分子生物学实验原理与技术论文.doc

CHITOSAN ON ANDRAEANUM TISSUEINHIBITION OF BACTERIACONTAMINATIONAbstract: The fungi was isolated and purified from polluted anthurium subculture medium, identified to be DeuteromycotinaHyphpmycetes Hyphomycetales Dematiaceae and trichoderma. The inhibition effect of the three kinds of chitosan on the fungi wasconducted.The results showed that the chitosan had inhibition effect on the fungi. The higher concentrations of chitosan had a betteinhibition effect than the lower ones.The inhibition effect of 50000 and 250000 molecular weight chitosan is better than that of the3000 molecular weight. It is preliminarily proved that chitosan had good inhibition effect on the fungi from polluted tissue culture , andwill have good application value in the fields of pollution prevention in flowers tissue culture. Key words: tissue culture； contamination； Chitosan； inhibition effect Plant tissue culture, not only in plant breeding, rapid propagation of seedlings and the production of useful secondary metabolites, such as the use of a wide range of plant genetic engineering is and Plant Molecular Biology, one of the important foundation for the study 1 . Pollution, browning, glass and tissue culture are the main problems, including pollution of the most common 2.Therefore take effective prevention and control measures to reduce the chance of contamination occurred, tissue culture is an important guarantee for success. Chitosan (chitosan) is the chitin in strong alkaline conditions from B.After the formation of acyl an important derivative is determined by a number of N-acetyl glucosamine through -(1-4) glycosidic bond linking the nature of 18 it more lively. Has natural antibacterial properties of chitosan, and a broad spectrum antimicrobial , in recent years, chitosan in various fields such as agriculture, medicine 3, manufacture 4, food 5 , etc. The use of great importance. Tissue culture of the chitosan in the application of inhibition has not been reported. This experiment to be from the contaminated medium subculture andraeanum extraction, purification and identification of bacteria contamination, with different molecular weight chitosan and different concentrations of their inhibition, and explore the best inhibitory effect of chitosan molecular weight and the best inhibitory concentration, in order to explore the chitosan as a new type of antimicrobial agent to provide specific information. 1 Materials and Methods 1.1 Test materials1.1.1 Isolates Streptomyces bacteria pollution (code-named WCHPA), separated from the laboratory contamination andraeanum subculture medium. 1.1.2 Test for Drugs Chitosan, a Zhejiang Xing Biotechnology Australia Limited, a molecular weight of 3kd (degree of deacetylation 80%), 50kd (degree of deacetylation 90%), 25kd (degree of deacetylation 90%) 6. 1.1.3 Medium PDA mediumPotatoes 200g, glucose 20g, agar 20g, distilled water 1000ml, pH value of the natural. 1.2 Test Method 1.2.1 Isolation and purification Clean out the work in Taichung andraeanum subculture medium pollution fungi, from PDA medium. Repeat several times until the colony characteristics and morphology changes are no longer, we can identify bacteria have been purified. 1.2.2 Identification of bacteria contamination Continuous observation of colony morphology after purification. Film production equipment to further observe the shape strain, mycelium, spores, spore cell spore cell morphology and conidiation of the ways and forms and so on, taking pictures with a digital microscope. 1.2.3 Bacteriostatic Test That the different molecular weight chitosan with 1M solution of acetic acid dissolved mixed with PDA medium to produce a final concentration of chitosan (0.5,1.0,1.5,2.0,2.5,3.0)mg/ml of culture medium, 1M solution of sodium hydroxide by adjusting pH value of 6.0, in addition to the control without chitosan, the other with the addition of chitosan in the same medium 7 . To eliminate all high-pressure cooker sterilization 30min under 121 and pour plate. Mycelium white to green after 24h and then into a brown, hyphae developed, velvet-like, 3d training around the middle of the brown and white flocculent mycelium of fimbriae produced. Hyphae are separated from a long stalk conidia to produce lateral branches whorled branches meristematic conidiation short bottles. Single-cell conidia oval, smooth. 2 Results and Analysis 2.1 Andraeanum symptoms subculture medium pollution Andraeanum subculture in growth medium brown Fimbriae, medium surface was black, brown and white mycelium on the flocculation between the hyphae. 2.2 fungal pure culture and identification of the results PDA medium in the upper colony diameter, edge neatly, spore germination of the PDA medium by adding chitosan, 4d clearly observed after the colony is surrounded by a black halo, black halo and colony growth as to the relocation of . The better the general inhibitory effect of the more obvious black halo 8. Colony has no control this phenomenon. Chitosan molecular weight and concentration of the bacteriostatic effect. 2.3 The same three concentrations of the bacteriostatic effect of chitosan molecular weight compared Concentration of 0.5mg/ml 3 species of molecular weight chitosan WCHPA colony inhibitory effect of six concentrations of three kinds of molecular weight of chitosan on the inhibition rate of colony WCHPA results 0.5mg/ml concentration of three kinds of molecular weight chitosan there is no clear law, the maximum inhibitory rate of 50,000 in molecular weight of chitosan for the culture medium for 20.45%. Inhibitory rate and the time and no close ties. The results show that the concentration of 0.5mg/ml of the three kinds of molecular weight of the antibacterial effect of chitosan has not been satisfactory9 . 5d prior to the first three kinds of chitosan molecular weight the greater the inhibitory rate of the higher molecular weight, and are extended over time to reduce the rate of inhibition. In paragraph 5d, 3000 molecular weight inhibitory rate of more than two other inhibitory rate of molecular weight, molecular weight and inhibition rate of 3000 also reached the highest value. At its bacteriostatic effect after 7d and 5d is similar to before, the greater the inhibitory rate of the higher molecular weight. 3 Discussion and conclusions 3.1 Chitosan as a potential new antibacterial agents Pollution plant tissue can be broadly divided into two types of operations may produce air pollution mainly mold contamination； explants is itself brought about by bacteria primarily by fungal and bacterial contamination 10. High inhibitory rate was as high as 79.55%. Antibiotics in tissue culture is now the most common molecular weight of chitosan and 3.2 have an important impact on antimicrobial properties of the experimental results from the 50,000 and 250,000 the bacteriostatic effect of chitosan molecular weight than that of 3000； the highest inhibitory rate in the 250,000 molecular weight concentration of 3.0mg/ml and 2.0mg/ml of the two. But on the whole, taking into account the factors of price and its operation to the conclusion that the experimental molecular weight of 50,000 as the best concentration of 1.5mg/ml. 3.2 Concentration on the antimicrobial properties of chitosan have a significant impact Qiu-ping 11, The results show that the antimicrobial effect of chitosan with its concentration increases, the experimental results support the results of the study. A wide range of applications of chitin, chitosan in the medical, food, environmental protection, in areas such as chemical products have wide-ranging and important applications. Chitosan flowers will be used for tissue culture would be very good prospects. References1MK.Razdan. Zun-Xiao-an, ZHU Yang translation. Introduction to Plant Tissue Culture M. Beijing: Chemical Industry Press ,2006:1-2.2Hu, ZHANG Li-jun,baixuemei, et al. Plant Tissue Culture Pollution Analysis and the disinfection of explants J. Anhui Agricultural Science, 2007,35 (3) :680-681. 3Yi-Ting Wang,wanglianping , Mou Hero, et al. Industrial production of tissue culture studies of pollution and its control J. Anhui Agricultural Science, 2005, 33 (12): 2357 - 2358. 4Chiang big. Chitin M. Beijing: China Environmental Science Press, 1996. 5Fu Tian Xia, LIU Xiao-yu, Liu Zhiheng. Chitosans antibacterial properties of the progress of applied research J. Liaoning Chemical, 2005,1234 (12) :541-544. 6Yu Hanshou, Wu Zhang, Yang Bing. Chitosan inhibit research progress of plant diseasesJ.Natural products research and development,1999,12 (3) :94-97. 7Li Fang,shishaowei. Chitosan Preparation and application in the food industry Mechanism J. Food and Drug, 2006 (03A) :19-22. 8Wu, Xia water. Non-biological factors on the antibacterial activity of chitosan J. Food Science, 2004,25 (7) :53-55. 9wuxiaoyong, Qing-Xiao Zeng, MOshaofang. Several of the antibacterial properties of chitosan J. Food and Fermentation Industry, 2005,206 (2) :18-21. 22 高等教育自学考试毕业(论文)说明书 10Hu, ZHANG Li-jun,baixuemei, et al. Plant Tissue Culture Pollution Analysis and the disinfection of explants J. Anhui Agricultural Science, 2007,35 (3) :680-681. 11Qiu-ping, Chitosan on mango Colletotrichum gloeosporioides, Diplodia antagonism bacteria J. Food and machinery, 2005,21, (1) :25-27. 23壳聚糖对红掌组培污染菌的抑制作用摘要:在污染的红掌继代培养基中提取污染菌并进行了分离及纯化,初步鉴定为半 知菌亚门(Deuteromycotina),丝孢纲(Hyphpmycetes),丝孢(Hyphomycetales ) , 淡色孢科(Dematiaceae),木霉属(trichoderma)。研究了壳聚糖对该菌的抑制作用,探索了 3 种分子量及 其 6 种浓度的壳聚糖对该菌的抑制效果。结果表明:壳聚糖对该污染菌有 抑制作用,5 万和 25 万分子量的抑菌效果高于 3000 分子量,浓度越高, 抑菌效果越好。初步证实了壳聚糖对组培污染菌类有很好的抑制作用,在花卉组织培养污染防治方面具有应用价值。 关键词:组织培养；污染；壳聚糖；抑制作用 植物组织培养不仅在植物育种, 苗木快速繁殖和有用次生代谢产物生 产等方面应用广泛,也是植物基因工程和植物分子生物学研究的重要基础 之一 1 。污染,褐化,玻璃化等是组织培养存在的主要问题,其中污染最普遍 2 。因此采取有效的防控措施,降低污染发生的机率,是组织培养成 功的重要保障。壳聚糖(chitosan)是甲壳素在强碱性条件下进行脱乙酰 基作用后形成的一种重要的衍生物,是由多个 N-乙酰氨基葡萄糖通过 -(1-4)糖苷键连接起来,但它的性质较为活泼。壳聚糖具有天然抑菌性能,且抑菌谱广,近年来壳聚糖在各个领域如农业,医学 3,制造4 食品5等方面中的运用受到很大的重视。有关壳聚糖在组织培养中的抑菌应用尚 未见报道。本实验拟从污染的红掌继代培养基中提取,纯化污染菌并进行 鉴定, 用不同分子量及其不同浓度的壳聚糖对其进行抑制实验,摸索出抑菌效果最好的壳聚糖分子量及最佳抑菌浓度,为探索壳聚糖成为新型的抑菌剂提供具体信息。 1 材料与方法 11 实验材料111 供试菌株 霉菌污染菌(代号 WCHPA) ,分离于实验室污染的红掌继代培养基中。 112 供试药品 壳聚糖,有浙江澳兴生物科技有限公司提供,分子量为 3kd(脱乙酰 度80%) ,50kd(脱乙酰度90%) ,25kd(脱乙酰度90%)6 。 113 培养基 PDA 培养基 土豆 200g,葡萄糖 20g,琼脂 20g,蒸馏水 1000ml,pH 值自然。 12试验方法121污染菌的分离及纯化在超净工作台中挑出红掌继代培养基中的污染真菌,接到 PDA 培养基中。重复若干次,直至菌落特征及其显微形态不再有变化,可确定菌种已纯化。 122 污染菌的鉴定 连续观察纯化后的菌落形态。制作装片,进一步形观察菌株,菌丝,孢子,产孢细胞的形态及产孢细胞的产孢方式和形态等,用数码显微镜进行拍照。 123 抑菌试验 称取不同分子量壳聚糖用 1M 的乙酸溶液溶解后与 PDA 培养基混合, 制成壳聚糖终浓度分别为(0.5,1.0,1.5,2.0,2.5,3.0)mg/ml 的培养基,用1M的氢氧化钠溶液调节 pH 值为 6。0,对照除不加壳聚糖外,其他与加有壳聚糖的培养基相同7。高压灭均锅 121下灭菌 30min后倒平皿。产生白色菌丝24h后变为绿色继而变为褐色,菌丝发达,绒状,培养3d左右,在褐色菌丝中间有白色絮状的菌毛产生。菌丝有隔,分生孢子 梗长,产生侧向分枝,分生枝上轮生短的产孢瓶体。分生孢子单胞卵圆形,光滑。 2 结果与分析 21 红掌继代培养基污染症状 在红掌继代培养基中长出褐色的菌毛,培养基表面呈黑色,褐色的菌丝上间有白色絮状的菌丝。 22 真菌纯培养的形态特征及鉴定结果菌落在 PDA 培养基上等径生长,边缘整齐,孢子萌发加入壳聚糖的PDA培养基,4d 后能明显观察到菌落周围有黑色晕圈,且黑色晕圈随着菌 25 高等教育自学考试毕业(论文)说明书落的生长而向外移。一般抑菌效果越好黑色晕圈越明显8 。对照菌落始终 没有出现这种现象。壳聚糖的分子量和浓度影响抑菌效果。 23 相同浓度的三种分子量壳聚糖抑菌效果的比较 浓度为0.5mg/ml 3 种分子量壳聚糖对 WCHPA 菌落的抑制效果6个浓度3种分子量壳聚糖对 WCHPA 菌落的抑菌率结果 0.5mg/ml 浓度的 3 种分子量壳聚糖之间没有明显的规律,最高抑菌率出现在