【作者】 杜研；

【导师】 张美萍；

【作者基本信息】 吉林农业大学 ， 作物生物技术， 2007， 硕士

【摘要】 本文以毛霉、木霉、青霉、散子囊菌和水杨酸作为诱导子，探讨了诱导子对黄芪细胞培养物生长和次生代谢的影响，研究结果如下：毛霉、木霉、青霉、散子囊菌作为诱导子处理黄芪愈伤组织，除木霉以外均能提高黄芪愈伤组织中黄芪总皂苷的含量。在黄芪细胞培养周期的对数生长末期加入浓度为80μg／mL的散子囊菌，黄芪总皂苷达到了11.51mg／g·DW，比对照组提高了31.99％。真菌诱导子的加入可以激发黄芪细胞的防御性应答反应，产生氧进发和一氧化氮进发，细胞的氧化还原态势发生了明显的变化，膜脂过氧化产物MDA的含量大幅度提高，POD、PPO、CAT的活力出现波动性的变化，并具有一定的时序性。同时，与次生代谢产物积累有关的PAL的活力也得到大幅度的提高。以水杨酸为诱导子添加到黄芪细胞培养体系后，观察其对黄芪细胞培养物次生代谢的影响。选择出了水杨酸的最佳添加浓度为10^-3mg／L，最佳的添加时间为继代培养的第28d，以这种方式处理后的黄芪愈伤组织中黄芪总皂苷的含量比对照组提高了29.47％。同时，跟踪测定了水杨酸作用过程中的氧化酶、PAL活力的变化，以及氧进发和NO进发、膜脂过氧化程度的变化情况。结果表明：水杨酸加入后引起了黄芪细胞的过氧化氢和一氧化氮含量瞬间的上升，也使膜脂过氧化产物MDA的含量大幅度提高，同时POD、PPO、PAL的活力始终高于处理组，而CAT的活力则受到抑制。经过80μg／mL的散子囊菌诱导子、10^-3mg／L的水杨酸、80μg／mL的散子囊菌诱导子+10^-3mg／L水杨酸3种处理，对黄芪愈伤组织的膜脂过氧化，代谢过程中的酶活力、活性氧和总皂苷含量的影响。结果表明：真菌诱导子和水杨酸单独使用均能引起黄芪细胞膜脂过氧化，而散子囊菌诱导子+10^-3mg／L水杨酸联合处理可以减轻真菌诱导子单独处理细胞所引起的膜脂过氧化程度，散子囊菌诱导子+10^-3mg／L水杨酸联合处理与二者单独使用相对比，较大地提高了POD和PAL的活力，同时提高细胞内过氧化氢的含量，而且过氧化氢含量升高的速度要快于二者单独使用。3种处理均可以提高黄芪总苷含量，特别以散子囊菌诱导子+10^-3mg／L水杨酸处理含量最高，达到12.957mg／g·DW，达最大值的时间也有所提前。本实验是首次以黄芪根际微生物和水杨酸作为诱导子来探讨诱导子对黄芪愈伤组织的生长和次生代谢的影响。更多还原

【Abstract】 In this article, used Mucor, Trichoderma, Penicillium, Eurotium and salicylic acid as elicitors. Studied on the effects of elicitor on the growth and second metabolism of Astragalus membranaceus cells culture. The results as follow:The saponin content of Astragalus membranaceus cells culture was enhanced by treating with six kinds of fungal elicitors separately ecxcept for Trichoderma, among them the Eurotium had the highest inducing efficiency. The saponin content was 31.99% higher then control. The best response of Astragalus membranaceus cells to elicitor treatment was in the late exponential Growth phase.Effects of fungal elicitoe on redox status and saponin production were studieded in cell cultures of Astragalus membranaceus in the late exponential stage. The results show that fungal elicitor oxygen and nitrogen monoxidum burst, changes of cell redox status, fluctuation of the activity of redox enzymes with a sequence.The content of MDA increased,while the activity of phenylalnine ammonialyase was promoted.The study investigated the influence of the salicylic acid elicitor on the gynthesis of saponin in Astragalus membranaceus callus culture. The result showed that salicylic acid enhanced greatly the accumulation of saponin. But the elicitation dosage and addition time brought different influence for saponin content. The saponin content was significantly increased in the presence of 10^-3mg/mL salicylic acid on day 28 and the maximum content was 29.47% higher than untreated callus repectively. While the content of nitrogen monoxidum, hydrogen peroxide and MDA were imprved. The activity of peroxidase, polyphenol oxidase, phenylalnine ammonialyase could be kept at a higher level in contrast to the control. But the activity of hydrogen peroxidase was inhibited.Effects of the treatents of 80μg/mL fungal elicitor, 10^-3mg/L salicylic acid（SA） and 80μg/mL fungal elicitor+10^-3mg/L SA on membranelipid preoxidation, activity of enzyme during the metabolic process, active oxygen and total saponin production in Astragalus membranaceus cell culture were studied. The result showed that either fungal elicitor or SA caused cell to membranelipid preoxidation. Combined treatment of fungal elicitor SA could degree of cell membranelipid preoxidation caused by only fungal elicitor treatment. Combined treatment induced higher POD, PAL activity and obtanied more biomass than that of only fungal elicitor treatment did. Meanwhile the content of H₂O₂ could be increased. Among three treatments, the combined treatment get the most saponin production, to achieve 12.957mg/g·DW, and the time of reaching maximum advanced.更多还原