【作者】 赵云生；

【导师】 万德光；

【作者基本信息】 成都中医药大学 ， 中药学， 2009， 博士

【摘要】 远志为远志科（Polygalaceae）植物远志Polygala tenuifolia Willd.或卵叶远志P. sibirica L.的干燥根,具有安神益智、祛痰、消肿、抗炎与抗诱变等作用,临床应用较广。本文从远志高产途径、质量评价、种质资源遗传多样性、远志鲨烯环氧酶（SE）基因克隆四个方面对远志进行了研究。1.远志高产途径研究以远志种子千粒重与发芽率为指标拟定了远志种子贮存年限与千粒重标准：一级种子,贮存年限≤2年,千粒重≥2.711g；二级种子,贮存年限≤4年,千粒重≥2.441g；三级种子为劣质种子。首次采用二次饱和D-最优设计建立了远志产量与N、P₂O₅、K₂O施用量的产量回归模型：Y=5121+36.113X₁+150.557X₂+71.157X₃-439.15X₁²-321.447X₂²-168.478X₃²-91.306X₁X₂-28.206X₁X₃+8.238X₂X₃。氮、磷、钾对远志产量效应顺序为：N（X₁）>P（X₂）>K（X₃）,交互作用顺序为：NP>PK>NK,获得5146.172kg/hm²远志需投入氮117.128kg,磷,120.333kg,钾,136.257kg/hm²,投入比例1∶1.03∶1.16。旱棚下采用盆栽方式进行了水肥耦合试验,土壤含水量14%的低水条件下,氮肥早施（开花期）优于晚施,低量氮肥比高量氮肥好,土壤含水量24%的高水条件下,返青期+开花期施N处理,产量极显著高于其它处理组合。远志对氮肥的耐受范围较窄,生长早期耐受性强,开花期是远志对水分与氮肥的需求高效期。远志需水规律与病虫害防治研究结果表明盛花期灌溉产量最高,乐果与苦参碱防治蚜虫效果显著高于鱼藤碱,而乐果与苦参碱间防治效果差异不显著,生物农药木霉制剂与化学农药退菌特结合施用比二者分别单独施用,对防治远志根腐病效果显著。首次对大田栽培远志群体生长动态进行了系统研究,2007～2008年远志主茎直径、鲜根直径、鲜根／干根比、鲜根重、根干物质重、茎叶干物质重、生物学产量均随生长期的延长而增加,分枝个数、支根数2007年较2008年为多,远志年生长期内株高变化呈抛物线形,2008年远志株高明显高于2007年。远志根干物质月增长最快时期在2007年7月20日至8月20日与2008年6月27日至7月27日,2008年远志根部干物质积累总量大于2007年。2.远志质量评价研究远志HPLC指纹图谱试验结果显示：23批远志指纹图谱相似度较高,远志野生变家栽后,有效物质群与野生药材相差较小,质量相当。不同采收月份与生长时期栽培远志指纹图谱结构相似,生长时间对远志有效物质群种类影响不大。远志根心对照图谱与药材指纹图谱结构相似,但峰面积较小、含量较低,栽培与野生远志根心指纹图谱与其对照图谱相似度均在0.807以上。远志酸含量影响因素试验结果表明：产地对远志酸含量影响达极显著水平；海拔高度与纬度对远志酸含量具有一定影响；2008年不同月份采收的远志,远志酸含量明显高于2007年同期采收的远志,远志中远志酸含量与远志生长年限与收获时期密切相关,而与远志生长发育阶段关系可能并不密切,远志在达到生长年限后应选择6、7月份采收较好；远志根心中含有一定量的远志酸,根心与药材远志酸积累具有负相关的趋势；不同产地远志（Polygala tenuifolia Willd）叶中几乎不含远志酸,但四川茂县与九龙山卵叶远志叶（P. sibirical L）中远志酸含量稍高。3.种质资源遗传多样性研究首次采用AFLP（amplified fragment length polymorphism）技术、毛细管电泳技术、植物等位酶分析技术在DNA、蛋白质与等位酶三个层次分别对远志种质资源遗传多样性进行了研究。对远志种质资源DNA采用AFLP技术进行研究,共扩增出清晰可重复的310个等位基因谱带,其中多态性条带261条,占84.19%,平均每个位点有21.75个等位变异,有效等位基因数1.49个,24个远志居群的遗传一致度在0.4000-0.8645之间,遗传距离在0.1456～0.9163之间,表明远志居群的遗传多样性较为丰富,远志AFLP分子标记的结果与经典形态学分类结论一致,也与地理分布的不同相一致。采用毛细管电泳技术对远志种质资源蛋白质遗传多样性研究结果表明,远志种质资源相似系数在0.316～0.895之间,遗传多样性丰富,通过远志种质资源蛋白质所反映的亲缘关系仍表现为地理位置相近的远志资源相似系数较高,亲缘关系较近,但远志栽培资源蛋白质性状差异较大,山西不同产地的栽培远志,在蛋白质的多样性表达上却表现出与地理位置较远的四川茂县或甘肃等地野生远志较高的相似性,远志蛋白质遗传多样性研究与远志DNA遗传多样性研究结果并不完全一致。远志可溶性蛋白与等位酶的研究结果表明,迁移率为0.136处的过氧化物酶谱带与迁移率为0.818处的酯酶谱带可以将卵叶远志与远志区别开来,迁移率为0.492处的过氧化物酶谱带可以将山西沁源远志与其它8种远志区别开来,远志等位酶电泳条带多态性在0.198～0.444之间,9份远志种质资源的遗传一致性在0.3～0.9之间,卵叶远志与远志间的遗传一致性明显低于远志种质资源间的遗传一致性,遗传距离较大,远志可溶性蛋白与等位酶酶谱相似度与远志地理位置的分布具有一定的相关性。首次对远志酸生物合成途径中关键酶鲨烯环氧酶基因（SE）进行了研究,利用5对简并引物,对远志鲨烯环氧酶DNA基因进行克隆,获得一条517bp的核苷酸,经GenBank Blastx鉴定,该核苷酸片段属于SE基因片断,根据Zea mays完整的cDNA SE基因序列（Accession:BT042800）推测,已克隆远志SE基因片段与5`端的距离有1426bp,与3`端的距离有110bp,在已克隆部位,Zea mays序列长度比远志SE基因片段长23bp。更多还原

【Abstract】 Radix Polygalae is the dried root of Polygala tenuifolia Willd. or P. sibirica L It has a wide clinical use for its effection of calm the nerves, beneficial intellect, expectorant, detumescence, antiinflammatory and antimutagenesis. This essay contains four research sections:channels of high yield, quality assessment, genetic diversity, gene cloning of squalene epoxidase partial cds which is the key enzyme of biological synthesis of polygalacic acid.1. Ways of high yieldStudy out the standard of storage period of polygala tenuifolia seed and thousand kernel weight according to 1000-grain weight and germination rate:for the firsr-class seeds, length of storage≤2 years,1000-grain weight≥2.711g; for the second-class seeds, length of storage≤4 years,1000-grain weight≥2.411g; the third-class seeds are in poor quality.For the first time, establish the yield regression model with D-saturation design between the yield and application amount of N, P₂O₅, K₂O:Y=5121+36.113X1+ 150.557X2+71.157X3-439.15X12-321.447X22-168.478X32-91.306X1X2-28.206X1X3+8.238X2X3. The sequence of yield effection of nitrogen, phosphor, kalium is:N （X1）> P（X2）> K（X3）. The sequence of interaction effection is:NP> PK > NK.117.128kg nitrogen,120.333 kg phosphor, and 136.257 kg/hm2 kalium are in need if the prospect polygala tenuifolia yield is 5146.172 kg/hm2. and the proportion is 1:1.03:1.16.Carry out the water and fertilizer coupling experiment with pot culture in rainprotection shed, under the low water condition with less than 14% water in soil, early fertilizer （anthesis） is comparesively better than late fertilizer, and low nitrogen is better than high nitrogen; under the high water condition with 24% water in soil, add nitrogen during the period of seedling establishment and anthesis, the yield is very significant difference for other groups. Polygala tenuifolia has a narrow tolerance range of nitrogen, and has a better tolerance in early growth, and it is in great demanding of water and nitrogen during anthesis.Study on water demanding rule and pest contorl shows polygala tenuifolia is in highest yield if immgrated during bloom stage. The control effect of rogor and matrine are significant difference from that of derris alkali in aphid control, while rogor and matrine are no significant differences. Joint use of biological pesticide trichoderma preparation and chemical pesticide of tuzet has more significant effection than respective use in prevention and cure of root rot disease.For the first time, carry out the systematic study on growth dynamics of Polygala tenuifolia in 2007～2008, and the results indicates that the diameter of main steam or fresh root, fresh root/dried root proportion, weight of fresh root, weight of dry root, weight of dry steam and leave, biological yield increased along with the prolong of growth period. Branch number or rootlet number in 2007 was bigger than that in 2008. The height of plant changed in parabola, and higher in 2008 than in 2007. The fastest monthly increasing period of dry root weight was between 20th July and 20th August 2007, and between 27th June to 27th July 2008. The total accumulation of dry matters in root in 2008 was higher than in 2007.2 Quality assessment of Radix PolygalaeThe experimental results of HPLC fingerprint on Polygala tenuifolia shows: fingerprints are in high similarity among 23 batch radixes of Polygala tenuifolia, and only slight differences are observed between wild and cultivated species; There is similar structure in HPLC fingerprints of cultivated species for different grow periods and collection month, growth time has few effects on the types of active matters; the fingerprints of root xylem are similar to those of Radix Polygalae, while the peak areas are comparatively low, so the content is low, and the fingerprint similarity of root xylem is above 0.807 between the cultivated and the wild.The experimental results of effect matters of polygalic acid shows:producing area influences the content of polygalic acid significantly. Altidude and dimensionality have some effect on the content of polygalic acid. There are higher content of polygala tenuifolia in the different month collected polygala tenuifolia in 2008 than that in the same month in 2007. Polygalic acid is highly interrelated with growth years and the collected period, but may be not interrelated with the developmental stages. It is considered better to collection in June or July after polygala tenuifolia reaches its growth years. Polygalic acid can be found in root heart of polygala tenuifolia, and its accumulation may be negatively correlated with drugs. Leaves of polygala tenuifolia contain poor quantity of polygalic acid except P. sibirical L has slightly higher.3. Study on genetic diversity of germplasm resourcesFor the first time, with AFLP, capillary electrophoresis technique and allozyme Analysis, genetic diversity is studied in the ways of DNA, protein, allozyme on Polygala tenuifolia Willd. or P. sibirica L.Carry out amplified fragment length polymorphism （AFLP） method to study on the DNA genetic polymorphism,310 clear and repeatable gene bands are amplified. Among those bands,261 are polymorphism bands, accounting for 84.19%, and 21.75 allelic variation are found in each locus, while effective ones account for 1.49. The genetic identity of 24 polygala populations is between 0.4000～0.8645, and the genetic distance is between 0.1456～0.9163, which indicates that the genentic diversity is abundant. The result of AFLP experiment is in accordance with classical morphology result, and also the geographical distribution.The experimental results of protein genetic diversity by capillary electrophoresis show that the similarity coefficient of germplasm resources for polygala tenuifolia is between 0.316～0.895, and the genetic diversity is full. The protein genetic diversity reflected that the resources similarity is higher and genetic relationship is closer for species in closer geographical position. The cultivation species, however, the protein traits are in greater difference. Cultivated polygala tenuifolia in Shanxi has higher similarity with wild ones in Sichuan Maoxian and Gansu. The result of protein genetic diversity research is not all in accordance with the result of genetic diversity study.The outcome of soluble protein and allozyme shows the bands of peroxidase with mobility of 0.136 and the bands of esterase with mobility of 0.818 can identify Polygala tenuifolia Willd. and P. sibirica L., while the bands of peroxidase with mobility of 0.492 can identify polygala tenuifolia origins from Shanxi Qinyuan and other eight species. The electrophoresis band polymorphisms are between 0.198～0.444, and the genetic identity of 9 species is between 0.3-0.9. The genetic identity between Polygala tenuifolia Willd. and P. sibirica L. is lower than that among the populations of Polygala tenuifolia Willd, and the genetic distance is greater. The similarity of soluble protein and allozyme is accordance with peographical distribution in certain level.4. Study on cloning of squalene epoxidase geneFirstly clone the DNA gene of squalene epoxidase with 5 degenerate primer, and obtain a nucleotide with 517bp. After identification of Blastx, the fragment of the nucleotide belongs to SE gene fragment. According to the conjecture of Zea mays with full cDNA gene sequence （Accession:BT042800）, the cloned SE fragment has 1426bp distance to 5’end, while 110bp to 3’end. At cloned part, the length of Zea mays is 23bp longer than SE gene fragment.更多还原