抗真菌活性粘细菌分离筛选与鉴定

【Abstract】wWw.shuoshilunwen.com Myxobacteria is a kind of bacterias which neglected by scientists for a long time, until the 20th century a study of its various properties has just began. Although the studies of myxobacterias were started relatively late, a lot of results had also made. First to be studied is the problem of separation and purification of myxobacterias, due to its peculiar life cycle and growth characteristics, making it difficult to purify, and also the time for purifying is so long, a lot of manpower and material resources were consumed. So far, the preservation of the strains of myxobacterias is still not enough all over the world, which greatly limit the classification and identification of myxobacterias and their standard definition of the evolutionary status. Since the discovery of antibiotics substances from the secondary metabolites of myxobacterias, the research focus of scientists was shift to the secondary metabolites, in order to discover and separate the bioactive substances, making a research hotspot to get the antibacterial, anti-tumor compounds from new secondary metabolites of myxobacterias.In this study, in order to expand the quantity of the strains of myxobacterias, so as to provide the material basis for basic research, and to establish the library of secondary metabolites of myxobacterias, and also for the purposes of screening of biologically active substances for the latter part,555 soil samples of rich type were collected from Shaanxi, Hebei, Guangdong, Henan, Inner Mongolia, Hubei, Liaoning, Hunan, Ningxia, Jiangsu, Guizhou, Chongqing, Heilongjiang, Jilin, Sichuan, Shandong, Hainan, Fujian, Xinjiang, Yunnan, Qinghai, Zhejiang, Anhui. We also improved and innovated the methods of traditional induction, isolation and purification of myxobacterias, this greatly reducing the purification of myxobacterias’ cycles.From the soil samples, we obtained 237 myxobacterias of varied colors and shapes, according to the classification of myxobacterias in the "Berger’s manual of bacterial identification" of eighth edition, the myxobacterias we isolated were identified in which 8 genus, among them, Myxococcus 31 strains, Archangiaceae 41 strains, Cystobacter 33 strains, Melittangium 14 strains, Stigmatella 10 strains, Polyangium 46 strains, Nannocystis 19 strains, Chondromyces 16, there are 27 known genera,237 myxobacterias were described and photographic records of their purified colones, the study enriching the basic research of myxobacterias.Myxobacteria that grew in liquid medium is not better than that in solid medium, and for the strain-specific, the secondary metabolites may be different in different mediums for one strain. So in our study, we used 4 different liquid mediums for its fermentation, and we get more secondary metabolites than before, and fermentation conditions were described, and for the subsequent fermentation of liquid foundation.We build the method that use the Candida albicans as a model to screening the myxobacterias with antifungal activities.9 strains of 237 purified myxobacterias that inhibited the growth of Candida albicans were obtained after first screening. After 3 time’s second screening, ZG15, R16-1 and ZJ2 were found, broad-spectrum antibiotic experiment was done for these 3 strains, and the result showed that the number of ZJ2 myxobacteria has the broad-spectrum antimicrobial activities. Then we extracted its total DNA with NaCl-CTAB, plastic recycling after the PCR amplification, and connect the T vector and tranormed into competent cells, selected positive clones plaid extraction, conducted the 16SrDNA sequencing, and identified the species in molecular levels. With its sequence in GenBank, show that the myxobacteria ZJ2 is Myxcoccus, and the similarity to Myxococcus fulvus EU262991.1 is 100%. Select the the other genera’s 11 myxobacteria strains, build the phylogenetic tree, determine its phylogenetic position.