【作者】 程媛媛；

【导师】 王一丁；

【作者基本信息】 四川师范大学 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 植物内生菌(Endophyte)是一类应用前景广阔的资源微生物,具有固氮、促进植物生长、增强宿主植物抗逆性等作用,可通过组织学方法或表面消毒等方法从植物组织中分离。重楼属植物是一类极具药用价值的植物,具有解毒、镇痛、抗菌、抗癌等功效,是许多中成药和新药的有效成分。随着医药产业对重楼的开发利用,野生重楼遭到了毁灭性的人为破坏,濒临枯竭。药用植物内生菌的研究表明,其内生菌具有合成与宿主植物相同或相似的活性成分的能力,有的内生菌还具有促进宿主植物合成活性成分的能力。本文选择对四川华重楼内生菌的研究,从分离药用植物的活性成分方面解决重楼资源不足的问题。通过表面灭菌,从药用植物华重楼(Paris polyphylla var.chinensis )的地下根状茎中分离得到82株内生菌。通过滤纸片扩散法初筛和平板对峙法复筛,有多株菌有抑菌活性,本文对其中一株具有较好抑制作用的内生细菌PCE45进行了研究。根据形态学特征、生理生化特征及16SrDNA序列分析,确定PCE45为一株枯草芽孢杆菌(Bacillus subtilis)。通过硫酸铵盐析、透析、丙酮低温沉淀,得到PCE45的粗提液,采用管碟法测得硫酸铵最佳饱和度为50%~70%。粗提液经SephadexG75柱、DE52纤维素柱和SephadexG25凝胶柱纯化得到一种抗菌肽,命名为PCP-1。稳定性测试表明,该抗菌肽对对蛋白酶不敏感,对高温、强酸、强碱有较好的耐受性,可造成稻瘟病菌菌丝畸形并抑制孢子萌发。抑菌谱表明该抗菌肽对玉米弯胞病菌等真菌和大肠杆菌等细菌有较强的抑菌效果。质谱测得其分子量为1058.3D。氨基酸组成分析表明该小肽主要由7种氨基酸组成。结合茚三酮反应呈阴性,酸水解后,茚三酮反应和双缩尿反应呈阳性的性质,推测PCP-1可能为低分子量的环状小肽。更多还原

【Abstract】 Plant endophyte is a group of resource microorganism with broud perspective, which have ability of nitrogen fixation, could encourage plant growth and reinforce antireversion force of the plant . It can be separated from the inner part of plant by histology or disinfection. Paris L is a class of plant with variety of medicine value ,which have ability of detoxication, analgesia, antibiosis, anticancer et al. It is the major material of many prepared Chinese medicines. However, with the exploitation of medicine industrial, we are facing the crisis of it’s gradually drying up.Base on the research results of medicine plants, it’s endophyte can synthetize the same active ingredient, some endophyte can encourage plant to synthetize active ingredient.We are study on endophyte in Paris polyphylla var.chinensis Hara which could produce active ingredient in order to resolve the crisis of wild paridis lacking.Through surface sterilization ,82 strains endophytes were isolated and purified from the fresh hypogeal stems of Paris polyphylla var.chinensis . Many endophytes showed antimicrobial activity through preliminary antimicrobial screening against pathogenic bacterias and further antimicrobial screening against pathogenic fungi. The strain PCE45 with best antimicrobial activity was selected to the next series of studies.Based on the characteristics of morphology,physiology,and biochemistry as well as 16SrDNA analysis, the strain PCE45was classified into Bacillus subtilis.We made the crude extract of PCE45 after the steps of ammonium sulfate salting-out, dialysis and acetone precipitation. Through the method of Oxford Plate,we find the best sulfate saturation is 50%~70% .After SephadexG75,DE52 and SephadexG25 column chromatography, we separated an antimicrobial peptide PCP-1 from the strain PCE45.The stability against high temperature and proteinase, and antimicrobial activity were also analyzed. The antimicrobial peptide PCP-1 was stable to proteinase and tolerated high temperature, strong acid and strong base. PCP-1 caused deformation of the hyphae of Pyricularia oryzae and prohibited the spore germination. It also inhibited fungi such as Curvularia lunata and bacteria such as Escherichia coli. Mass spectrogram measurement revealed its molecular weight of 1058.3 Da. The amino acid composition of the peptide composed of 7 amino acids. Ninhydrin reaction showed negative trait whereas after acid hydrolysis with positive ninhydrin reaction and biuret reaction. The ninhydrin reaction and biuret reaction imply that the peptide PCP-1 is a cyclic lipeptide.更多还原