【作者】 文涛；

【导师】 杨文钰；

【作者基本信息】 四川农业大学 ， 作物栽培与耕作学， 2009， 博士

【摘要】 虎杖(Polygonum cuspidatum Sieb.et Zucc.)为多年生蓼科草本植物,是我国传统中药,其主要活性成分白藜芦醇(Resveratrol)是一种天然抗毒素,同时具有多种药理作用,被誉为最有前途的抗癌药物之一。为了实现该药用植物资源的可持续利用,以获得高含量的白藜芦醇为目标,本研究建立了虎杖愈伤组织无性系。从愈伤组织的光、温培养条件、前体物质苯丙氨酸的添加,以及真菌诱导子、水杨酸与紫外线诱导方面对其展开了系统研究,并通过对诱导培养过程中虎杖愈伤组织生理生化变化的分析,探讨了调控措施对愈伤组织中白藜芦醇生物合成的影响,建立了提高虎杖愈伤组织白藜芦醇含量的调控技术。并得出以下主要结论:1.通过研究外植体和培养基对愈伤组织的影响,了解到以茎段作为外植体,其愈伤组织生长比叶片快,且其中的白藜芦醇含量为叶片中的5倍;MS培养基上的愈伤组织增殖量最多,其茎段和叶片的鲜重增长率均极显著高于N6和B5培养基,分别达到了141.0%和73.8%,但鲜样中白藜芦醇含量略低于B5培养基。因此,以MS为基本培养基,茎段为外植体诱导出的愈伤组织成愈较高,生长旺盛,维持时间长,白藜芦醇含量高,优于其他处理。2.通过分析愈伤组织生长与白藜芦醇含量的关系,可见在愈伤组织生长最快时期,白藜芦醇含量增加也达到最高,因此对于虎杖愈伤组织的收获应选择在愈伤组织快速生长达到饱和时的时期,整个培养可在同一种培养基中完成。3.通过正交实验研究了溶剂种类、时间、温度对愈伤组织中白藜芦醇提取率的影响,极差分析结果显示,提取工艺过程中最重要的因素为提取温度,其次是提取时间,而提取溶剂对白藜芦醇提取率的影响最小。综合评价各指标,用乙酸乙酯在4℃下提取48 h效果最佳。4.将茎段诱导出的愈伤组织,分别置于暗光(0 lx)、弱光(1340～1560 lx)、强光(3110～3310 lx)三个条件下进行培养,结果显示,愈伤组织的生物量随着光照强度的增强而增加,强光培养下愈伤组织的生物量达到了53.682 g/LFW。但白藜芦醇含量与光照强度不成正相关,弱光培养下,愈伤组织中白藜芦醇的含量显著高于另外两个处理,为140.074μg/gDW。因此虎杖愈伤组织对光敏感,以弱光培养效果最好。25℃培养条件下茎段愈伤组织生长速度适宜,愈伤组织出现较早,愈伤组织中白藜芦醇含量最高。5.通过不同浓度前体物质苯丙氨酸饲喂虎杖愈伤组织,研究发现10～30 mg/L浓度的苯丙氨酸能促进愈伤组织生物量的增殖,20 mg/L处理愈伤组织生物量增殖最多。用Richards方程模拟愈伤组织的生物量在一个生长周期内的动态积累过程,可以稳健、可靠地估计不加前体物质愈伤组织的生长动态变化,发现苯丙氨酸在一定程度上调节了愈伤组织的增长情况。由生长速率参数(K)可知,随着苯丙氨酸浓度的增加,愈伤组织增长速度加快,极显著高于对照16.5倍以上。且白藜芦醇含量也随着苯丙氨酸浓度的增加而增加,30mg/L处理白藜芦醇的含量达到最大值,鲜样、干样中分别为18.025μg/g和241.489μg/g,较对照高出16.8%和5.7%。6.前体物质苯丙氨酸能诱导苯丙氨酸裂解酶(PAL)活性的增高,PAL的活性表现出随着添加的苯丙氨酸浓度的增大而升高。各处理PAL活性的变化趋势基本一致,都在培养的第7d达到最大值,表明培养前期,苯丙氨酸浓度的增加促进了PAL活性的增强,为白藜芦醇的合成提供了充足的底物,促进了白藜芦醇的合成。此外添加适量的前体物质能延缓虎杖愈伤组织细胞的衰老,增强愈伤组织的抗性。7.通过以黑曲霉、青霉、啤酒酵母和灰葡萄孢的粗提物为诱导子对愈伤组织进行诱导,发现黑曲霉诱导子对白藜芦醇合成的促进作用最为显著,比对照提高了50.2%,表明虎杖对真菌诱导子种类具有选择性。同时研究发现以100 mg/L的黑曲霉诱导子效果最好,比对照高128.9%。同时,诱导子添加时间不同对愈伤组织和白藜芦醇含量的影响不同,以第0d加入白藜芦醇含量最高,但对愈伤组织生物量的积累有抑制作用,而在第14d和21d加入诱导子对愈伤组织生物量的积累没有影响。在诱导培养过程中,分析白藜芦醇含量的变化规律发现,培养到第6天白藜芦醇含量达到最高,为227μg/g,而在第6天后白藜芦醇含量开始下降。综合分析可得到虎杖愈伤组织真菌诱导优化方案:在接种后第14d加入100mg/L的黑曲霉诱导子,诱导培养6d后收获,可以使处理中白藜芦醇的含量比对照提高2.25倍。8.对黑曲霉诱导子组分分离研究结果表明,黑曲霉诱导子提取液中主要是真菌壁多糖诱导了愈伤组织中白藜芦醇的生物合成,而蛋白质对愈伤组织中白藜芦醇的合成有抑制作用。9.通过分析真菌诱导子对虎杖愈伤组织中苯丙氨酸解氨酶(PAL)、超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)酶活性的影响、内源激素脱落酸(ABA)的变化以及POD和CAT同工酶变化,研究发现真菌诱导子刺激后在培养第1d到第4d内源ABA含量大量产生,呈急剧上升趋势,在第4d达到峰值,PAL活性迅速增加,也在第4d达到最高;同时在培养的前3d,SOD和POD酶活性下降,CAT在第2d出现第一次高峰:当培养到第5d至第6d,对照与诱导处理之间出现很大的差异,SOD与POD活性诱导处理突然出现下降再急剧上升的趋势,而对照则维持较高水平;CAT在第5天出现第二次高峰;POD同工酶有新酶带的出现。10.通过对水杨酸添加浓度、加入时间、诱导时间的筛选,发现在接种后第14d加入7mg/L的水杨酸,诱导6d的效果最好,可以使白藜芦醇比对照高近1倍,但诱导效果不及黑曲霉诱导子。黑曲霉诱导子与水杨酸、苯丙氨酸的进行不同组合,研究发现各组合对白藜芦醇的合成均有一定促进作用但与单独添加黑曲霉诱导子相比变化不大,黑曲霉诱导子、水杨酸和苯丙氨酸三种物质组合效果最好。11.紫外光(30W)下20cm处照射,每次照射15 min,停止5 min,重复照射6次,2h照射时间处理,不能提高愈伤组织中自藜芦醇含量。12.建立了提高虎杖中白藜芦醇含量的调控技术,以虎杖茎段为外植体,在MS+6-BA 1.0+KT 0.2～0.5+NAA 0.2(mg/L)培养基上建立愈伤无性系,转接于液体培养基上,在继代后第14天加入黑曲霉粗提物100 mg/L为诱导子。同时饲喂20mg/L苯丙氨酸和微量水杨酸,在弱光1300lx,25℃培养,加入后第6天收获,可提高虎杖愈伤组织中白藜芦醇含量达3倍以上。更多还原

【Abstract】 Polygonum Cuspidatum is a kind of herbage belongs to polygonaceae.It is a Chinese traditional medicine.The mostly activity component is Resveratrol in Polygonum Cuspidatum. Resveratrol is consider of a promising natural chemical anticarcinogen.In order to achieve the sustainable use of Polygonum Cuspidatum resources,regard obtaining high-load resveratrol as goal,this research set up Polygonum callus clones.From the callus of light,temperature culture conditions,precursors of phenylalanine added,as well as fungal elicitor,Salicylic acid and UV-induced,we Carried out systematic research.Through to inducing the analysis of training physiological biochemistry of Polygonum cuspidatum callus in the course to change,have canvassed the measure of adjusting and controlling to the biosynthesis influence of resveratrol in the callus,have set up the technology system of improving the resveratrol content of Polygonum cuspidatum.And draw the following main conclusion:1.Through studying the impact on callus culture from explant and medium,understand that regards stem as explant,its callus grows faster than the blade,and the concentration of resveratrol among them is 5 times in the blade;The callus on MS culture medium is the most in proliferation,fresh heavy growth rate of its section of stem and blade is higher than N6 and B5 culture medium extremely notably,up to 141.0%and 73.8%separately,but the alcohol content of the white black false hellebore is slightly lower than B5 culture medium,but the concentration of resveratrol is slightly lower than B5 culture medium.So regard MS as basic culture medium,stem as explant induce appear callus relatively high,grow vigorously,maintain long time,the concentration of resveratrol is high,superior to other treatment.2.Passing analyzes the relation between the callus grows and the concentration of resveratrol,it is obvious the callus grows in the fastest period,while the concentration of resveratrol reach the most high too,so Polygonum callus harvest should be selected in the callus Organization of rapid growth when it reached saturation period,the entire culture can be completed in the same medium.3.Orthogonal experimental study the impact of solvent kind,time and temperature on the extraction rate of resveratrol,the variance analysis show that the most important factor is the extraction temperature,secondly the extraction time,and it is minimum to show the impact on drawing extraction rate of the resveratrol of solvents.Comprehensive all,using the ethyl acetate extract 4℃under 48 h is the best results.4.The callus inducing out the stem section,locate in dark mere(0 lx),weak light(1340-1560 separately Lx),strong light(3110-3310 Train under lx) three conditions,the result reveals,the biomass of the callus increases with enhancement of illumination intensity,the strong light trains the biomass of the lower callus to be up to 53.682 g/LFW.But the resveratrol content and illumination intensity of the resveratrol do not become positive correlation,only train weakly,the content of resveratrol is higher than other two to deal with notably in the callus,it is 140.074μg/ gDW.So the Polygonum cuspidatum callus is sensitive to the light,by only training the result to be best weakly.25℃trains one section of calluses growth speed of stem under the condition to be suitable,the callus relatively appears early,resveratrol has the highest content,is superior to other treatment in the callus.5.Feed Polygonum Callus on the different concentrations of phenylalanine precursor substances, the study found that 10～30 mg/L concentrations of phenylalanine can promote callus proliferation of biomass,20 mg/L deal Callus proliferation of the largest biomass.Richards equation with simulated callus biomass growth cycle in a process of dynamic accumulation,found no precursor substances in the circumstances,can get steady and reliable estimates,phenylalanine in a certain extent,the regulation of callus increased.By the rate of growth parameters(K),we can see that with the increase in the concentration of phenylalanine,callus growth rate accelerated significantly higher than the control,higher than the 16.5 times.The resveratrol also with the content and phenylalanine concentration increases,30mg/L deal with the maximum concentration of resveratrol,fresh,dried samples were 18.025μg / g and 241.489μg/g,controls more than 16.8%and 5.7%.6.Precursor substances phenylalanine can induce phenylalanine pyrolysis of enzyme activity,PAL activity of the show along with the added phenylalanine concentrations higher increase.To deal with all the activity of PAL’s basically the same trend,both in the development of the first 7 d maximum,pre-show culture,phenylalanine concentration promoted an increase in PAL enzyme activity increased,for the synthesis of resveratrol the substrate to provide adequate and promote the synthesis of resveratrol.Therefore,add the appropriate amount of precursor substances to delay the Polygonum callus cell senescence,increased resistance to the callus.7.Through Aspergillus niger,Penicillium,brewer’s yeast and the Botrytis cinerea extracts for the induction of sub callus induction carried out and found niger-induced synthesis of resveratrol on the most significant role in the promotion,improved control 50.2%that of Polygonum-type fungi induced selective.At the same time,in order to study found that 100 mg/ L of niger-induced best, 128.9%higher than the control group.When elicitor was added at different times,it was to different influence on callus and content of resveratrol.The content of resveratro was highest at the beginning,but the callus on the accumulation of biomass is a disincentive and in the first 14d and 21d join the sub-induced callus on the accumulation of biomass did not affect.In the process of induction training,the content of resveratro changes in the law of the analysis.It found that the cultured content of resveratro reached highest in the first 6d and the content was 227μg/g,but after the first 6d the content of resveratro started to decline.As a result,by a comprehensive analysis,we receive a best program: Of fungi induced callus.On the 14 day of Polygonum callus was inoculated, elicitor(100mg/L)from Aspergillus niger was added into the medium,the introduction of elicitor from Aspergillus niger the content of resveratrol in Polygonum callus was 2.25 times higher than that of the control.8.Aspergillus Niger inducer separation results show that Aspergillus niger-induced extract the main wall is a fungal polysaccharide induced a callus in the biological synthesis of resveratrol,and the composition of protein in callus inhibit the synthesis of resveratrol.9.Through fungal elicitor of Polygonum callus in PAL(PAL),superoxide dismutase(SOD), peroxidase(POD) and catalase(CAT) activity,Endogenous hormone changes in the ABA,as well as CAT and POD isozyme analysis found that the fungal elicitor stimulated the first Culture in 1d to 4d endogenous ABA have a lot of content,showed a sharp upward trend in the first 4d peak, PAL activity increased rapidly,reaching a peak in the first 4d;at the same time in training before the 3d,SOD and POD activity decreased,CAT in the first 2d appeared for the first peak;when the first train to 5d to 6d,a great deal of difference between Control and induced,SOD activity with POD induced processing suddenly dropped sharply,then a sharp upward trend,while the control is at a fairly high level;CAT at the 5th day of the second peak;POD isozyme have with the emergence of a new enzyme;and CAT MB is no significant change.10.Filtrating the adding the concentration of salicylic acid,adding time and screening time,By adding salicylic acid of 7mg/L by the last 14d,induced 6d of the best,we can make resveratrol nearly 1 time higher than the control group,but the effects induced by Aspergillus niger as elicitor. A.Niger elicitor and salicylic acid,phenylalanine in different combinations,the combination of resveratrol synthesis have a certain role,but with a separate add-in comparison A,niger induced change little,induced by A..niger,salicylic acid and phenylalanine is the most effective combination of three types of material.11.By exposuring UV(30W) under the department 20cm at per 15 min,stopped 5 min and repeated exposure to 6 times,the content of resveratro can not raise in 2h exposure processing.12.Set up the technology system to improve the resveratrol of Polygonum cuspidatum,regard stem section of Polygonum cuspidatum as explant,establishing callus clones on MS十6-BA 1.0十KT 0.2～0.5十NAA 0.2(mg/L) culture medium,Switching adds crude extract of dark Aspergillus 100 mg/L as Niger inducer on the 14th day after taking the place of on liquid culture medium,feeds 20 mg/L phenylalanine and minim salicylic acid at the same time,trains in weak mere 13001x,25℃.On the 6th day after adding,we can improve by more than 3 times of resveratrol in Polygonum cuspidatum callus.更多还原