【作者】 张春荣；

【导师】 李玲；

【作者基本信息】 华南师范大学 ， 植物学， 2002， 硕士

【摘要】 野葛，Pueraria lobata（Willd.）Ohwi，富含葛根素、大豆苷、大豆苷元等异黄酮类化合物，在我国及其他东亚国家广泛用作医药业、食品业和轻工业原料。本研究以野葛幼叶愈伤组织建立悬浮细胞培养体系，研究了几种有机附加物、诱导子、有机溶剂等对野葛细胞生长和异黄酮类化合物生产的影响，为野葛细胞工程生产葛根素等异黄酮类化合物奠定了基础。 野葛未成熟种子萌发获得的无菌苗幼叶在含1mg／L NAA，2mg／L BA的固体培养基上诱导愈伤组织，愈伤组织在含0.5 mg／L 4-PU的MS固体培养基上继代形成结构疏松、生长迅速的淡黄色愈伤组织，接入含1mg／L 2,4-D，1mg／L NAA，0.5 mg／L KT，0.2％水解酪蛋白（CH），20.g/L,蔗糖(简写为B₅+2,4-D₁+NAA₁+KT_0.5+0.2％CH)液体培养基中振荡培养建立了颗粒均一、分散良好、生长迅速的野葛幼叶细胞悬浮培养体系。悬浮培养条件为（25±2）℃、全天光照，光强5.34μmol·m^-2·s^-1，摇床转速110～130r／min。 野葛悬浮细胞的生物量在培养的第8d达最大值，为12.58mgDW／L，细胞产率为72.18％。悬浮细胞中葛根素的合成与细胞生长呈紧密偶联型，第8d时，细胞和培养液中的葛根素含量达最大值，分别为7.98mg／g DW和3.46mg／L，此时细胞和培养液中葛根素总产量为103.85mg／L。野葛悬浮细胞及培养液中总异黄酮类化合物积累的动力学曲线不同于葛根素积累的动力学曲线。野葛细胞接入新鲜培养液后，总异黄酮类化合物随细胞的生长而不断合成、积累，细胞进入稳定期后，总异黄酮类化合物的积累速度反而加快，第10d达最大值，为129.05mg／g DW，此时细胞和培养液中异黄酮类化合物的总产量为1.59g/L。野葛幼叶悬浮细胞中总异黄酮类化合物的最高含量超过野生葛根含量（68.7mg／gDW），而葛根素最高含量则低于野生葛根含量（12.6 ms／g DW）。野葛幼叶悬浮细胞及培养液中葛根素的总含量占异黄酮类化合物总含量的9.22％。 过滤灭菌的椰乳和高压灭菌的椰乳的作用效应不同。10％（v／v）椰乳对野葛幼叶悬浮细胞的生长及次生代谢物的积累具有一定的抑制作用。0.2％的水解酪蛋白（CH）对野葛幼叶悬浮细胞的生长及葛根素和总异黄酮类化合物的积累和释放均有促进作用，可使葛根素和总异黄酮类化合物的产量分别提高34.05％和40.76％，因此野葛细胞悬浮培养宜用的培养基为B₅+2,4-D₁+NAA₁+KT_0.5+0.2％CH。 酵母提取物（YE）和脱乙酚几丁质（DC）对野葛幼叶悬浮细胞的生长均 无促进作用，较高浓度（0．5 g／L）的 DC处理较长时间6 d和 sd)抑制细胞的 生长。YE加到悬浮细胞培养液中后，引起细胞轻微褐化。YE不利于野葛悬浮 细胞中葛根素产量的提高。以 0刀 lgh DC处理 8 d对葛根素的增产效果最大（增 产28．40％人YE和DC对野葛幼叶悬浮细胞中异黄酮类化合物的积累及总产量 的提高均无促进作用。 SA、MJ和乙烯利对野葛幼叶悬浮细胞的生物量没有明显促进作用。SA和 MJ能引起细胞褐化，而乙烯利则能较好地保持细胞的活力。各种浓度的SA和 低浓度＊．ling／L和 lwt）的 MJ对葛根素总产量的影响不大，ling／L MJ 处理 8 d和 5 d略微促进葛根素的生产，而 0．ling／L乙烯利处理 3 d能显著提高 葛根素的总产量（增产27．37％）。SA、MJ和乙烯利对野葛悬浮细胞异黄酮类 化合物的生产没有促进作用。 浓度在1％－5％（v／V）范围内的DMSO即使处理5 d也不影响细胞的活力，浓 度在佃－5％（v／v）范围内的Tween－20和Titon X－100处理3 d内对细胞活力的影 响较小。三种有机溶剂均能促进细胞中葛根素及其它异黄酮类化合物的释放，但 均不利于葛根素的生产。5％（v／v）的Titon X－10o处理3d能够显著促进总异黄酮 类化合物产量的提高，增产率达40．56％。更多还原

【Abstract】 Pueraria lobata (Willd.) Ohwi, rich in puerarin, daidzein, daidzine and other isoflavones, has been wildly used in China and other East Asia countries as resources in curatorial, food and light industries. In this study, the P. lobata cell suspension culture system was constructed from callus of seedling leaves. The effects of some organic appendices, elicitors and organic solutions on cell growth and isoflavones production were investigated. The purpose of the study is to provide technical support for P. lobata cell engineering to produce puerarin and other isoflavones.Callus of P. lobata have been established on MS solid medium supplemented with 3% sucrose, 1.0 mg/L NAA, 2.0 mg/L BA using leaf explants of seedlings germinated from immature seeds. The callus grew fast and became yellowish and friable after subcultured on MS solid medium supplemented with 3% sucrose and 0.5 mg/L 4-PU. After these callus were inoculated and then subcultured for more than 8 times in B5 liquid medium containing 2% sucrose, 1.0 mg/L 2,4-D, 1.0 mg/L NAA, 0.5 mg/L KT and 0.2% CH, the cell cultures became homogeneous and grew fast. Hence the cell suspension culture system was established at (25?)癈, in 5.34 JJ mol ?m2 ?s"1 full day light, on gyratory shaker (110-130 r/min).The maximal biomass accumulation of P. lobata cell suspension cultures was obtained on day 8 for 12.58 mg DW/L. Cell productivity was 72.18%. The puerarin production was closely coupled with cell cultures’ growth. It reached highest on day 8 both in cell cultures and medium with 7.98 mg/g DW and 3.46 mg/L, respectively. The total yield of puerarin reached peak (103.85 mg/L) on day 8. The time-courses of total isoflavones accumulation in P. lobata cell cultures and medium were different from those of puerarin. It coupled with cell cultures growth on the first 8 days and then became faster in cell stable growth phase. On day 10, it reached highest with 129.05 mg/g DW in cell cultures, and 1.59 g/L in cell cultures and medium, totaly. This content of total isoflavones in cell cultures surpassed the content in wild root of P. lobata that was 68.7 mg/g DW, while the content ofpuerarin was lower than that in wild root of P. lobata that was 12.6 mg/g DW. In P. lobata cell cultures and medium, the total content of puerarin occupied 9.22% of total isoflavones.The effect of coconut juice sterilized by autoclaving was different from that sterilized by filtration. 10% coconut juice restrained the growth of cell cultures and the accumulation of total isoflavones, while 0.2% casin acid hydrolysates (CH) promoted the growth of cell cultures and the accumulation and release of puerarin and total isoflavones. The total yield of puerarin and isoflavones were 34.05% and 40.76% highter than control, respectively. It was concluded that the optimizing medium for cell cultures in leaves of P. lobata seedlings was 85 liquid medium supplemented with 2% sucrose, 1.0 mg/L 2,4-D, 1.0 mg/L NAA, 0.5 mg/L KT and 0.2% CH.Both yeast extract (YE) and deacetylated chitin (DC) had no promotion effects on the growth of P. lobata cell cultures. 0.5 g/L DC restrained the cell growth when treated for 5 d and 8 d. YE could cause the cell brown slightly, and didn’t benefit the production of puerarin. 0.01 g/L DC improved the total yield of puerarin over 28.40% after treatment of 8 days. YE and DC had no effects on production of total isoflavones.SA, MJ and ethrel had no promotion effects on the biomass accumulation of P. lobata cell cultures. SA and MJ both caused the cell brown, while ethylene had no negative effect on cell vitality. 0.1-5 mg/L SA and 0.1 mg/L MJ had not much effects on the production of puerarin. 1 mg/L MJ improved the yield of puerarin slightly after 8 d and 5 d of elicitation, while 0.1 mg/L ethrel had a better effect which improved 27.37% higher than control after treatment of 3 days. SAs MJ and ethrel had no effects on improving the yield of total isoflavones in cell cultures and medium.DMSO at concentration 1%~5% had no effect on cell vitality even treated for 5 d更多还原