【作者】 金燕华；

【导师】 裘娟萍；

【作者基本信息】 浙江工业大学 ， 微生物学， 2008， 硕士

【摘要】 大观霉素是一种氨基糖苷类广谱抗生素,对革兰氏阳性菌及革兰氏阴性菌均有抗菌作用,应用前景广阔。因此,对大观霉素进行菌种选育以提高其产量具有重要的社会及经济价值。本文以试验室筛选的一株高产大观霉素的壮观链霉菌为研究菌株,研究了菌体培养时间、酶浓度、酶解时间、酶解温度、预处理方式等对壮观链霉菌原生质形成和再生的影响,确立壮观链霉菌原生质体形成与再生的最佳条件为:在S培养基中加入0.5%的甘氨酸,以30℃,220 r/min恒温振荡培养36 h(二级培养)的菌丝体为材料,用0.3 mol/L的蔗糖作为酶处理前的菌体洗涤液,溶菌酶浓度为2 mg/mL,37℃酶解120 min;分析比较了HPLC、GC、管碟法、微生物比浊法的优缺点,确定并建立了一种快速、高效、准确的检测方法.微生物比浊法,用于大观霉素发酵液的检测;以NCBI核酸数据库中的spcS2基因序列为模板,运用primerpremier 5.0软件设计引物,PCR扩增了壮观链霉菌中的谷氨酰胺.氨基环醇氨基转移酶基因(spcS2),全长为1460 bp,对其测序并使用Blast与NCBI核酸数据库中的spcS2基因进行比对,两者的相似性达到95%,将其与大肠杆菌.链霉菌穿梭质粒pHZ1272连接,构建重组质粒pHZ1272-spcS2,转化大肠杆菌E.coliDH5α,未得到阳性克隆。更多还原

【Abstract】 Spectinomycin is a broad-spectrum antibiotic which belongs to aminoglycosides and show great efficiency against a broad range of microorganisms including G~+ and G~- germ and have a wide application future. Therefore, it will be of great social and economic value for us to screen a Spectinomycin-overproducing strain.In this paper, as an initial strain, whose potency is as high as 2000 U/mL. This strain was studied at variety of condition including cell culture time, lysozyme concentration, enzymatic hydrolysis time, hydrolysis temperature, pretreatment methods to judge facts effecting protoplast formation and regeneration of Streptomyces spectabilis so as to formate protoplasts. And the best conditions was concluded as following: the strain was incubated in medium S adding 0.5% glycine for 36 h at the constant temperature 30℃, 220 r / min, washing mycelium with 0.3 mol / L sucrose as a pretreating method before got hydrolysed, then hydrolysing for 120 min with a lysozyme concentration of 2 mg / mL at 37℃. The technique mentioned above could lay the foundation for exogenous DNA transducting protoplast, protoplast mutation and fusion.In addition to the study on protoplast formation and regeneration, the comparison was carried out as well between HPLC, GC, cup-plate method and turbidimetric method which were for the use of detecting spectinomycin. And turbidimetric method was considered as the best one owing to its accurency, high effection and short-time consuming.Besides, spcS2 coding a glutamine-aminocyclitol transferase was amplificated with PCR technique using the spcS2 nucleic acid sequence from NCBI database as a template and applying primer premier 5.0 software as primers designing, and then sequenced to be 1460 bps long and blasted with spcS2 from NCBI to get an identity of 95%.After that, the cloned spcS2 was conjucted to Escherichia coli-Streptomyces shuttle expression vector pHZ1272 formming a recombinant plasmid pHZ1272-spcS2, then transtucted E.coliDH5αand growen on LB adding 100μg/mLAmp.Unluckily no bateria of positive clone was picked from a number of clones.更多还原