远志高产途径、种质资源与鲨烯环氧酶基因克隆研究
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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、P_2O_5、K_2O施用量的产量回归模型：Y=5121+36.113X_1+150.557X_2+71.157X_3-439.15X_1~2-321.447X_2~2-168.478X_3~2-91.306X_1X_2-28.206X_1X_3+8.238X_2X_3。氮、磷、钾对远志产量效应顺序为：N(X_1)>P(X_2)>K(X_3),交互作用顺序为：NP>PK>NK,获得5146.172kg/hm~2远志需投入氮117.128kg,磷,120.333kg,钾,136.257kg/hm~2,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。
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 yield
Study 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, P_2O_5, K_2O: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 Polygalae
The 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 resources
For 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 gene
Firstly 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.
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、P_2O_5、K_2O施用量的产量回归模型：Y=5121+36.113X_1+150.557X_2+71.157X_3-439.15X_1~2-321.447X_2~2-168.478X_3~2-91.306X_1X_2-28.206X_1X_3+8.238X_2X_3。氮、磷、钾对远志产量效应顺序为：N(X_1)>P(X_2)>K(X_3),交互作用顺序为：NP>PK>NK,获得5146.172kg/hm~2远志需投入氮117.128kg,磷,120.333kg,钾,136.257kg/hm~2,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。
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 yield
Study 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, P_2O_5, K_2O: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 Polygalae
The 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 resources
For 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 gene
Firstly 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.
引文
1.国家药典委员会.中华人民共和国药典[M].2005年版,一部.北京：化学工业出版社,2005：107～108.
2.李世全.中药采购技术手册[M].西安：陕西科学技术出版社,1998,411～412.
3.丁乡.远志人气旺后市稳中升[J].中药研究与信息,2005,7(9)：47～48.
4.李敏主编.中药材规范化生产与管理(GAP)方法及技术[M].北京：中国医药科技出版社,2005,20-60.
5.刘旭.中国生物种质资源科学报告[M].北京：科学出版社,2003：124-139.
6.孙元枢.小黑麦基因库的组建[J].作物品种资源,1993(1)：12～14。
7.曾亚文等.云南稻种资源核心种质库构建及其利用前景[J].植物遗传资源科学,2000,1(3)：12～16.
8.贺玉林,李先恩,淡红梅.远地种子质量分级标准研究[J].种子,2007,26(1)：106～107.
9.张辰露,梁宗锁,王渭玲,等.绞股蓝氮磷钾肥效反应模式研究[J].西北农业学报,2005,14(4)：48～52.
10.赵云生,毛福英.远志需肥特性研究[J].西北药学杂志,2006,21(5)：206～207.
11.全国农业技术推广中心.全国测土配方施肥技术规范(试行)[M].北京：中国农业出版社,2005,7～10.
12.孙文涛,孙占祥,王聪翔.滴灌施肥条件下玉米水肥耦合效应的研究[J].中国农业科学,2006,39(3)：563～568.
13.翟丙年,李生秀,齐亚婷.不同水分状况下追施氮肥对冬小麦产量及其构成因素的影响[J].西北植物学报,2001,21(3)：462～467.
14.王静,朱九生,乔雄梧.6%吡·丁硫微乳剂防治苹果蚜虫田间药效试验[J].植物保护,2006,32(5)：117～119.
15.徐燕,李中幸.18%高渗氧乐果乳油对麦蚜的防治效果[J].农药,1997,36(8)：35～36.
16.朱九生,乔雄梧,秦曙,等.20%丁硫克百威乳油防治苹果蚜虫田间药效试验[J].农药,1998,37(10)：35～36.
17.王峰,刘西莉,高仁君,等.20%克·多·甲种衣剂防治棉花苗期病虫害的研究[J].种子,1999,105(6)：6～8.
18.陈晓明,李克森,胡劲松.17%克多酮种衣剂防治小麦病虫害的药效试验[J].安徽农业科学,1999,27(3)：332.
19.张雯晴,杨秀峰,颜兴贵,等.不同药剂防治白术根腐病和白绢病试验研究[J].种子,2004,23(5)：49-50.
20.徐昭玺.中草药种植技术指南[M].北京：中国农业出版社,2000：266～269.
21.夏厚林,董敏,吴希,等.远志蜜炙前后HPLC指纹图谱对比研究[J].中草 药,2006,37(11)：1657～1659.
22.姜勇,张娜,崔振,等.远志药材的HPLC指纹图谱[J].药学学报,2006,41(2)：179～183.
23.刘友平,万德光,宋英.HPLC法测定远志中去羟基远志皂甙元含量[J].中草药,2001,32(9)：786～787.
24.刘友平,万德光,刘涛,等.分光光度法测定不同产地远志总皂甙的含量[J].成都中医药大学学报,2000,23(2)：46～47.
25.邹喻苹,葛颂,王晓东,等.系统进化植物学中的分子标记[M].科学出版社,2001,16～17.
26.张阵阵.应用AFLP技术筛选与红花品质相关的分子标记[M.第二军医大学硕士学位论文,2006：28～30.
27.Bassam B J. Caetano-Anolles G, resshoff P M, et al. Fast and sensitive silver staining of DNA in polyacrylamide gels. Anal Biochem,1991,96(1):80～83.
28.岳志芹,王伟继,孔杰,等.用AFLP方法分析中国对虾抗病选育群体的遗传变异.水产学报.2005,29(1)：13～19.
29.李文英,顾万春,周世良.蒙古栎天然群体遗传多样性研究的AFLP[J].林业科学.2003,41(1)：49～56.
30.李琴韵,王智华,洪筱坤.]HPCE-FPS技术对吴茱萸质量的研究[J].中成药,2003,25(7)：570～573.
31.许重远,张煜,李亦蕾,等.高效毛细管蛋白电泳法对鸡内金和穿山甲的鉴别[J].解放军药学学报,2007,23(6)：464～466.
32.胡平,罗国安,王如骥,等.中药菟丝子的高效毛细管电泳法鉴别[J].药学学报,1997,32(7)：549～552.
33.刘丽,陈新民,何中虎,等.毛细管电泳图谱在小麦品种鉴定中的应用及其与品质的关系[J].麦类作物学报,2007,27(2)：229～236.
34.王中仁.植物等位酶分析[M].北京：科学出版社,1996,95～102.
35.郝建军,刘延吉.植物生理学实验技术[M].沈阳：辽宁科学技术出版社,2001：128～136.
36.邹琦.植物生理学实验指导.北京：中国农业出版社,2000.131-135.
37.萨姆布鲁克J,弗里奇EF,曼尼阿蒂斯 T.分子克隆实验指南(第三版)[M].北京：科学出版社,2002,100～102.
38.Suzuki,H., Achnine,L.,Xu,R., Matsuda,S.P. and Dixon,R.A. A genomics approach to the early stages of triterpene saponin Plant J.,2002,32(6):1033-1048.
39 He,F., Zhu,Y., He,M. and Zhang,Y. Molecular cloning and characterization of the gene encodingsqualene epoxidase in Panax notoginseng DNA Seq.2008:19 (3),270-273.
40.Ono T, Nakazono K, Kosaka H:Purification and partial characterization of squalene epoxidase from rat liver microsomes.Biochim Biophys Acta 1982,709(1):84-90.
41.Abe I,Seki T,Umehara K,Miyase T,Noguchi H, Sakakibara J. Ono T:Green tea polyphenols: novel and potent inhibitors of squalene epoxidase. Biochem Biophys Res Commun 2000, 268(3):767-771.
42.Stefanie L.Butland, Monica L. Chow and Brian E. Ellis, A diverse family of phenylalanine ammonia-lyase genes expressed in pine trees and cell culture, Plant molecular Biology,1998,37: 15～24.
43.徐昭玺.中草药种植技术指南[M].北京：中国农业出版社,2000：266～269.
44.北京市人民政府农林办公室科教处,北京市农林科学院农业科技信息研究所.中草药栽培及经济虫类养殖技术[M].北京：中国农业科技出版社,1999：43.
45.中国科学院中国植物志编辑委员会主编.中国植物志[M].北京：科学出版社,43(3)：142.
46.张丽萍.远志[M].北京：中国中医药出版社,2001：1～2.
47.赵云生,李占林,毛福英,等.远志种子贮存特性研究[J].中国中医药学刊,2006,24(8)：1485～1486。
48.宋长水,赵云生.远志种子采集时间与方法研究[J].中国农村小康科技.2007,10(10)：53～54。
49.薛辉.远志种子繁殖实验观察[J].中国中药杂志,1989,14(8)：15.
50.陈瑛.实用中药种子技术手册[M].北京：人民卫生出版社,1999：306～307.
51.谢凤勋.中草药栽培实用技术[M].北京：中国农业出版社,2001：220～223.
52.李恒森,徐同印,杨霞.远志栽培技术简介[J].中草药,2002,33(3)：271～272.
53.阎龙民,张利华,周萍.山东远志丰产栽培技术[J].时珍国药研究,1998,9(3)：276.
54.白效令.中药材栽培与采集[M].山西：山西科学技术出版社,1992：27～29.
55.赵云生,李占林,毛福英,等.远志栽培密度试验报告[J].中药材,2006,29(7)：652～653.
56.赵云生,毛福英.远志需肥特性研究[J].西北药学杂志,2006,21(5)：206～207。
57.张虎占,董泽宏,余靖.中药现代研究与应用(第三卷)[M].北京：学苑出版社,1998：2121～2131.
58.彭汶泽,许实波.四种远志皂甙的镇咳和祛痰作用[J].中国药学杂志,1998,33(8)：491.
59.彭汶泽.远志皂甙H对离体平滑肌与心脏的作用[J].中国药学杂志,1999,34(4)：241～243.
60.刘友平,万德光,宋英.HPLC法测定远志中去羟基远志皂甙元含量[J].中草药,2001,32(9)：786～787.
61.刘友平,万德光,刘涛,等.分光光度法测定不同产地远志总皂甙的含量[J].成都中医药大学学报,2000,23(2)：46～47.
62.刘友平,万德光,黄荣,等.薄层扫描法测定远志中远志皂苷元的含量[J].中草药,2000,31(7)：512-514.
63.姜勇,屠鹏飞.远志研究进展[J].中草药,2001,32(8)：759～761.
64.杨学东,徐丽珍,杨世林.远志属植物中口山酮类成分及其药理研究进展[J].天然产物研究与开发,1999,12(5)：88～94.
65.金宝渊,朴政一.远志生物碱成分的研究[J].中国中药杂志,1993,18(11)：675～677.
66.杨国红,孙晓飞.反相高效液相色谱法测定远志中远志皂甙元的含量[J].药物分析杂 志,2001,21(4)：260～263.
67.国家食品药品监督管理局.中药注射剂指纹图谱研究的技术要求(暂行)的通知[J].中成药,2000,22(10)：671～674.
68.罗国安,王义明.中药指纹图谱的分类和发展[J].中国新药杂志,2002,11(1)：46～51.
69.Natalie L.Use of fingprinting and compounds for identification and standardization of botanical drugs strategies for applying pharmaceutical HPLC analysis to herbal products[J].Drug Inf J,1998,32(4):497-512.
7O.Peter J H.Establishing identification criteria for botanicals[J].Drug Inf J,1998,32(4):461～469.
71.ECRD of FDA. Guidance for industry Botanical Drug products(Draft guidance).2000.
72.WHO. Guidelines for the Assessment of Herbal Medicines.1996.
73.罗国安,王义明.中药复方有效部分研究方法及理论初探[J].中成药,1997；19(8)：44.
74.罗国安,王义明.中药复方的化学研究体系[J].世界科学技术---中药现代化,1999,1(1)：16.
75.罗国安,王义明.中药复方物质基础和药效相关性研究[J].世界科学技术---中药现代化,1999；1(1)：11.
76.苏薇薇,吴忠.中药指纹图谱及计算机信息处理世界科学技术[J].中药现代化,2001,3(2)：30.
77.严琳,高健,侯莉莉.中药指纹图谱的方法学研究概述[J].泸州医学院学报,2004,27(1)：87-89.
78田徽,王建,夏厚林,等.蜜远志与生远志水煎液的HPLC指纹图谱对比研究[J].中草药,2007,38(8)：1180～1183.
79.夏厚林,董敏,吴希,等.远志蜜炙前后HPLC指纹图谱对比研究[J].中草药,2006,37(11)：1657～1659.
80.马克平.试论生物多样性的概念[J].生物多样性,1993,1(1)：20～22.
81 刘萍,马宏玮,王掌军.我国药用植物种质资源遗传多样性及其研究进展[J].农业科学研究,2008,29(3)：66～70.
82.相宁,洪焰.扩增片段长度多态性技术及其在植物研究中的应用[J].植物生理学通讯,36(3)：236～240.
83.Ham rick JL, Godt M J. Allozyme Diversity in Plant Species In Brown. A H D etal r(eds) Plant Population Genetics Breeding and Genetic Resources[M], Sunderland:Sinaues 1990,43～63.
84.Moritz C, Hilli D M. Molecular Systematics; Contex and Controversies. In Hillis D M and C Moritz (eds.)Molecular systematics. 1990, Sunderland; Sinauer.1～11.
85.沈振国,刘友良.重金属超量积累植物研究进展[J].植物生理学通讯,1998,34(2)：133～1391.
86.Baker A J M. Accumulators and excluders:Stradegies in response of plants to heavy metals. J. Plant Nutr.1981,:643～654.
87.时明芝,宋会兴.植物遗传多样性研究方法概述[J].世界林业研究,2005,(18)：27～31.
88.陈灵芝.中国的生物多样性--现状及其保护对策[M].北京：科学出版社,1993,99～113.
89.Schaal, B. A.,W. J. Leverich and S. H. Rogstad. Comparison of methods for assessing genetic variation in plant conservation biology. In Falk, D. A. and K. E. Holsinger(eds.). Genetics and Conservation of Rare Plants. New York:Oxford University Press,1991,123-134.
90.沈浩,刘登义.遗传多样性概述[J].生物学杂志,2001,18(3)：5～8.
91.马小军,肖培根.种质资源遗传多样性在药用植物开发中的重要意义[J].中国中药杂志,1998,23(10)：579～582.
92.赵擞平.植物基因组：构建、表达和调节[J].北京：首都师范大学出版社,1996.
93.丘德有.利用转基因植物生产有用的次生代谢产物.植物科学综论[M].哈尔滨：东北林业大学出版社,1993：20.
94.李承森,植物科学进展(第一卷)[M].北京：高等教育出版杜,1998：293.
95.夏铭.遗传多样性研究进展[J].生态学杂志,1999,1；(3)：59～65.
96.Merrell, D. J.(黄瑞复等译).生态遗传学[M].北京：科学出版社,1991.
97.Robinson N J., Tommey A M. et al. Plant metallothioneins. Biochem. J.,1993,295:1-10.
98.任旭琴.遗传多样性及其研究方法[J].淮阴工学院学报,2002,11(5)：6～8.
99.王中仁.植物等位酶分析.北京：科学出版社,1996：10～11.
100. Nei, M. Analysis of gene diversity in subdivided populations. Proc. Natl. Acad. Sci, USA, 1973,70:3321～3323.
lO1.Hamrick J L, Linhart Y B, Mitton J B.Relationships between life history characteristics and .electrophoretically detectable genetic variation in plant.Ann Rev Ecol Syst,1979,10:173～200.
102.葛颂等.遗传多样性及其检查方法[M]..北京：中国科技出版社,1994,123-140.
103.潘莹,赵桂仿.分子水平的遗传多样性及其测量方法[J].西北植物学报,1998,18(4)：645～653.
104.邱芳,伏健民,金德敏,等.遗传多样性的分子检测[J].生物多样性,1998,6(2)：143～150.
105.Bostein D,Whiter L, Skdnick M, et al. Construction of a genetic linkage map in man using restriction fragment length po lymo rph ism s. Am. J. Hum. Genet.,1980,32:314～331.
106.Zebeau M,Vos P 1993.Selective Restrication Fraglnent Amplification a General Method for DNA Fingerprinting European Patent Application No.0534855
107.高翔,庞红喜,裴阿卫.分子标记技术在植物遗传多样性研究中的应用[J].河南农业大学学报,2002,36(4)：356～359.
108.伍莲,邓洪平,徐洁,等.金毛狗居群遗传多样性的AFLP分析[J].中国中药杂志,2007,32(14)：1468～1469.
109.戴住波,朱常成,钱子刚,等.金铁锁种质资源的遗传多样性分析[J].中草药,2007,38(7)：1070～1074.
111.孟庆文,崔卫东,白光红,等.红景天种内遗传多样性分析AFLP方法建立[J].新疆农业科学,2008,45(1)：88～92.
111.葛淑俊,李广敏,马峙英,等.甘草野生种群遗传多样性的AFLP分析[J].中国农业科学,2009,42(1)：41～54.
112.川牛膝(Cyathula officinalis Kuan.) RAPD和AFLP标记的多态性聚类分析[J].安徽农业科学,2008,36(16)：6682～6686.
113.关萍,石建明,陈放.天麻AFLP反应体系的建立及优化[J].中国中药杂志,32(20)：263～265.
114.药用灵芝遗传多样性的AFLP分析[J].中国中药杂志,2007,32(17)：163～166.
115.珍稀濒危药用植物黄檗野生种群遗传多样性的AFLP分析[J],生物多样性2006,14(6)：488～497.
116.Bach, T. J. Some new aspects of isoprenoid biosynthesis in plants:areview[J]. Lipids.1995,30: 191～202.
117.Mcgarvey D, Croreau R.1995. Terpenoid metabolism[J]. Plant Cell.7:1015～1026.
118.Lichtenthaler HK, Schwender J, Disch A, Rohmer M. Biosynthesis of isoprenoids in higher plant chloroplasts proceeds via a mevalonate-independent pathway[J]. FEBS Lett.1997, 400(3):271～274.
119.Kajiwara S, Fraser PD, Kondo K, Misawa N. Expression of an exogenous isopentenyl diphosphate isomerase gene enhances isoprenoid biosynthesis in Escherichia coli[J]. Biochem J. 1997,324(2):421～426.
120.Zairen Sun, Francis X. Cunningham, Elisabeth Gantt. Differential Expression of Two Isopentenyl Pyrophosphate Isomerases and Enhanced Carotenoid Accumulation in a Unicellular Chlorophyte[J]. Proceedings of the National Academy of Sciences of the United States of America,1998,95(19):11482～11488.
121.Rodriguez-Concepcion. Genetic evidence of branching in the isoprenoid pathway for the production of isopentenyl diphosphate and dimethylallyl diphosphate in Escherichia coli[J]. M.FEBS Lett,2000,473(3):328～332.
122.Choi D, Ward BL, Bostock RM. Differential induction and suppression of potato 3-hydroxy-3-methylglutaryl coenzyme A reductase genes in response to Phytophthora infestans and to its elicitor arachidonic acid[J]. Plant Cell.1992 Oct;4(10):1333～1344.
123.Wang G Y, Keasling J D. Amplification of HMG-CoA reductase production enhances carotenoid accumulation in Neurospora crassa[J]. Metab Eng,2002,4(3):193-201.
124.孙彬贤,翁颖琦,刘涤,等.代谢中间产物和诱导子对南方红豆杉培养细胞生长和紫杉醇含量的影响[J].上海中医药大学学报,2000,14(3)：54～56.
125.JUNG J D, PARK H W, HAHN Y, et al. Discovery of genes for ginsenoside biosynthesis by analysis of ginseng expressed sequence tags [J]. Plant Cell Rep,2003,22(3):224～230.
126.邢朝斌,王一曼,陈正恒,等.三萜皂苷的生物合成[J].生命的化学,2005,25(5)：420-422.
127.赵明文,钟家禹,王南,等.鲨烯合酶的研究进展[J].微生物学报,2003,43(5)：676～670.
128.彭剑,龚炳永.角鲨烯环氧化酶抑制剂的研究进展[J].国外医药抗生素分册,1998,19(3)：177～183.
129.CHOI D W, JUNG J, HA YI, et al. Analysis of transcripts in methyl jasmonate-treated ginseng hairy roots to identify genes involved in the biosynthesis of ginsenosides and other secondary metabolites [J].Plant Cell Rep,2005,23(8):557～566.
130.SUZUKI H, ACHNINE L, XU R, et al. A genomics approach to the early stages of trirerpene saponin biosynthesis in Medicago truncatula [J]. Plant J,2002,32(6):1033-1048.
131.陈莉,吴耀生.三萜皂苷生物合成途径及相关酶[J].国外医药·植物药分册,2004,19(4)：156～161.
132HAYASHIH, HUANG P, INOUE K. Up regulation of soyasaponin biosynthesis by methyl jasmonate in cultured cells of Glycyrrhiza glabra [J].Plant Cell Physiol,2003,44(4):404～411.
133.Kalb VF, Loper JC. Proteins from eight eukaryotic cytochrome P-450 families share a segmented region of sequence similarity [J]. Proc Natl Acad Sci US A.1988,85(19): 7221～7225.
134.Durst F, Nelson DR. Diversity and evolution of plant P450 and P450-reductases[J]. Drug Metabol Drug Interact.1995; 12(3-4):189～206.
135.Kurosawa, Y., Takahara, H. and Shiraiwa, M. UDP-glucuronic acid:soyasapogenol glucuronosyltransferase involved in saponin biosynthesis in germinating soybean seeds[J]. Planta,2002,215:620-629.
2.李世全.中药采购技术手册[M].西安：陕西科学技术出版社,1998,411～412.
3.丁乡.远志人气旺后市稳中升[J].中药研究与信息,2005,7(9)：47～48.
4.李敏主编.中药材规范化生产与管理(GAP)方法及技术[M].北京：中国医药科技出版社,2005,20-60.
5.刘旭.中国生物种质资源科学报告[M].北京：科学出版社,2003：124-139.
6.孙元枢.小黑麦基因库的组建[J].作物品种资源,1993(1)：12～14。
7.曾亚文等.云南稻种资源核心种质库构建及其利用前景[J].植物遗传资源科学,2000,1(3)：12～16.
8.贺玉林,李先恩,淡红梅.远地种子质量分级标准研究[J].种子,2007,26(1)：106～107.
9.张辰露,梁宗锁,王渭玲,等.绞股蓝氮磷钾肥效反应模式研究[J].西北农业学报,2005,14(4)：48～52.
10.赵云生,毛福英.远志需肥特性研究[J].西北药学杂志,2006,21(5)：206～207.
11.全国农业技术推广中心.全国测土配方施肥技术规范(试行)[M].北京：中国农业出版社,2005,7～10.
12.孙文涛,孙占祥,王聪翔.滴灌施肥条件下玉米水肥耦合效应的研究[J].中国农业科学,2006,39(3)：563～568.
13.翟丙年,李生秀,齐亚婷.不同水分状况下追施氮肥对冬小麦产量及其构成因素的影响[J].西北植物学报,2001,21(3)：462～467.
14.王静,朱九生,乔雄梧.6%吡·丁硫微乳剂防治苹果蚜虫田间药效试验[J].植物保护,2006,32(5)：117～119.
15.徐燕,李中幸.18%高渗氧乐果乳油对麦蚜的防治效果[J].农药,1997,36(8)：35～36.
16.朱九生,乔雄梧,秦曙,等.20%丁硫克百威乳油防治苹果蚜虫田间药效试验[J].农药,1998,37(10)：35～36.
17.王峰,刘西莉,高仁君,等.20%克·多·甲种衣剂防治棉花苗期病虫害的研究[J].种子,1999,105(6)：6～8.
18.陈晓明,李克森,胡劲松.17%克多酮种衣剂防治小麦病虫害的药效试验[J].安徽农业科学,1999,27(3)：332.
19.张雯晴,杨秀峰,颜兴贵,等.不同药剂防治白术根腐病和白绢病试验研究[J].种子,2004,23(5)：49-50.
20.徐昭玺.中草药种植技术指南[M].北京：中国农业出版社,2000：266～269.
21.夏厚林,董敏,吴希,等.远志蜜炙前后HPLC指纹图谱对比研究[J].中草 药,2006,37(11)：1657～1659.
22.姜勇,张娜,崔振,等.远志药材的HPLC指纹图谱[J].药学学报,2006,41(2)：179～183.
23.刘友平,万德光,宋英.HPLC法测定远志中去羟基远志皂甙元含量[J].中草药,2001,32(9)：786～787.
24.刘友平,万德光,刘涛,等.分光光度法测定不同产地远志总皂甙的含量[J].成都中医药大学学报,2000,23(2)：46～47.
25.邹喻苹,葛颂,王晓东,等.系统进化植物学中的分子标记[M].科学出版社,2001,16～17.
26.张阵阵.应用AFLP技术筛选与红花品质相关的分子标记[M.第二军医大学硕士学位论文,2006：28～30.
27.Bassam B J. Caetano-Anolles G, resshoff P M, et al. Fast and sensitive silver staining of DNA in polyacrylamide gels. Anal Biochem,1991,96(1):80～83.
28.岳志芹,王伟继,孔杰,等.用AFLP方法分析中国对虾抗病选育群体的遗传变异.水产学报.2005,29(1)：13～19.
29.李文英,顾万春,周世良.蒙古栎天然群体遗传多样性研究的AFLP[J].林业科学.2003,41(1)：49～56.
30.李琴韵,王智华,洪筱坤.]HPCE-FPS技术对吴茱萸质量的研究[J].中成药,2003,25(7)：570～573.
31.许重远,张煜,李亦蕾,等.高效毛细管蛋白电泳法对鸡内金和穿山甲的鉴别[J].解放军药学学报,2007,23(6)：464～466.
32.胡平,罗国安,王如骥,等.中药菟丝子的高效毛细管电泳法鉴别[J].药学学报,1997,32(7)：549～552.
33.刘丽,陈新民,何中虎,等.毛细管电泳图谱在小麦品种鉴定中的应用及其与品质的关系[J].麦类作物学报,2007,27(2)：229～236.
34.王中仁.植物等位酶分析[M].北京：科学出版社,1996,95～102.
35.郝建军,刘延吉.植物生理学实验技术[M].沈阳：辽宁科学技术出版社,2001：128～136.
36.邹琦.植物生理学实验指导.北京：中国农业出版社,2000.131-135.
37.萨姆布鲁克J,弗里奇EF,曼尼阿蒂斯 T.分子克隆实验指南(第三版)[M].北京：科学出版社,2002,100～102.
38.Suzuki,H., Achnine,L.,Xu,R., Matsuda,S.P. and Dixon,R.A. A genomics approach to the early stages of triterpene saponin Plant J.,2002,32(6):1033-1048.
39 He,F., Zhu,Y., He,M. and Zhang,Y. Molecular cloning and characterization of the gene encodingsqualene epoxidase in Panax notoginseng DNA Seq.2008:19 (3),270-273.
40.Ono T, Nakazono K, Kosaka H:Purification and partial characterization of squalene epoxidase from rat liver microsomes.Biochim Biophys Acta 1982,709(1):84-90.
41.Abe I,Seki T,Umehara K,Miyase T,Noguchi H, Sakakibara J. Ono T:Green tea polyphenols: novel and potent inhibitors of squalene epoxidase. Biochem Biophys Res Commun 2000, 268(3):767-771.
42.Stefanie L.Butland, Monica L. Chow and Brian E. Ellis, A diverse family of phenylalanine ammonia-lyase genes expressed in pine trees and cell culture, Plant molecular Biology,1998,37: 15～24.
43.徐昭玺.中草药种植技术指南[M].北京：中国农业出版社,2000：266～269.
44.北京市人民政府农林办公室科教处,北京市农林科学院农业科技信息研究所.中草药栽培及经济虫类养殖技术[M].北京：中国农业科技出版社,1999：43.
45.中国科学院中国植物志编辑委员会主编.中国植物志[M].北京：科学出版社,43(3)：142.
46.张丽萍.远志[M].北京：中国中医药出版社,2001：1～2.
47.赵云生,李占林,毛福英,等.远志种子贮存特性研究[J].中国中医药学刊,2006,24(8)：1485～1486。
48.宋长水,赵云生.远志种子采集时间与方法研究[J].中国农村小康科技.2007,10(10)：53～54。
49.薛辉.远志种子繁殖实验观察[J].中国中药杂志,1989,14(8)：15.
50.陈瑛.实用中药种子技术手册[M].北京：人民卫生出版社,1999：306～307.
51.谢凤勋.中草药栽培实用技术[M].北京：中国农业出版社,2001：220～223.
52.李恒森,徐同印,杨霞.远志栽培技术简介[J].中草药,2002,33(3)：271～272.
53.阎龙民,张利华,周萍.山东远志丰产栽培技术[J].时珍国药研究,1998,9(3)：276.
54.白效令.中药材栽培与采集[M].山西：山西科学技术出版社,1992：27～29.
55.赵云生,李占林,毛福英,等.远志栽培密度试验报告[J].中药材,2006,29(7)：652～653.
56.赵云生,毛福英.远志需肥特性研究[J].西北药学杂志,2006,21(5)：206～207。
57.张虎占,董泽宏,余靖.中药现代研究与应用(第三卷)[M].北京：学苑出版社,1998：2121～2131.
58.彭汶泽,许实波.四种远志皂甙的镇咳和祛痰作用[J].中国药学杂志,1998,33(8)：491.
59.彭汶泽.远志皂甙H对离体平滑肌与心脏的作用[J].中国药学杂志,1999,34(4)：241～243.
60.刘友平,万德光,宋英.HPLC法测定远志中去羟基远志皂甙元含量[J].中草药,2001,32(9)：786～787.
61.刘友平,万德光,刘涛,等.分光光度法测定不同产地远志总皂甙的含量[J].成都中医药大学学报,2000,23(2)：46～47.
62.刘友平,万德光,黄荣,等.薄层扫描法测定远志中远志皂苷元的含量[J].中草药,2000,31(7)：512-514.
63.姜勇,屠鹏飞.远志研究进展[J].中草药,2001,32(8)：759～761.
64.杨学东,徐丽珍,杨世林.远志属植物中口山酮类成分及其药理研究进展[J].天然产物研究与开发,1999,12(5)：88～94.
65.金宝渊,朴政一.远志生物碱成分的研究[J].中国中药杂志,1993,18(11)：675～677.
66.杨国红,孙晓飞.反相高效液相色谱法测定远志中远志皂甙元的含量[J].药物分析杂 志,2001,21(4)：260～263.
67.国家食品药品监督管理局.中药注射剂指纹图谱研究的技术要求(暂行)的通知[J].中成药,2000,22(10)：671～674.
68.罗国安,王义明.中药指纹图谱的分类和发展[J].中国新药杂志,2002,11(1)：46～51.
69.Natalie L.Use of fingprinting and compounds for identification and standardization of botanical drugs strategies for applying pharmaceutical HPLC analysis to herbal products[J].Drug Inf J,1998,32(4):497-512.
7O.Peter J H.Establishing identification criteria for botanicals[J].Drug Inf J,1998,32(4):461～469.
71.ECRD of FDA. Guidance for industry Botanical Drug products(Draft guidance).2000.
72.WHO. Guidelines for the Assessment of Herbal Medicines.1996.
73.罗国安,王义明.中药复方有效部分研究方法及理论初探[J].中成药,1997；19(8)：44.
74.罗国安,王义明.中药复方的化学研究体系[J].世界科学技术---中药现代化,1999,1(1)：16.
75.罗国安,王义明.中药复方物质基础和药效相关性研究[J].世界科学技术---中药现代化,1999；1(1)：11.
76.苏薇薇,吴忠.中药指纹图谱及计算机信息处理世界科学技术[J].中药现代化,2001,3(2)：30.
77.严琳,高健,侯莉莉.中药指纹图谱的方法学研究概述[J].泸州医学院学报,2004,27(1)：87-89.
78田徽,王建,夏厚林,等.蜜远志与生远志水煎液的HPLC指纹图谱对比研究[J].中草药,2007,38(8)：1180～1183.
79.夏厚林,董敏,吴希,等.远志蜜炙前后HPLC指纹图谱对比研究[J].中草药,2006,37(11)：1657～1659.
80.马克平.试论生物多样性的概念[J].生物多样性,1993,1(1)：20～22.
81 刘萍,马宏玮,王掌军.我国药用植物种质资源遗传多样性及其研究进展[J].农业科学研究,2008,29(3)：66～70.
82.相宁,洪焰.扩增片段长度多态性技术及其在植物研究中的应用[J].植物生理学通讯,36(3)：236～240.
83.Ham rick JL, Godt M J. Allozyme Diversity in Plant Species In Brown. A H D etal r(eds) Plant Population Genetics Breeding and Genetic Resources[M], Sunderland:Sinaues 1990,43～63.
84.Moritz C, Hilli D M. Molecular Systematics; Contex and Controversies. In Hillis D M and C Moritz (eds.)Molecular systematics. 1990, Sunderland; Sinauer.1～11.
85.沈振国,刘友良.重金属超量积累植物研究进展[J].植物生理学通讯,1998,34(2)：133～1391.
86.Baker A J M. Accumulators and excluders:Stradegies in response of plants to heavy metals. J. Plant Nutr.1981,:643～654.
87.时明芝,宋会兴.植物遗传多样性研究方法概述[J].世界林业研究,2005,(18)：27～31.
88.陈灵芝.中国的生物多样性--现状及其保护对策[M].北京：科学出版社,1993,99～113.
89.Schaal, B. A.,W. J. Leverich and S. H. Rogstad. Comparison of methods for assessing genetic variation in plant conservation biology. In Falk, D. A. and K. E. Holsinger(eds.). Genetics and Conservation of Rare Plants. New York:Oxford University Press,1991,123-134.
90.沈浩,刘登义.遗传多样性概述[J].生物学杂志,2001,18(3)：5～8.
91.马小军,肖培根.种质资源遗传多样性在药用植物开发中的重要意义[J].中国中药杂志,1998,23(10)：579～582.
92.赵擞平.植物基因组：构建、表达和调节[J].北京：首都师范大学出版社,1996.
93.丘德有.利用转基因植物生产有用的次生代谢产物.植物科学综论[M].哈尔滨：东北林业大学出版社,1993：20.
94.李承森,植物科学进展(第一卷)[M].北京：高等教育出版杜,1998：293.
95.夏铭.遗传多样性研究进展[J].生态学杂志,1999,1；(3)：59～65.
96.Merrell, D. J.(黄瑞复等译).生态遗传学[M].北京：科学出版社,1991.
97.Robinson N J., Tommey A M. et al. Plant metallothioneins. Biochem. J.,1993,295:1-10.
98.任旭琴.遗传多样性及其研究方法[J].淮阴工学院学报,2002,11(5)：6～8.
99.王中仁.植物等位酶分析.北京：科学出版社,1996：10～11.
100. Nei, M. Analysis of gene diversity in subdivided populations. Proc. Natl. Acad. Sci, USA, 1973,70:3321～3323.
lO1.Hamrick J L, Linhart Y B, Mitton J B.Relationships between life history characteristics and .electrophoretically detectable genetic variation in plant.Ann Rev Ecol Syst,1979,10:173～200.
102.葛颂等.遗传多样性及其检查方法[M]..北京：中国科技出版社,1994,123-140.
103.潘莹,赵桂仿.分子水平的遗传多样性及其测量方法[J].西北植物学报,1998,18(4)：645～653.
104.邱芳,伏健民,金德敏,等.遗传多样性的分子检测[J].生物多样性,1998,6(2)：143～150.
105.Bostein D,Whiter L, Skdnick M, et al. Construction of a genetic linkage map in man using restriction fragment length po lymo rph ism s. Am. J. Hum. Genet.,1980,32:314～331.
106.Zebeau M,Vos P 1993.Selective Restrication Fraglnent Amplification a General Method for DNA Fingerprinting European Patent Application No.0534855
107.高翔,庞红喜,裴阿卫.分子标记技术在植物遗传多样性研究中的应用[J].河南农业大学学报,2002,36(4)：356～359.
108.伍莲,邓洪平,徐洁,等.金毛狗居群遗传多样性的AFLP分析[J].中国中药杂志,2007,32(14)：1468～1469.
109.戴住波,朱常成,钱子刚,等.金铁锁种质资源的遗传多样性分析[J].中草药,2007,38(7)：1070～1074.
111.孟庆文,崔卫东,白光红,等.红景天种内遗传多样性分析AFLP方法建立[J].新疆农业科学,2008,45(1)：88～92.
111.葛淑俊,李广敏,马峙英,等.甘草野生种群遗传多样性的AFLP分析[J].中国农业科学,2009,42(1)：41～54.
112.川牛膝(Cyathula officinalis Kuan.) RAPD和AFLP标记的多态性聚类分析[J].安徽农业科学,2008,36(16)：6682～6686.
113.关萍,石建明,陈放.天麻AFLP反应体系的建立及优化[J].中国中药杂志,32(20)：263～265.
114.药用灵芝遗传多样性的AFLP分析[J].中国中药杂志,2007,32(17)：163～166.
115.珍稀濒危药用植物黄檗野生种群遗传多样性的AFLP分析[J],生物多样性2006,14(6)：488～497.
116.Bach, T. J. Some new aspects of isoprenoid biosynthesis in plants:areview[J]. Lipids.1995,30: 191～202.
117.Mcgarvey D, Croreau R.1995. Terpenoid metabolism[J]. Plant Cell.7:1015～1026.
118.Lichtenthaler HK, Schwender J, Disch A, Rohmer M. Biosynthesis of isoprenoids in higher plant chloroplasts proceeds via a mevalonate-independent pathway[J]. FEBS Lett.1997, 400(3):271～274.
119.Kajiwara S, Fraser PD, Kondo K, Misawa N. Expression of an exogenous isopentenyl diphosphate isomerase gene enhances isoprenoid biosynthesis in Escherichia coli[J]. Biochem J. 1997,324(2):421～426.
120.Zairen Sun, Francis X. Cunningham, Elisabeth Gantt. Differential Expression of Two Isopentenyl Pyrophosphate Isomerases and Enhanced Carotenoid Accumulation in a Unicellular Chlorophyte[J]. Proceedings of the National Academy of Sciences of the United States of America,1998,95(19):11482～11488.
121.Rodriguez-Concepcion. Genetic evidence of branching in the isoprenoid pathway for the production of isopentenyl diphosphate and dimethylallyl diphosphate in Escherichia coli[J]. M.FEBS Lett,2000,473(3):328～332.
122.Choi D, Ward BL, Bostock RM. Differential induction and suppression of potato 3-hydroxy-3-methylglutaryl coenzyme A reductase genes in response to Phytophthora infestans and to its elicitor arachidonic acid[J]. Plant Cell.1992 Oct;4(10):1333～1344.
123.Wang G Y, Keasling J D. Amplification of HMG-CoA reductase production enhances carotenoid accumulation in Neurospora crassa[J]. Metab Eng,2002,4(3):193-201.
124.孙彬贤,翁颖琦,刘涤,等.代谢中间产物和诱导子对南方红豆杉培养细胞生长和紫杉醇含量的影响[J].上海中医药大学学报,2000,14(3)：54～56.
125.JUNG J D, PARK H W, HAHN Y, et al. Discovery of genes for ginsenoside biosynthesis by analysis of ginseng expressed sequence tags [J]. Plant Cell Rep,2003,22(3):224～230.
126.邢朝斌,王一曼,陈正恒,等.三萜皂苷的生物合成[J].生命的化学,2005,25(5)：420-422.
127.赵明文,钟家禹,王南,等.鲨烯合酶的研究进展[J].微生物学报,2003,43(5)：676～670.
128.彭剑,龚炳永.角鲨烯环氧化酶抑制剂的研究进展[J].国外医药抗生素分册,1998,19(3)：177～183.
129.CHOI D W, JUNG J, HA YI, et al. Analysis of transcripts in methyl jasmonate-treated ginseng hairy roots to identify genes involved in the biosynthesis of ginsenosides and other secondary metabolites [J].Plant Cell Rep,2005,23(8):557～566.
130.SUZUKI H, ACHNINE L, XU R, et al. A genomics approach to the early stages of trirerpene saponin biosynthesis in Medicago truncatula [J]. Plant J,2002,32(6):1033-1048.
131.陈莉,吴耀生.三萜皂苷生物合成途径及相关酶[J].国外医药·植物药分册,2004,19(4)：156～161.
132HAYASHIH, HUANG P, INOUE K. Up regulation of soyasaponin biosynthesis by methyl jasmonate in cultured cells of Glycyrrhiza glabra [J].Plant Cell Physiol,2003,44(4):404～411.
133.Kalb VF, Loper JC. Proteins from eight eukaryotic cytochrome P-450 families share a segmented region of sequence similarity [J]. Proc Natl Acad Sci US A.1988,85(19): 7221～7225.
134.Durst F, Nelson DR. Diversity and evolution of plant P450 and P450-reductases[J]. Drug Metabol Drug Interact.1995; 12(3-4):189～206.
135.Kurosawa, Y., Takahara, H. and Shiraiwa, M. UDP-glucuronic acid:soyasapogenol glucuronosyltransferase involved in saponin biosynthesis in germinating soybean seeds[J]. Planta,2002,215:620-629.