河南科技大学硕士学位论文河南道地药用植物辛夷内生真菌Y111的初步研究姓名：刘笑洁申请学位级别：硕士专业：生物化学与分子生物学指导教师：何佳20091201摘 要 I论文题目：河南道地药用植物辛夷内生真菌论文题目：河南道地药用植物辛夷内生真菌 Y111 的初步研究的初步研究 专专 业：生物化学与分子生物学业：生物化学与分子生物学 研研 究究 生：刘笑洁生：刘笑洁 指导教师：何指导教师：何 佳佳 教授教授 摘 要摘 要 内生真菌是指那些在其生活史中某一段时期生活在活的植物组织中，并不引起植物组织产生明显病害症状的真菌。近年来，研究表明内生真菌可以产生抗菌，抗肿瘤等的活性物质。因此，作为一种新型的具有潜在开发价值的微生物资源，内生真菌已得到人们的广泛认同。现阶段对药用植物抗菌内生真菌的研究才刚刚起步，对其了解也不够深入，无法对它们充分利用。因此，研究药用植物抗菌内生真菌不仅能带来巨大的社会效益，还具有现实的理论意义。 本文选用药用植物辛夷内生真菌Y111为对象进行研究，研究结果如下： 1通过对不同固体培养基菌丝长势的分析，筛选出适合Y111菌株生长的固体培养基为PDA培养基；通过对不同液体培养基中Y111的生物量及抑菌效果的分析，筛选出适合Y111生长的液体培养基为PDA培养基。以PDA培养基为基础，用单因素及正交试验对Y111菌株的发酵培养基进行优化，得到适宜菌丝生长 的 最 佳 培 养 基 为 ： 在 20% 土 豆 汁 中 加 入 4% 葡 萄 糖 、 0.3% 蛋 白 胨 、0.1%K2HPO4；抑菌效果最好的发酵培养基为：在20%土豆汁中加入4%葡萄糖、0.3%蛋白胨、0.2%K2HPO4。用单因素和正交试验对Y111菌株发酵条件进行优化，抑菌效果为指标，得到最佳的培养条件为：接种量为12%、装液量为100mL/250mL、温度28、培养时间12天。 2通过测定Y111对部分细菌和真菌的抗菌效果，得到Y111的部分抗菌谱。进一步研究发现Y111发酵液对pH、温度和紫外线都有很好的稳定性。Y111抗菌效果，可能与发酵后期产生的次生代谢产物有关；对Y111抗真菌的显微及直观观察，发现抗菌作用可能与重寄生作用和生长竞争作用有关。这些还有待进一步研究。 3以形态学和分子生物学手段对辛夷内生真菌Y111进行鉴定。形态学的鉴定得到Y111菌株是多节孢属。通过分子鉴定结果得到，Y111的ITS序列和炭团菌属真菌相似性较高，达94，与多节孢属的相似性91%。根据rDNA ITS区序列分析的一般准则，Y111菌株为炭团菌属真菌。真菌的鉴定以形态学鉴定为主，因此Y111菌株为多节孢属。 摘 要 II4通过对辛夷内生真菌Y111挥发油化学成分的气相色谱-质谱联用分析，发现了辛夷内生真菌Y111与其宿主植物辛夷含有相同的化学成分，他们分别是石竹烯、E,E-金合欢醛、桉树脑等，并且这些成分大都是辛夷植物中的主要成分。 关关 键键 词：词：内生真菌，辛夷，发酵条件，抗菌，菌种鉴定，挥发油成分 论文类型：论文类型：应用基础研究 摘 要 IIISubject: Primary studies on endophytic fungi Y111 in Lily Magnolia from HeNan genuine medicinal plant Specialty: Biochemistry and Molecular Biology Name: Liu Xiaojie Supervisor: Prof. He Jia ABSTRACT Endophytic fungi are the fungi associated with living tissue of plants without causing any noticeable symptoms of disease. Recent years, studies on entophytic fungi showed that some antibacterial and antitumor products made by them. It is consent that endophytic fungui are new microbes resource which has latent value for exploitation. At the present stage, our study on medicinal plant endophytic fungi is just beginning. We can not take full advantages of entophytic fungi because many of their characters have not been understood. So study on entophytic fungi in medicinal plant not only can bring great social benefits, but also have the realistic theoretical significance. In this scientific dissertation, studies on the object that endophytic fungui Y111 in medicinal plant Lily Magnolia. The results obtained as following: 1. According to the growth rates of mycelia on different media, an optimum solid medium suitable strain Y111 was PDA. Through analysing the amount of biomass and the effect of inhibit bacteria on different liquid media, an optimum liquid medium suitable Y111 was selected out then, it was PDA. On the basis medium of PDA, the fermentation media of strain Y111 were further optimized by the single factor and orthogonal experiment. The results showed that 20% of the potato juice by adding 4% glucose, 0.3% peptone, 0.1% K2HPO4 were the optimum conditions for its mycelial growth, 20% of the potato juice by adding 4% glucose, 0.3% peptone, 0.2% K2HPO4 were suitable for inhibit bacteria. With antibacterial activity as an indicator, using the single factor and orthogonal experiment optimized the fermentation conditions of strain Y111.The best culture conditions were as below: inoculum size 5%, 250mL flask containing 100mL medium, temperature 28, fermentation time 12d. 2. Through testing Y111 antibacterial effect on some of the bacteria and fungi, the part of the antimicrobial spectrum of Y111 obtained. Through further analysis showed that Y111 fermentation liquid had good stability on the pH, temperature and UV. The antibacterial effect may be related to the secondary metabolite produced by 摘 要 IVY111. Through microscopical observation of the Y111 inhibit fungal found that the effect of antifungal may be related to the hyperparasitism and competitive growth. It needs further study. 3. Y111 was identify by the morphology and the molecular biology method. The morphologial identification indicated that Y111 is Nodulisporium.species. The molecular identification indicated that Y111 ITS sequence with the similarity of 94% to Hypoxylon sp. and 91% to Nodulisporium. According to the general guidelines of the rDNA ITS sequence analysis, we supposed that Y111 is Hypoxylon sp. species. In identification of fungi, morphologial identification is dominant, so Y111 strain is Nodulisporium.species. 4. Using GC-MS to analysis chemical constituents in the essential oil from Lily Magnolia endophytic fungi Y111. Results showed that the Lily Magnolia endophytic fungi Y111 produced the same chemical constituents with its host plant. They are Caryophyllene, E,E-Farnesal, Eucalyptol. These constituents are mostly the main components of Magnolia. KEY WORDS: entophytic fungi, Lily Magnolia, fermentation condition