【作者】 穆凌霄；

【导师】 吴元华；

【作者基本信息】 沈阳农业大学 ， 植物病理学， 2011， 博士

【摘要】 嘧肽霉素是我校自主研发的生物农药,具有高效、低毒、低残留等优点,可以防治多种农作物病毒病害和真菌病害,已获得国家发明专利。本文以生物活性为检测手段,利用多种色谱方法获得了嘧肽霉素中两种抗真菌物质的纯品组分1和组分2,一种抗细菌物质的纯品组分3,和一种抗病毒物质的粗品组分4；通过波谱法对组分1和组分2进行了结构解析；开发了组分1和组分3的高效液相色谱分析方法；研究了组分1的抗真菌机理和组分3的抗病毒机理,结果如下：1.组分1以烟草赤星病菌(Alternaria alternata)为指示菌分离而得。嘧肽霉素发酵液经离心得上清,草酸酸化预处理充分去除蛋白质等杂质,依次经过大孔吸附树脂Diaion HP20吸附,50%甲醇洗脱；TOSOH SP650M离子交换柱吸附,0-1mol/L NaCl梯度洗脱；Daisogel ODS-B反相柱纯化,20%甲醇作为流动相,冻干得纯品。经紫外光谱、红外光谱、质谱和核磁共振试验最终得到了组分1的分子式为C12H13N5O4,并推测了其结构式。这是首次获得嘧肽霉素抗真菌组分的纯品和结构式。2.组分2同样以烟草赤星病菌(Alternaria alternata)为分离指示菌,经过大孔吸附树脂吸附后的活性馏分用硅胶柱层析进行进一步分离,氯仿：正丁醇：甲醇：醋酸=42：2：4：2作为流动相,并用Sephadex LH-20以20%甲醇作为流动相做最后的纯化,可得到纯品。质谱检测表明组分2的分子量是244。质谱、核磁、紫外等多种试验结果表明组分2可能是组分1合成过程的中间体,也可能是组分1由于发酵时间过度或其它原因发生降解的产物。3.组分3以蜡样芽孢杆菌(Bacillus cereus. Frankland)作为指示菌分离得到。预处理后的发酵液先后经过大孔吸附树脂Diaion HP20和离子交换树脂SK1B去除杂质后,用732阳离子交换树脂配合0.1-0.5 mol/L氨水梯度洗脱进行浓缩分离,最后依次通过HW40和LH-20分子筛层析柱纯化,得到分子量为574的纯品。4.组分4具有抗TMV活性,通过枯斑抑制试验分离。嘧肽霉素发酵液通过大孔吸附树脂吸附,用50%丙酮洗脱并浓缩蒸干之后用ODS-BP反相柱分离,0-100%甲醇梯度洗脱,得到组分4粗品。5.开发了嘧肽霉素组分1的高效液相色谱定性和定量分析方法,最佳色谱条件为：色谱柱选用DIKMA DiamonsilⅡC18柱(200mm×4.6mm,φ5μm);检测波长为278nm；流动相为20%甲醇水溶液,柱温为30℃。此色谱条件下组分1保留时间约10.2min,此方法的准确度和精密度较高,通过此方法测得的嘧肽霉素发酵液中抗真菌组分含量为4.11μg/mL。组分2的性质与组分1相近,可以采用同样的分离条件进行分离。6.明确了组分3的色谱分离条件：色谱柱选用DIKMA DiamonsilⅡC18柱(200mm×4.6mm,Φ5μm检测波长为230nm；流动相采用30mmol/L磷酸盐缓冲液,含有10mmol/L辛烷磺酸钠,pH=3。柱温为30℃。此方法可以进一步验证用于定性和定量分析。7.抗菌谱测试表明组分1对番茄灰霉病菌(Botrytis cinerea)、西瓜炭疽病菌(Colletotrichum orbiculare)、番茄早疫病菌(Alternaria solani)黄瓜菌核病菌(Sclerotinia sclerotiorum)、烟草赤星病菌(Alternaria alternata)等16种植物病原真菌有抑菌效果。组分1的抑菌防病机理研究表明：组分1能够显著抑制赤星病菌菌丝生长和孢子萌发,并导致菌丝和孢子萌发后的芽管明显畸形,但杀菌作用很弱或基本无杀菌效果。组分1能导致菌丝体内电解质泄漏,细胞膜上麦角固醇的含量降低,菌丝内和培养液中脂质过氧化产物—丙二醛的含量升高,证明其对病菌细胞膜有一定破坏作用。与此同时,组分1还可显著抑制菌丝体内可溶性蛋白的含量。8.在嘧肽霉素各组分针对烟草花叶病毒的防效试验中,组分4在心叶烟上对TMV的防治效果最好,枯斑抑制率达100%,组分3次之,枯斑抑制率为74.9%,而组分1和组分2没有明显的防治效果。当测试四种组分对普通烟的治疗效果时,组分4的治疗效果最好,防效达到62.8%,组分3次之,为49.8%。组分1也表现出一定的防治效果,防效为18.3%。组分2对TMV在系统寄主普通烟NC89上没有治疗作用。抗病毒机理研究表明,嘧肽霉素组分可以在体外直接作用于病毒粒子,使其丧失侵染能力,从而保护心叶烟免受TMV的侵染。生物学方法、紫外分光光度计法以及ELISA测定结果均表明,嘧肽霉素组分3处理后烟草植株体内的病毒浓度均显著降低,这说明组分3能够降低植株体内的病毒浓度,充分说明嘧肽霉素对病毒的复制和增殖有较强的抑制作用。应用实时荧光定量PCR技术对TMV RNA在烟草中蓄积量测定结果表明组分3可以在TMV侵入寄主早期抑制其基因组RNA的复制。更多还原

【Abstract】 Cytosinpeptidemycin is a high efficient, low-toxic and low-residue bio-pesticide used to control multiple crop fungi and virus diseases. Cytosinpeptidemycin was independently developed by Shenyang Agricultural University, and it was registered as a national patent. Under the guidance of its biological activities, two pure antifungal compounds 1 and 2, one pure anti-bacterial compound 3 and one impure antiviral compound 4 were obtained by different separation techniques such as column chromatography; The molecular formula of the compound 1 and compound 2 was elucidated by spectra analysis; The HPLC qualitative or quantitative analysis method was developed, and the anti-fungal and antiviral mechanism of compound 1 and 3 was investigated respectively. The results were:1. Compound 1 was purified under the guidance of anti- Alternaria alternata activity. The crude fermentation broth was purified to eliminate protein and other mess, absorbed by Diaion HP20 and eluted by 50% methanol. Then the bio-active fractions were further purified through TOSOH SP650M ion exchange chromatography with 0-1 mol/L NaCl as eluent. Finally, the pure compound 1 was obtained by Daisogel ODS-B reverse phase chromatography in 20% methanol and lyophillzed. The molecular formula was elucidated as C12H13N5O4 by interpretation of UV, IR, MS and NMR data. It was the first time to obtain the pure antifungal compound and elucidate its molecular structure.2. The anti- Alternaria alternata compound 2 was obtained through absorption of SP825L and eluted by 50% methanol, followed by silica gel and Sephadex LH-20 chromatography with Chloroform:1-butanol:methanol:acetic acid=90:5:3:2 (by vol.) and 70% methanol as mobile phase respectively. MS analysis showed the compound 2 molecular weight is 244. Results from UV and NMR analysis indicated compound 2 may be an intermediate during the compound 1 synthesis or a degradation substance in the compound 1 fermentation process due to the over time fermentation or other reason.3. Compound 3 was obtained based on the antibacterial activity on Bacillus cereus. Frankland. Some impurities were eliminated by macros absorption resin Diaion HP20 and ion exchange resin SK1B, then bio-active substance was enriched by 732 strong cation exchange resin and gradient eluted by 0.1-0.5 mol/L ammonia water. The molecular weight of pure compound 3 is 574 after the purification by HW40 and LH-20.4. Compound 4 has the activity of anti-virus and was isolated by bioassay. The crude compound 4 was isolated through a simple procedure of absorption and reverse phase chromatography. 5. The study developed the qualitative and quantitative analysis method of compound 1. The HPLC method mainly uses DIKMA DiamonsilⅡC18 column and UV detector by using methanol/water (20:80, by vol) as mobile phase with 1 mL/min flow rate and UV detection at 278 nm. The column temperature was 30℃, and the retention time was 10.2 min. The liner correlation of this method was 0.9998. The average recovery rate and coefficient variation was 99.4 and 0.25%, respectively.6. The conditions for HPLC separating compound 3 are studied:DIKMA DiamonsilⅡC18 column (200mm×4.6mm,Φ5μm) with UV detection at 230nm,30mmol/L phosphate buffer contain 10mmol/L sulfonic acid sodium salt as moble phase with column temperature at 30℃. It can be used as a quantitative and qualitative analysis method.7. Antimicrobial spectrum test showed that compound 1 was against 20 phytopathogenic fungi such as Botrytis cinerea, Colletotrichum orbiculare, Alternaria solani, Sclerotinia sclerotiorum and Alternaria alternate. Compound 1 has a obvious inhibition effect on mycelial growth and spore germination. It also can cause the hypha and the tube to grow in abnormal shapes. The test of antibiosis showed that compound 1 has inhibition action on Alternaria alternata, but has little or even no fungicide function. Studies of compound 1 active mechanism were conducted, and the result showed that compound 1 can cause the protoplasm leaked out from hypha. At the same time, the ergosterol content in mycelia of Alternaria alternata decreased remarkably and MDA content in mycelia and cultural filtrate increased significantly. These results revealed the action site of compound 3 is on the cell membrane, and it changed the content of protein in mycelia.8. The TMV control effect tests were conducted on Nicotiana glutinosa in green house, the results showed that compound 4 had a significant control effect on TMV at almost100% on Nicotiana glutinosa, compound 3 had a control effect at 74.9%. Compound 1 and 2 didn’t have a clear control effect. When examining the Nicotiana tobacum curative effects, compound 4 showed a result of 62.8%, compound 3 was 49.8%, compound 1 was 18.3, and compound 2 didn’t have any curative effect.Research on anti-viral mechanism showed that Cytosinpeptidemycin can directly act on TMV particles and depress its ability of infection. Bioassay, ultraviolet spectrophotometry and ELISA test showed compound 3 can significantly reduce the concentration of TMV virions. In addition, the Real-time RT-PCR revealed that compound 3 can inhibit TMV genome replication in early stage.更多还原