【作者】 潘翠；

【导师】 宋晓平；

【作者基本信息】 西北农林科技大学 ， 动物生物技术， 2012， 硕士

【摘要】 瑞香狼毒（Stellera Chamaejasme L.）为瑞香科（Thymelaeaceae）狼毒属（Stelllearlinn）多年生草本植物，瑞香狼毒全株有毒，家畜误食可致中毒，母畜接触可致流产，严重时引起死亡。瑞香狼毒产种量大，种子生命力强，在天然草地植物群落中可形成优势种，与优良牧草争夺肥、水、光照和空间，抑制优良牧草生长，加速天然草原退化。目前，草原狼毒防除主要采用化学防除法，虽然化学防除的效果是肯定的，但该法对环境存在潜在危害。内生真菌生活史的一定阶段或全部阶段在植物体内，与宿主植物互利共生，即植物为内生真菌提供营养，内生真菌的代谢产物又能刺激植物生长和提高植物抵抗力。据此推测，有毒植物体内应存在可降解其毒性成分的内生真菌。为了给瑞香狼毒及草原其他有毒植物的脱毒利用提供新资料和新思路，本试验通过对一株分离自瑞香狼毒的内生真菌降解宿主黄酮类和香豆素类成分的作用研究，取得了以下结果：1瑞香狼毒内生真菌C1Y74的形态和分子生物学鉴定：通过对C1Y74在PDA、CA、CYA、CYA20S及MEA培养基上的菌落及孢子形态学观察，进一步用含0.05%茴香醛察氏培养基来进行区别，菌落和分生孢子不呈现红色，将其初步鉴定为黄曲霉（Aspergillus flavus）。通过对C1Y74的DNA纯度检测：经测定A₂₆₀/A₂₈₀=1.86，表明提取的DNA纯度好，没有蛋白质、RNA杂质，浓度为50.5ng/μL，符合进行下一步PCR扩增条件。将提取的DNA在0.8%琼脂糖电泳（100V，50min）后，在紫外光下观察到清晰条带，说明已成功提取真菌基因组DNA。以菌株C1Y74基因组DNA为模板，以特异性引物ITS1/4为引物，扩增出602bp的DNA片段，GenBank登录号为JN648706。ITS序列系统发育分析，将测得序列输入Genebank中比对，同源性为100%的为黄曲霉种（Aspergillus flavus）和米曲霉种（Aspergillus oryzae），结合形态学最终将菌株C1Y74确定为曲霉属黄绿组的黄曲霉种（Aspergillus flavus）。2瑞香狼毒内生真菌C1Y74降解宿主黄酮和香豆素类成分的研究：将瑞香狼毒内生真菌孢子悬液接种于以瑞香狼毒提取物为主要碳源的PDA培养基中，分别在培养的第0，3，5，7，9，12天时，采用可见紫外分光光度测定体系中黄酮和香豆素类成分含量。考察不同接种量（10%、20%、30%）、初始pH（5、6、7、8、9、10）、外加碳源（葡萄糖0、0.2%、0.5%、1.0%、1.5%、3.0%）和表面活性剂（吐温800、1%、2%、3%、4%）对降解性能的影响。在培养第3天时，瑞香狼毒内生真菌C1Y74株对黄酮和香豆素类成分的降解作用明显，培养体系中残留量分别为432.24mg/L和1.25mg/L，极显著低于对照组474.68mg/L和1.61mg/L，在12d时达最低值，分别为303.78mg/L和0.43mg/L。正交试验结果表明，菌株C1Y74降解瑞香狼毒提取物中黄酮类化合物的最优条件为：接种量10%，pH=5，葡萄糖浓度0.5%，吐温80浓度2%，各因素影响大小为：pH>接种量>葡萄糖浓度>吐温80浓度，降解率为81.23%；菌株C1Y74降解瑞香狼毒提取物中香豆素类化合物的最优条件为：接种量20%，pH=7，葡萄糖浓度1.0%，吐温80浓度2%，各因素影响大小为：吐温80浓度>葡萄糖浓度>pH>接种量，降解率为89.21%。更多还原

【Abstract】 The plant Stellera Chamaejasme L （Thymelaeaceae） is perennial herbal. It is toxicwhich can cause livestock poisoning and pregnant female miscarriage, severe cases canresult in death. S. Chamaejasme can produce amount of seeds, have strong vitality, competewith grass for fertilizer, water and light, thus inhibiting the growth of forage grass, resultinga vicious cycle of grassland ecosystem to accelerate the deterioration of natural grassland.At present, it is still difficult to control S. chamaejasme in the grassland. Although the effectof chemical control is conformed, the potential harm on the environment is also existed.Endophytic fungi are fungi which spend the whole or part of their life cycle inside thehealthy plant tissues without any discernible infectious symptoms. They are mutualism thatthe plant provides nutrition to endophytic fungi, and metabolites of endophytic fungi canstimulate plant growth and improving plant resistance. It can conclude that the toxic plantsmay be biodegradable toxic components by its endophytic fungi. In order to provide newinformation and new ideas to use to of S. chamaejasme and grasslands and other toxicplants detoxification of the test by an endophytic fungi isolated from S. chamaejasmedegradation of the host flavonoids and coumarins. The results were as follows:1. The morphological and molecular biological identification of endophytic fungistrain C1Y74from S. chamaejasme: From colony morphology and morphology wereanalyzed, endophytic fungi strain C1Y74preliminarily identified as Aspergillus surroundsubgenus, yellow Green Group （Aspergillus section Flavi）. The method of extract DNA isCTAB, and the testing of DNA purity, determination of the A₂₆₀/A₂₈₀=1.86, concentrationis50.5ng/μL.The extraction efficiency is higher. Being took strain C1Y74genomic DNAas the template and specific primers of ITS1/4as the primers, a602bp DNA fragmentwhose GenBank accession number was JN648706that was amplified. BLASTn analysisshowed that the sequence with Aspergillus ITS sequences with high origin, to prove that theamplified fragment is indeed the ITS sequences. The result of morphological identificationis just same as the molecular identification of, it can conclude that strain C1Y74isAspergillus flavus. 2. Study on degradation of host flavonoids and coumarins by endophytic fungistrain C1Y74from S. chamaejasme: The spore suspension of the endophytic fungi wasincubated to the PDA culture medium with adding the extract of S. chamaejiasme as maincarbohydrate source, and the content of residual flavonoids and coumarins in the culturalsystems was determined by ultraviolet-visible spectrophotometer, and the degradationeffects of the initial inoculum size （10%,20%,30%）, initial pH （5,6,7,8,9,10）,additional carbon source （glucose,0,0.2%,0.5%,1.0%,1.5%,3.0%） and surfactant （Tween80,0,1%,2%,3%,4%） were investigated. At the3rdday, the degradation effects offlavonoids and coumarins were obvious, and the content of the residues was432.24mg/Land1.25mg/L, respectively, which was significantly lower than that of the control group（474.68mg/L and1.61mg/L）. At the12thday, the volume of that was303.78mg/L and0.43mg/L, respectively. The orthogonal experiment results showed that the strains C1Y74optimal conditions of degradation in Stellera chamaejasme extract flavonoids as follows:10%of the inoculum size, pH=5, the glucose concentration of0.5%and Tween80concentration of2%, the impact of various factors were as follows: pH> inoculum size>glucose concentration> Tween80concentration, the degradation rate is81.23%, strainsC1Y74optimal conditions for degradation of extract coumarins as follows:20%of theinoculum size, pH=7, the glucose concentration of1.0%and Tween80concentration of2%,the impact of various factors were as follows: Tween80concentration> glucoseconcentration> pH> vaccination, the degradation rate is89.21%.更多还原