【作者】 刘伟；

【导师】 郑应华；

【作者基本信息】 华中农业大学 ， 微生物学， 2005， 硕士

【摘要】 细胞分裂素（Cytokinin,CTK）是目前人们已经知道的六大天然植物激素（即生长素、赤霉素、细胞分裂素、脱落酸、乙烯和芸苔素内酯）之一。其生理作用是:（1）对细胞分裂与分化有明显刺激效果;（2）促进细胞生长;（3）能解除顶端优势,促进侧芽生长;（4）对某些种子和芽有促进萌发打破休眠的作用;（5）减少叶绿素的水解,延迟叶片衰老。细胞分裂素可广泛应用于大田作物,蔬菜、水果等经济作物,能显著提高产量和改善品质。细胞分裂素在植物体内普遍存在、含量极微,但它是植物生长所必需的物质。 从北京地区的土壤中分离到一株芽孢杆菌,该菌株经中科院微生物所鉴定,为地衣芽孢杆菌（Bacillus licheniformis）。该菌株通过发酵可产生细胞分裂素物质。用苋红素合成法,即利用尾穗苋黄化苗在供给细胞分裂素和酪氨酸的条件下,子叶能合成苋红素,然后经反复冰冻浸出全部苋红素用于比色测定,来进行活性跟踪定量,选取玉米粉、棉饼粉、酵母浸提物和CaCO₃作为试验因素,采用L₉（3⁴）正交表设计四因素、三水平的正交试验,确定了最适发酵培养基,其组成为豆饼粉0.8%,玉米粉1%,酵母浸粉0.2%。 本文利用溶剂萃取、薄层层析对发酵液中的细胞分裂素进行了初步提取和分离,通过薄层层析的相对迁移率（R_f值）、红外吸收光谱（IR）、高效液相色谱（HPLC）及核磁共振光谱（NMR）得到了该组分的相关信息。其流程为:用薄层色谱法（TLC）作先导,采用荧光硅胶GF₂₅₄来确定柱层析分离条件。粉剂经过80%甲醇浸提后减压浓缩,再用正丁醇进行萃取,有效成分被分配到正丁醇相中。浓缩正丁醇相,得到的粗提物上硅胶层析柱,用氯仿/甲醇按体积比92:8～90:10进行梯度洗脱,相同组分合并得到有效活性流份B,把流份B再上一次硅胶柱,按氯仿/甲醇体积比9:1的比例进行洗脱,用苋红素合成法跟踪活性,得到比活性高的细胞分裂素。 为了考察含有此类细胞分裂素的发酵液对黄瓜产量的影响,对发酵液在黄瓜上进行田间应用试验。黄瓜生长期用发酵液100、200、300倍液进行叶面喷雾三次,结果表明对黄瓜安全无药害,不仅有很好的促生长作用,而且还具有很好的防病作用。其中处理区对对照区分别增产34.8%、25.3%、19.8%,与对照区差异显著。对黄瓜霜霉病的抑制率最高可达到36.1%,对黄瓜炭疽病抑制率最高可达61.5%,均达显著水平。更多还原

【Abstract】 Cytokinin is known as one of the six natural plant hormones, including IAA,GAs CTK, ABA, Eth and BR. Its physiological role is as following: 1) have good stimulative effect on cell fission and cytodifferentiation; 2) accelerate cell growth; 3) promote ateral buds; 4)break dormancy of some seeds and buds; 5) reduce the hydrolysis of chloroplast and postpone blade aging. It can be applied extensively in crops such as vegetable,fruits and other economic crops. This substannce ,for increasing the output and improving quality notably, exist generally in the body of plant. Though little in content, is it indispensable.A Bacillus licheniformis, isolated from soil in Beijing, was identified by Chinese Academy of Sciences and was found to produce cytokinin by fermention. namely, if Amaranthus caudatus was provided with cytokinin and tyrosine, the cotyledone of etiolation seedling could synthesize betacyanin, All betacyanins, soaked by reiteration forst, could be measured with colorimetry, so its activity was tracked and rationed at the same time. Studies on four elements such as soy bean meal, maize powder, yeast extract and CaCO3 were conducted in three levels using the orthogonal form of L9(34) to investigate the effects on biosynthesis. The result demonstrated the optimized fermentation medium was: 0.8% of soybean meal, 1% of maize powder, 0.2% of yeast extract.Solvent-extraction and chromatography were used to preliminarily extract and separate cytokinin in fermentation culture broth. The structure was analysized by comparing the Rf value of the thin layer chromatogram (TLC) , the UV-spectrum, the retention time of RP-HPLC and the spectrum of NMR. TLC was carried out on plates precoated with silical gel F254, for better separating methods of column chromatography. Powder of spraying drying was extracted with 80% of methanol, followed by extraction with n-butanol at room temperature. The combined extracts were concentrated in vacuo to give a crude extract, which was partitioned between H2O and n-butanol to provide n-butanol extract and water soluble fraction. The n-butanol extract was concentrated and subjected to column chromatography eluted with solvent mixture of methanol/chloroform (92:8-9:1, by vol) to give three fractions. Fraction B, which had effective activity ,was concentrated and subjected to column chromatography (methanol /chloroform 9:1, by vol) again, get cytokinin, which showed a high specific activity.the cucumber, The bioactive component obtained could promote the growth of the cucumber and enhance the disease resistance of cucumber. Compared with their control, the output was increased by 34.8%, 25.3%, 19.8%, respectively, reaching notable effect on increasing cucumber yield. Moreover, efficiency of inhibition to cucumber downy mildew and anthracnos could be up to 36% and 61.5%, respectively.更多还原