【作者】 王景艳；

【导师】 刘兆普；

【作者基本信息】 南京农业大学 ， 植物营养学， 2009， 博士

【摘要】 长春花(Catharanthus roseus (L.) G. Don)是夹竹桃科长春花属一种重要的药用植物,其所含的吲哚生物碱(Terpenoid Indole Alkaloids, TIAs)是一类重要的次生代谢物质,具有世界公认的抗肿瘤、抗癌等功能。长春花吲哚生物碱的代谢和积累具有组织和发育阶段特异性；环境因子,比如光、温度、水分、盐分等既极大影响植物的初生代谢,在生物碱的代谢过程中也起着积极的调控作用；植物生长调节物质,如茉莉酸甲酯(methyle jasmonate, MeJA))、脱落酸(abscisic acid, ABA)等都在植物次生代谢调控中起着重要作用。本论文通过温室砂培试验,研究了不同浓度NaCl和海水处理分别对长春花幼苗及长春花不同生长发育时期、不同部位生物碱含量及产量的影响,为通过人工栽培措施提高生物碱含量、降低生产成本和后续大田栽培研究提供理论依据。在此基础上,研究了代谢前体(色氨酸)、不同生长调节物质(CaCl2、ABA、MeJA)与盐分对长春花生长和生物碱代谢的耦合效应,以进一步探索长春花生物碱在逆境胁迫下的代谢调控效应及其机制。主要结果如下：1.用不同浓度NaCl(0、50、100、150、200和250mmol·L-1)和等Na+含量的海水(0、10%、20%、30%和40%)分别浇灌长春花幼苗7天,长春花幼苗生长受到不同程度抑制。在NaCl处理下,植株光合速率降低。色氨酸脱羧酶(TDC)活性及文多灵、长春质碱、长春碱和长春新碱的含量及目标碱(长春碱和长春质碱)单株产量在50mmol·L-1NaCl处理下比淡水浇灌处理显著增加；即使在200mmol·L-1NaCl处理下,长春花干重虽然显著降低,但长春花中主要的目标生物碱产量仍显著高于淡水灌溉植株的生物碱产量,这一代谢调控措施可以显著提高生物碱产量,并大幅降低长春花中生物碱的分离提取成本。海水处理抑制长春花幼苗生长,其生物量下降。在10%海水灌溉下,长春花幼苗TDC活性及文多灵、长春质碱和长春碱含量和单株产量比淡水浇灌植株相比显著升高；长春新碱的含量和单株产量在海水处理下比淡水处理显著降低,表明低浓度海水处理能显著地促进长春花幼苗中目标生物碱中间产物(文多灵和长春质碱)的积累。2.研究了NaCl及海水处理分别对不同生长发育时期的长春花生长、离子分布及不同部位生物碱含量和单株产量的变化,探索长春花对盐胁迫响应机制及初生与次生代谢调控效应的变化。结果表明,盐处理抑制长春花生长,增加其体内Na+和Cl-的含量,降低K+含量。在所有处理的植株中,长春花四种生物碱的含量都是在叶片中最高,根次之,茎最低。与淡水浇灌处理相比,盐处理能够增加长春花中生物碱的含量和生物碱单株产量；5%海水和25mmol·L-1NaCl处理下长春花四种生物碱的产量显著高于10%海水和50mmol·L-1NaCl处理。这一研究结果为在我国南方海涂地区种植和利用海水资源浇灌长春花提供了理论依据。正常生长的长春花植株在播种后100天(果盛期)叶中四种生物碱含量最高,这为在实际生产中掌握长春花合适的收获日期、提高其生物碱提取率和降低生产成本奠定基础。3.通过砂培长春花幼苗试验,研究了外源CaCl2对淡水浇灌及盐处理下长春花生长和吲哚生物碱代谢的影响。结果表明：2.5和7.5mmol·L-1外源CaCl2对长春花幼苗生长没有显著影响,12.5mmol·L-1外源CaCl2抑制长春花生长。与淡水处理相比,外源CaCl2处理下长春花幼苗TDC活性显著升高,文多灵、长春质碱和长春碱的含量及产量显著增加。长春新碱含量及单株产量在2.5和7.5mmol·L-1外源CaCl2处理下与淡水处理相比差异不显著,在12.5mmol·L-1CaCl2处理下显著降低。说明适当浓度外源CaC12对长春花幼苗生物碱代谢有较好的调控作用。7.5mmol·L-1外源CaCl2能显著减轻NaCl对长春花幼苗的胁迫危害,提高盐处理植株TDC酶活性,显著增加长春花中文多灵、长春质碱和长春碱含量及单株生物碱产量。表明,7.5mmol·L-1外源CaCl2能促进盐胁迫下长春花幼苗生物碱的积累。4.向淡水处理的长春花幼苗喷施外源色氨酸后,其生长受到抑制,吲哚生物碱含量增加,单株生物碱产量变化不大。100mg·L-1外源色氨酸能够显著缓解100mmol·L-1NaCl对长春花幼苗造成的伤害,并且提高长春花体内文多灵、长春碱和长春新碱的单株产量。5.外源ABA对淡水处理的长春花幼苗生长有一定的抑制作用,其TDC酶活性在50和100μmol·L-1ABA作用下比淡水处理植株显著上升；除长春碱的单株产量比淡水处理显著增加外,其余3种生物碱的单株产量与淡水处理相比都无显著差异。外源ABA(10和50μmol·L-1)能减轻盐分对植物的胁迫危害,增加100mmol·L-1NaCl处理下长春花体内四种生物碱的产量。喷施外源MeJA能够显著提高淡水处理的长春花幼苗TDC活性和四种吲哚生物碱的含量及单株产量。盐处理植株再喷施外源MeJA后,NaCl对长春花幼苗的胁迫危害有不同程度的降低,其中以100μmol·L-1MeJA缓解盐害效果最好。外源2001μmol·L-1MeJA能显著提高100mmol·L-1NaCl处理下长春花幼苗体内文多灵、长春质碱、长春碱及长春新碱的含量和单株产量。与ABA相比,MeJA对淡水处理和盐处理下长春花中生物碱的代谢的促进作用更显著。更多还原

【Abstract】 Catharanthus roseus (L.) G. Don is a perennial, important medicinal and evergreen herb in the dogbane family (Apocynaceae). The terpenoid indole alkaloids (TIAs) in the plant’s composition such as catharanthine, vinblastine and vincristine are very important secondary metabolic productions owing to their wide pharmaceutical applications for human health.The metabolism and regulation of C. roseus alkaloids have been intensively investigated and that consequently the metabolizing ways were under coordinate regulations. Alkaloids are under organ-, development-and environment-specific and plant growth regulators control.In present study, the effects of different concentrations of NaCl and seawater stress, precursor, plant growth substance on the growth and alkaloid contents were determined to explicit relationship between alkaloid biosynthesis and environmental factors, moreover, provide useful data for enhancing the yield of the alkaloid by artificial cultivation. The main results obtained as follows.1. Seedlings of C. roseus were treated with different concentrations of NaCl (from0to250mmol·L-1) and seawater (from0to40%) stress respectively. Under salt stress, the fresh weight and dry weight of C. roseus seedlings decreased whereas the TBARS contents increased. The TDC activity was highest under50mmol-L"1NaCl stress, and then decreased with the increasing of NaCl concentrations. The vindoline, catharanthine, vinblastine and vincristine contents and yield of per plant were highest under50mmol·L-1NaCl treatment.The activity of TDC was highest with10%seawater treatment. The contents of vindoline and catharanthine were highest under10%stress compared with other treatments. And vinblastine contents increased with the increasing of seawater concentrations. Consequently, salt stress could restrain the growth of C. roseus seedlings, while promote the metabolite of alkaloid and enhance the alkaloid contents. Results from this experiment showed that under the salt treatment, the biomass of C. roseus seedlings decreased significantly, but the primary goal alkaloids yield were significantly higher than that of fresh water irrigated plants, especially in50mmol·L-1NaCl and10%seawater-treated plants.2. A sand culture experiment of C. roseus was conducted to determine the effects of different concentrations of NaCl (25and50mmol·L-1) and seawater (5%and10%) treatments on various durations of plant growth, inorganic ions and indole alkaloid concentrations in C. roseus. The salt-stressed plants had decreased the total fresh and dry weight and tissue K+concentration of C. roseus, and increased Na+and Cl-concentrations in roots, stems and leaves when compared to the control. The vindoline, catharanthine, vinblastine and vincristine concentrations and yield of per plant increased under seawater treatment. Under5%seawater and25mmol·L"1NaCl treatments, the concentrations and yield of the four kinds alkaloid were higher than that of10%seawater and50mmol·L-1NaCl treatments. Considering the industrial alkaloid production, it could reduce the cost of producing of alkaloid. In control plants, the highest of alkaloid concentrations were measured on100days after planting (DAP); hence, plant harvest must be optimized in terms of growth duration.3. Applied exogenous2.5and7.5mmol·L-1CaCl2had no effects on the growth of non-salt-stressed plants where as12.5mmol·L-1CaCl2decreased their biomass. TDC activity, vindoline, catharanthine and vinblastine contents and yield of per plant increased when exposed to exogenous CaCl2. Supplemental2.5and7.5mmol·L-1CaCl2had no effects on the vincritine content and yield while12.5mmol·L-1CaCl2treatment decreased the vincristine content and yield against control.Applied7.5mmol·L-1CaCl2to NaCl treated plants increased fresh and dry weight whilst decreased TBARS contents. TDC activity increased significantly under NaCl with or without CaCl2(7.5and12.5mmol·L-1) treatment when compared with the control. The vindoline, catharanthine, vincristine and vinblastine concentrations increased under salt stress. Exogenous7.5mmol·L-1CaCl2to100mmol·L-1NaCl-stressed plants increased indole alkaloid concentrations and yield of per plant significantly when compared with control. Exogenous CaCl2(7.5mmol·L-1) could promote the indole alkaloid metabolism under salt stress. 4. Exogenous tryptophan restrained the growth of non-salt-stressed C. roseus seedlings and promoted the alkaloid accumulation, but the yield of per plant had no significant change when compared with control. Supplemental100mg·L-1tryptophan alleviated the salt stress (100mmol·L-1NaCl) damage on the C. roseus seedlings and increased vindoline, vinblastine and vincristine yield of100mmol·L-1NaCl-stressed plant.5. Exogenous abscisic acid (ABA) decreased the biomass of C. roseus seedlings. The TDC activity increased significantly with50and100μmol·L-1ABA treatments against control. With ABA treatment, the vinblastine yield increased when compared with control. Exogenous ABA (10and5050μmol·L-1) could alleviate the effects of salt stress on the C. roseus seedlings. Applied ABA to100mmol·L-1NaCl-treated plants increased the contents and yield of vindoline, catharanthine, vinblastine and vincristine.Supplemental methyle jasmonate (MeJA) to non-salt-stressed plants reduced the biomass of the seedlings but enhanced the TDC activity and increased the alkaloid contents and yield. Applied exogenous MeJA (100μmol·L-1) alleviated the effects of salt stress on the C roseus seedlings. Supplemental200μmol·L-1MeJA to100mmol·L-1NaCl-treated plants increased the contents and yield of vindoline, catharanthine, vinblastine and vincristine. Compared with the ABA, MeJA was more preferable to promote the alkaloid metabolism of C. roseus seedlings.更多还原