble sugar content was significantly decreased but higher than the CK can not be restored to the level of CK. Changes in the trend of overallproline content is that has been an upward trend with the stress intensity and time and at rehydration process that its content is stress showed the downtrend but with the rehydration time prolonging the content and slowly rising. Leaf blade MDA change the general trend is that has been an upward trend with the stress intensity and time and rehydration process, except in severe stress, seven days stress after rewatering conditions the content of MDA continues to rise the content of MDA is relatively stress showed decline. Change tendency of SOD activity in leaf is that has been an upward trend with the stress intensity and time and rewatering process its activity continues to rise and the late activity decreased slowly. Change tendency of CAT activity in leaf is that increased first and then decreased with increasing stress intensity and at rewatering process with the extension of time and slow rise. Change tendency of POD activity in leaf is that has been an upward trend with the stress intensity and time and at rewatering process decreased rapiadly in short time with the time prolonging fell down but could not recover to the level of CK.
(5) Evoida rutaecarpa' s Cu/Zn-SOD and Mn/Fe-SOD gene cloning experiment has obtained the following experimental results. The full-length cDNA sequence of Cu/Zn-SOD gene is717bp and the open reading frame is459bp that encoding152amino acids. Its Genbank accession number is JQ285851This amino acid molecular weight is15142.7Da and isoelectric point is5.47, belonging to the hydrophilic acidic amion acid and stable protein. The full-length cDNA sequence of Mn/Fe-SOD gene is1048bp and the open reading frame is687bp that endoding229amino acids. Its Genbank accession number is JQ285852. This amino acid molecular weight is25434.1Da and isoelectric point is7.16, belonging to the hydrophilic basic protein and stable protein.
Conslusion
Analysis of senenty-one samples of germplasm of genus Evoida rutaecarpa in different varieties and different source of the genetic diversity of ISSR analysis and DNA barcoding region ITS2experiments proved that the genetic relationship of the original plants is closer but still affected areas, environment and reproduction method but has the differentiation. HPLC chemical fingerprint proved that Shihu and Shumuwuzhuyu contained in the chemical composition and content. The chemical composition of both confirmed the close genetic relationship. This result is common that the experimental result of molecular marker.But HPLC fingerprinting culster analysis and ISSR clustering and ITS2culstering results were not entirely consistent that showed themedicinal plant's secondary metabolite accumualtion may be affected by the objective factors such as environmental conditions and culture level.HPLC determination results showed that Evodia rutaecarpa's alkaloid content had the tendency that related to altitude and cultivation.
The change of cell osmotic substances and ROS system in cutting seedling at drought stress and rewatering time proves that the permeability of the somotic and ROS system is not synchronous operation under adverse conditions and the changing law is complicated. Soluble protein, free proline content and POD activity were increased firstly and then decreased. MDA content and SOD activity were increased. CAT activity showed "N"type curve change, namely the first increase then decrease and increase trend.
SOD gene cloning experiments proved that Cu/Zn-SOD and Mn/Fe-SOD gene have high similarity with other plants of similar genes. This experiment lays a foundation for breeding of drought resistance and SOD gene regulation.
引文
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