【作者】 王渭玲；

【导师】 山仑； 梁宗锁；

【作者基本信息】 西北农林科技大学 ， 植物学， 2008， 博士

【摘要】 膜荚黄芪Astragalus membranaceus（Fisch．）为豆科紫云英属多年生草本植物，以根入药，为传统的大宗药材。随着对黄芪药理作用及保健作用认识的不断深入，黄芪除供药用外，还广泛用于饮料、补品、化妆品、茶等工业生产中，市场需求用量逐年增加。人工栽培黄芪已成为主要的药材来源途径，但由于对黄芪矿质营养理论方面缺乏研究，黄芪的施肥技术尚无系统的资料，导致栽培黄芪的产量和质量偏低，严重制约了黄芪产业的发展。本论文采用田间试验和毛状根培养试验，系统研究了膜荚黄芪矿质营养特性、优化施肥模式、不同营养元素条件下黄芪多糖及黄芪皂苷的积累动态变化、毛状根培养系统的建立及毛状根的生长及次生代谢的调控。这些研究结果不仅具有重要的科学意义，也将为黄芪高产优质人工栽培技术的建立、对保护野生黄芪资源、对减轻生态环境破坏、实现中药现代化和中药国际化具有重要意义。主要研究结果如下：1．膜荚黄芪干物质累积规律通过对黄芪干物质累积动态分析，得出黄芪干物质累积的Logistic方程为：W₁=（33170）/(（1+298126e）^-0.1087x)，R²=-0．9939^**，相关性达到极显著水平，因此可用此模型计算黄芪生长期间的干物质积累量。黄芪植株不同部位干物质累积与分配随生长季节而变化，出苗后100～132d是地上部分干物质积累最快的时期，占总积累量的88．22％；根系干物质累积晚于地上部分，出现在出苗后163d～185d。全株干物质的累积率在出苗后100～132d，快速增加，是植株干物质累积最快的时期，主要是茎叶干物质组分。出苗后132～163d，全株干物质累积量最多，主要是根部干物质组分。2．黄芪植株N、P、K、Fe、Mn、Zn、Cu的吸收、分配特性与最大效率期黄芪植株对NPK的吸收量为N＞K＞P。不同部位的含量为茎叶＞根系。N、P、K含量苗期最高，随生育进程而下降。不同生育时期黄芪的养分积累状况是不均衡的，整株养分的积累量前期少，中期增加迅速，后期缓慢增加。地上N、P、K的积累量从苗期持续增加到出苗后163d左右达到高峰后，随后积累量增加缓慢。根系中N、P、K的积累量一直呈上升趋势，到收获期达到最大值。各个生育时期的地上部分和根系中，N的积累量最大，K次之，P最小。临界期和最大效率期以养分相对积累速率(RARN，g·g^-1·d^-1)分析养分累积高峰期，N、P的最大相对积累强度在100d左右，K的最大相对强度在100～132d之间。出苗后70～100d是N、P、K营养临界期；出苗后100～132d，N、P、K相对吸收强度基本都维持在最高值，是黄芪N、P、K养分最大效率期。黄芪植株Fe、Zn、Cu、和Mn的吸收与累积特性为：Fe、Mn、Zn、Cu元素的含量呈前期逐渐增加后期逐渐下降的趋势。黄芪根系中元素含量为Fe＞Zn＞Cu＞Mn。植株中元素的累积量均为Fe＞Zn＞Mn＞Cu。3．N、P、K对黄芪生长及次生代谢物累积的影响与优化施肥模式研究黄芪的生长及产量形成因子明显受N、P、K肥的供应水平所影响，影响程度由大到小依次为NPK配合＞单施N＞单施K＞单施P。N、P、K各元素及其配比明显影响了黄芪多糖、黄酮和总皂苷的累积，P、K促进了黄芪多糖、黄酮的累积，N、K促进了总皂苷的累积。对黄芪的需肥特性研究表明，黄芪根系中不同元素吸收积累总量顺序为：N＞K＞P＞Fe＞Zn＞Mn＞Cu。每生产100kg黄芪药材需要从土壤和肥料中吸收2．32kgN；0．323kg P₂O₅；1．62kg K₂O；37．77gFe；2．24g Zn；0．66g Mn；0．65gCu。N：P₂O₅：K₂O为7．18：1：5．02。建立了黄芪产量、黄芪根总皂苷与N、P、K三因素的回归模型，采用频率分析法对模型寻优，目标产量为6300～7300 kg／hm²及目标总皂苷含量为12．0～13．5mg／g时，优化施肥方案为N138～156Kg／hm²，P₂O₅79～85 Kg／hm²，K₂O122～43Kg／hm²，配比为1：0．5：0．87。4．黄芪毛状根培养体系的建立及次生代谢的调控用发根农杆菌R1601感染，以下胚轴作为的外植体，菌液A600 nm为0．55～0．60，侵染时间15min，外植体与发根农杆菌共培养3 d，MS培养基附加NAA 0．5mg／L，IBA0．2mg／L及以羧苄青霉素为除菌剂。黄芪毛状根生长可分为延迟期（0～5 d）、恒速生长期（5～25d）和衰亡期（25～40 d）。培养25d毛状根的生长量到达最大值。恒速生长期中毛状根干重的平均增长速率符合方程：（dx）/（dt）=（3．37g）/（F．d） R²=0．841^**相关性达极显著水平。IAA、IBA及NAA在0．1-0．5mg／L浓度范围时促进毛状根生长；2．4-D浓度时抑制黄芪毛状根生长。IAA和NA A促进黄芪毛状根多糖和总皂苷的累积，IBA和2．4-D对黄芪毛状根多糖和总皂苷的积累有负作用。茉莉酸甲酯和水杨酸对黄芪毛状根生长及多糖和总皂苷累积具有促进作用。更多还原

【Abstract】 Astragalus membranaceus （Fisch.） Bge.is a plant of the family leguminous and aperennial herb.Its roots usually used as a traditional staple medicinal material.As ceaselessresearches in the rule of pharmacology and health care ofAstragalus membranaceus （Fisch.）Bge.,it is not only applied as medicinal materials but as materials in many industrialproduction such as beverage,invigorant,cosmetic and tea.The market demand of Astragalusmembranaceus （Fisch.） Bge.is increase with years.Accordingly,the cultivation Astragalusmembranaceus （Fisch.） Bge.is the primary source of medicinal materials.However the yieldand quality is lower than wilding Astragalus membranaceus （Fisch.） Bge.Because there isshort of the basic research in mineral nutrition and the information of fertilization inAstragalus membranaceus （Fisch.） Bge.is unsystematic.In this thesis,Astragalusmembranaceus （Fisch.） Bge.Was taken as experimental materials,the growing rules andnutrient absorption characteristics were explored by field experiments and chemical analysis.The accumulation dynamic of polysaccharide and total saponin in different fertilization leverand the optimization fertilization model for its growth,high-yield and good-quality wasascertained by three factor guadratic D-saturation optimization design in field experiment.The growth rule and secondary metabolism of hair root ofAstragalus membranaceus （Fisch.）Bge.was explored.These results not only have theoretical significance for plant nutrition andplant physiology but have important significance in planting technology of Astragalusmembranaceus （Fisch.） Bge.It will be beneficial to protecting natural resource of Astragalusmembranaceus （Fisch.） Bge.and to alleviating ecological environmental disruption.Theresults are also having great significance for modernization and internationalization ofModernization of Traditional Chinese Medicine.The main results were summarized asfollows:1.The accumulative law of dry matter was researched using the field experiment.Thedry matter accumulative curve could be described with Logistic equation and was“S”shape.The Logistic equation is W_t=(33170/1+298169e^-0.1087x).The distribution rates of dry matter of itsorgans changed greatly during its growth period.The most rapid period of dry matteraccumulation in shoots is between 100 to 132 days after the seedlings emergence but is between 163 to 185 days in root.2.Absorbing capacity and accumulative law of nutrient elements was systematicallystudied by the field experiment,chemical analysis and biological statistic methods.the resultsshow that absorbing capacity of nitrogen（N）,phosphorus（P）,potassium（K） in plant was insequence of N＞K＞P In its full-growing stage.The nutrient absorption amount of N,P,and Kin shoots is much than in root.Content of iron（Fe）,manganese（Mn）,zinc（Zn）,copper（Cu） inroot was in sequence of Fe＞Zn＞Cu＞Mn but in plant it was Fe＞Zn＞Mn＞Cu。N,P,K accumulation increased continuously and reaches the peak amount at the days of163d after seedlings emergence then it returned to a slow increase until harvest.Accumulationof N,P,and K in root is increase all along and reached its maximum at harvest time.Accumulation of N in plant is the largest,next is K accumulation and P accumulation is thethird.Using the concept of relative accumulation rate,the nutrient critical period and thehighest efficiency period was ascertained.Nutrient critical period of N,P,and K is in the daytime of 70-100d after the seedlings emergence and the highest efficiency period is in the dayof 100-132 d after the seedlings emergence.In its full-growing stage,the amount of nutrientaccumulation in roots was in sequence of N＞K＞P＞Fe＞Zn＞Mn＞Cu.The nutrient requirementfor 100kg roots of Astragalus membranaceus （Fisch.） Bge.was 2.32kgN;0.323kg P₂O₅;1.62kg K₂O;37.77gFe;2.24g Zn;0.66g Mn and 0.65gCu.If compared with N,P,and Knutrients by proportion,the formula is as follows:N:P₂O₅:K₂O=7.18:1:5.02.3.The results of field experiment that using three factor D-saturation optimization designshow that the growth,yield and secondary metabolism were strongly impacted by rate of N,P,K fertilizer.The influence degree of N,P,K for yield was in order of N＞K＞P,forpolysaccharide accumulation was K＞P＞N,for flavonoids accumulation was K＞N＞P.According to frequency analysis and through simulated optimization,optimum fertilizerapplication scheme was N138-156Kg/ha,P₂O₅79-85 Kg/ha,K₂O122-143 Kg/ha in targetyield of root yield is 6300-7300 kg/ha and target content of total saponins is 12.0-13.5 mg/g.The nutrients proportion of N,P,K is as follows:N:P₂O₅:K₂O=1:0.5:0.87.4.Culture system of hair root of Astragalus membranaceus （Fisch.） Bge.withAgrobacterium rhizogene R1601 was established.The results indicated that transformationfrequency on hypocotyl explants was higher than that on the explants of leaf,stems andcotyledon.Hairy roots could come out 3-4 d earlier than usual treatment if explants wereprecultivated for 3 d.The best infection time was 15 minutes and the best bacteria remover iscarbenicilin.The results showed that the growth period of hair root can be divided intodelayed phase（0-5 d）,constant speed growth phase（5-25 d） and decline phase（25-40 d）.Thegrowth amount of hairy root up to the maximum value after 25 days of culture.The growth rate of dry weight in constant speed growth phase could be described with follow equation:（dx/dt）=（3.37g/F.d） R²=0.841**The growth of hairy root and biosynthesis of polysaccharide and total saponins werewere promoted by IAA and NAA.The growth of hairy root and biosynthesis ofpolysaccharide and total saponins were restrained by 2,4-D.IBA promoted the growth butinhibited the biosynthesis of polysaccharide and total saponins of hairy root.Methyljasmonate and Salicylic Acid could stimulate the growth and accumulation of polysaccharideand total saponins in hair root ofAstragalus membranaceus （Fisch.） Bge.更多还原