青蒿毛状生长,青蒿素合成以及营养物消耗的动力学

诱导产生的青蒿毛状根培养物置于ＭＳ培养基（含３０ｇ／Ｌ蔗糖）进行悬浮培养,并对悬浮培养过程中毛状根生长、青蒿素合成、蔗糖、磷酸盐和不同氮源的消耗、ＰＨ和电导率的动力学过程进行分析。经３０天培养,生物量干重和青蒿纱产量分别达到１３．７ｇ／Ｌ和０．２３ｇ／Ｌ,碳源和氮 源在培养过程中被逐渐利用,而磷酸盐的利用速率最快,培养至１５天所有的磷酸均被吸收,ＰＨ在培养初期降低,后又通宵四升, 率由于毛状根生长     

温度对青蒿毛状根生长和青蒿素生物合成的影响

本实验研究了不同温度(15℃～35℃)对青蒿毛状根生长和青蒿素生物合成的影响,发现25℃有利于毛状根生长,30℃促进了青蒿素生物合成。通过温度改变的二步培养技术(培养前20d温度控制在25℃,后10d温度提高到30℃),青蒿素的产量得到明显提高,高于在恒温培养时(25℃或30℃)的结果。     

青蒿毛状根合成青蒿素的培养条件研究

对影响青蒿（ＡｒｔｅｍｉｓｉａａｎｎｕａＬ．）毛状根生长及青蒿素合成的培养条件进行了研究,确定最适的培养条件为：初始ｐＨ５．８～６．０,摇瓶转速１３０～１５０ｒ／ｍｉｎ,摇瓶装液量体积分数为２５％,光照周期为１６ｈ／ｄ,温度为３０℃。在此条件下,经过２５ｄ培养获得青蒿素产量为２２３．３ｍｇ／Ｌ。     

青蒿素在露水草毛状根中的生物转化

露水草毛状根培养系中加入青蒿素培养８ｄ后,青蒿素转化去氧青蒿素。根据光谱数据,对去氧青蒿素的结构进行了鉴定。结果表明,通过水草毛状根能将青蒿素进行选择性还原为去氧青蒿素。     

利用气升式内环流生物反应器培养青蒿毛状根生产青蒿素

利用自制的气升式内环流生物反应器进行青蒿（ArtemisiaannuaL．）毛状根多层培养生产青蒿素。毛状根培养物在培养过程中均匀分布在生物反应器的筛网间，或以不锈钢网为附着点向四周生长，在25℃和12h／d光照周期下，经20d分批培养获得生物量干重22．57g／L，青蒿素产量374．4mg／L，并对培养过程中蔗糖、磷酸盐、硝酸盐和氨盐消耗的动力学进行了分析。     

利用气升式内环流生物反应器培养青蒿毛状根生产青蒿素

利用自制的气升式内环流生物反应器进行青蒿(Artemisia annua L.)毛状根多层培养生产青蒿素。毛状根培养物在培养过程中均匀分布在生物反应器的筛网间,或以不锈钢网为附着点向四周生长,在25 ℃和12 h/d光照周期下,经20 d分批培养获得生物量干重22.57 g/L,青蒿素产量374.4 mg/L,并对培养过程中蔗糖、磷酸盐、硝酸盐和氨盐消耗的动力学进行了分析。     

栝楼毛状根的生长与营养消耗动态研究

研究了栝楼毛状根在摇瓶培养条件下的生长与营养消耗规律。栝楼毛状根的生长过程可以分为四个生长阶段。生长迟滞期,缓慢生长期,快速生长期和衰亡期。在迟滞期,培养液中的蔗糖,氮,磷,钾首先被快速吸收,然后又有所回升;在缓慢生长期,培养液中的葡萄糖,果糖,氮,磷和钾的浓度开始降低,到了快速生长期,培养液中的碳源,氮,磷,钾等营养物质被快速消耗,到了衰亡期,以上的营养物质基本被消耗殆尽,Ca^2 从接种开始缓慢下降,当毛状根进入衰亡期以后,由于老化细胞的损伤而有部分钙离子外泻,栝楼毛状根的蛋白质也开始快速分泌。     

银型磷酸锆盐抗菌粉对青蒿根生长的影响及生长动力学研究

在植物组织和细胞培养过程中,尤其是在生物反应器培养中的染菌问题,一直是制约植物细胞培养工业化的难题,通过比较各种防腐剂的抑菌效果。确定银型磷酸锆盐抗菌粉为青蒿根培养的最佳防腐剂,银型磷酸锆盐抗菌粉在浓度为30mg/L时,既能降低培养液的染菌几率,又不明显抑制青蒿根的生长及青蒿素的生物合成,在添加30mg/L抗菌粉的培养液中进行的青蒿根生长,pH值变化以及残糖,铵离子和硝酸根离子消耗的动力学研究表明,在40d内青蒿根在培养液中生长良好,营养成分的消耗和对照呈相似的趋势。     

银型磷酸锆盐抗菌粉对青蒿根生长的影响及生长动力学研究

在植物组织和细胞培养过程中,尤其是在生物反应器培养中的染菌问题,一直是制约植物细胞培养工业化的难题.通过比较各种防腐剂的抑菌效果,确定银型磷酸锆盐抗菌粉为青蒿根培养的最佳防腐剂.银型磷酸锆盐抗菌粉在浓度为30 mg/L时,既能降低培养液的染菌几率,又不明显抑制青蒿根的生长及青蒿素的生物合成.在添加30 mg/L抗菌粉的培养液中进行的青蒿根生长、pH值变化以及残糖、铵离子和硝酸根离子消耗的动力学研究表明,在40 d内青蒿根在培养液中生长良好,营养成分的消耗和对照呈相似的趋势.     

生长调节物质对栝楼毛状根生长和天花粉蛋白合成的影响

添加GA3和CCC虽然影响栝楼毛状根的生物量积累 ,但是有利于促进天花粉蛋白 (TCN)的合成 ,当GA3的添加量为 2mg/L时 ,天花粉蛋白含量增加了 18 9% ,添加 1mg/L～ 2mg/LCCC的天花粉蛋白提高了 2 8%。分别添加维生素B1或B5会延长毛状根的生长迟滞期 ,但是 ,后期的生长非常迅速 ,并可以促进天花粉蛋白的合成。同时添加维生素B1和B5可以缩短生长迟滞期 ,并能促进毛状根的生长 ,同时添加1mg/LB1 4mg/LB5有利于促进天花粉蛋白总量的增加。     

Modulation of artemisinin biosynthesis by elicitors,inhibitor, and precursor in hairy root cultures of Artemisia annua L.

Artemisinin is frequently used in the artemisinin-based combination therapy to cure drug-resistant malaria in Asian subcontinent and large swath of Africa. The hairy root system, using the Agrobacterium rhizogenes LBA 9402 strain to enhance the production of artemisinin in Artemisia annua L., is developed in our laboratory. The transgenic nature of hairy root lines and the copy number of transgene (rol B) were confirmed using polymerase chain reaction and Southern Blot analyses, respectively. The effect of different concentrations of methyl jasmonate (MeJA), fungal elicitors (Alternaria alternate, Curvularia limata, Fusarium solani, and Piriformospora indica), farnesyl pyrophosphate, and miconazole on artemisinin production in hairy root cultures were evaluated. Among all the factors used individually for their effect on artemisinin production in hairy root culture system, the maximum enhancement was achieved with P. indica (1.97 times). Increment of 2.44 times in artemisinin concentration by this system was, however, obtained by combined addition of MeJA and cell homogenate of P. indica in the culture medium. The effects of these factors on artemisinin production were positively correlated with regulatory genes of MVA, MEP, and artemisinin biosynthetic pathways, viz. hmgr, ads, cyp71av1, aldh1, dxs, dxr, and dbr2 in hairy root cultures of A. annua L.     

Improved growth of Artemisia annua L hairy roots and artemisinin production under red light conditions

Hairy root cultures of Artemisia annua L were cultivated for 30 days under either white, red, blue, yellow or green light. Red light at 660 nm gave the highest biomass of hairy roots (5.73 g dry wt cells l^–1 medium) and artemisinin content (31 mg arteminsinin g^–1 dry cells) which were, respectively, 17% and 67% higher than those obtained under white light.     

Isolation and production of artemisinin and stigmasterol in hairy root cultures of Artemisia annua

Scaled-up hairy root culture of Artemisia annua L. was established in three-liter Erlenmeyer flask. Both artemisinin and stigmasterol that derive from the common precursors of isopentenyl diphosphate and farnesyl pyrophosphate were isolated from hairy roots. The production rate of artemisinin isolated by column chromatography from hairy root cultures was 0.54% (mg.gDW⁻¹). Stigmasterol was identified by mass spectrometry and nuclear magnetic resonance analysis. The production of stigmasterol isolated by column chromatography from hairy root cultures was 108.3% (mg.gDW⁻¹). In hairy root cultures, the production rate of stigmasterol was estimated to be 201 times greater than that of artemisinin. Our results suggest that investigation of secondary metabolites may provide a new insight to study artemisinin production in hairy root cultures. This revised version was published online in August 2006 with corrections to the Cover Date.     

Nitric oxide potentiates oligosaccharide-induced artemisinin production in Artemisia annua hairy roots

The purpose of the present study was to characterize the generation of nitric oxide （NO） in Artemisia annua roots induced by an oligosaccharide elicitor （OE） from Fusarium oxysporum mycelium and the potentiation role of NO in the elicitation of artemisinin accumulation. The OE （0.3 mg total sugar/mL） induced a rapid production of NO in cultures, which exhibited a biphasic time course, reaching the first plateau within 1.5 h and the second within 8 h of OE treatment. Artemisinin content in 20-day-old hairy roots was increased from 0.7mg/g dry wt to 1.3 mg/g dry wt by using the OE treatment for 4d. In the absence of OE, the NO donor sodium nitroprusside （SNP） at 10, 50 ~1 and 100 ~1 enhanced the growth of hairy roots, but had no effect on artemisinin synthesis, The combination of SNP with OE increased artemisinin content from 1.2 mg/g drywt to 2.2 mg/g dry wt, whereas the maximum production of artemisinin in cultures was 28.5 mg/L, a twofold increase over the OE treatment alone. The effects of SNP on the OE-induced artemisinin were suppressed strongly by the NO scavenger 2-（4- carboxyphenyl）-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide （cPTIO）. The results suggest that NO can strongly potentiate elicitor-induced artemisinin synthesis in A. annua hairy roots.     

青蒿发根生长及青蒿素生物合成动态的研究

从747条发根农杆菌ATCC15834转化的青蒿株系025发根中,筛选出7个生长较快的发根系,这7个系在生长速度和青蒿素含量上均有显著差异,其中发根系HR9青蒿素产率最高,达到每月3325mg/L。青蒿发根的生长量和青蒿素含量极显著高于未转化根和愈伤组织。青蒿发根在分批培养中没有明显的迟滞期,接种后第7天进入指数生长期,第11天生长最快,第20天进入稳定期。青蒿发根中青蒿素含量呈明显的“与生长相关”特性,在指数生长期,青蒿素含量缓慢下降,生长速度减缓后,青蒿素含量上升,发根生长停止后,继续延长培养时间,青蒿素含量也不再提高。在分批培养中,青蒿发根适宜的培养时间为21d。     

Artemisinin production in Artemisia annua hairy root cultures with improved growth by altering the nitrogen source in the medium

Murashige & Skoog medium was modified for enhancing artemisinin production in Artemisia annua hairy root cultures by altering the ratio of NO ₃ ^– /NH ₄ ⁺ and the total amount of initial nitrogen. Increasing ammonium to 60 mM decreased both growth and artemisinin accumulation in hairy root cultures. With NO ₃ ^– /NH ₄ ⁺ at 5:1 (w/w), the optimum concentration of total initial nitrogen for artemisinin production was 20 mM. After 24 days of cultivation with 16.7 mM nitrate and 3.3 mM ammonium, the maximum artemisinin production of hairy roots was about 14 mg l^–1, a 57% increase over that in the standard MS medium.     

Improved artemisinin accumulation in hairy root cultures of Artemisia annua by (22S, 23S)-homobrassinolide

When (22S, 23S)-homobrassinolide (SSHB) was added at 1 g l^–1 to hairy root cultures of Artemisia annua, the production of artemisinin reached to 14 mg l^–1, an increment of 57% over the control. SSHB treatment led concomitantly to an increased biomass production of 15 g l^–1. A stimulatory activity of SSHB on nucleic acids and soluble protein content in hairy roots was also observed at the growth stage.     

Quantification of artemisinin in Artemisia annua extracts by 1H-NMR

Artemisinin is a polycyclic sesquiterpene lactone that is highly effective against multidrug-resistant strains of Plasmodium falciparum, the etiological agent of the most severe form of malaria. Determination of artemisinin in the source plant, Artemisia annua, is a challenging problem since the compound is present in very low concentrations, is thermolabile and unstable, and lacks chromophoric or fluorophoric groups. The ain of this study was to develop a simple protocol for the quantification of artemisinin in a plant extract using an (1)H-NMR method. Samples were prepared by extraction of leaf material with acetone, treatment with activated charcoal to remove chlorophylls and removal of solvent. (1)H-NMR spectra were measured on samples dissolved in deuterochloroform with tert-butanol as internal standard. Quantification was carried out using the using the delta 5.864 signal of artemisinin and the delta 1.276 signal of tert-butanol. The method was optimised and fully validated against a reference standard of artemisinin. The results were compared with those obtained from the same samples quantified using an HPLC-refractive index (RI) method. The (1)H-NMR method gave a linear response for artemisinin within the range 9.85-97.99 mm (r(2) = 0.9968). Using the described method, yields of artemisinin in the range 0.77-1.06% were obtained from leaves of the A. annua hybrid CPQBA x POP, and these values were in agreement with those obtained using an HPLC-RI.