Construction of genetic analysis system in Nocardia brasiliensis

1. 偶联体系：4株巴西芽孢杆菌的不同营养缺陷突变体间偶联均未发现重组体。巴西孢子虫对溶菌酶的敏化作用：巴西孢子虫对溶菌酶表现出较高的抗性。但在32℃下与甘氨酸孵育使菌株对溶菌酶敏感。质粒的分离：用甘氨酸在32℃孵育得到的菌丝体进行裂解。7株菌株中有5株携带质粒（pNB1、2、3和4）。原生质体的制备与再生：利用菌丝制备原生质体。不同菌株的门质体产量不同（10^6-10^8/50ml培养）。原生质体在含有0.4M甘露醇和少量牛血清白蛋白或马血清的再生培养基上再生。在含有蔗糖或琥珀酸钠的培养基上未观察到再生。寄主菌株的发育：从IFM0236中分离出温敏突变体和从IFM15中分离出类胡萝卜素不产生突变体。后一突变体原生质体产量高（10^9/50ml）。聚乙二醇对原生质体再生的影响：用20-25%的聚乙二醇1000或4000处理1分钟，再生速率降低。到1/2以下。有必要研究一下其他的转换方法。载体质粒：选择类胡萝卜素基因（car）作为载体的标记基因。首先，在塞顿链霉菌中克隆了链霉菌的car。内切酶分解后的巴西乳猴染色体DNA与setonii的car DNA杂交呈强带。该DNA片段将与巴西白僵菌的质粒DNA连接。

1. Conjugation system: There could not find any recombinant by conjugation between various auxotrophic mutants derived from 4 strains of N. brasiliensis.2. Sensitization of N. brasiliensis to lysozyme: N. brasiliensis shows high resistance to lysozyme. But the incubation at 32C with glycine changed the strains to lysozyme sensitive.3. Isolation of plsmid: Mycelia prepared from the incubation at 32C with glycine were lysed. Five of 7 strains harboured plasmids (pNB1,2,3 and 4).4. Preparation and regeneration of protoplasts: Protoplasts were prepared from the mycelia. Yields of portoplasts varied from strain to strain (10^6-10^8/50ml culture).Regeneration of the protoplast occured on the regeneration medium containing 0.4M mannitol and small amount of bovine serum albumin, or horse serum. No regeneration was observed on the media containg sucrose or Na-succinate.6. Developement of host strain: Temperature sensitive mutants from IFM0236 and carotenoid non producing mutant from IFM15 were isolated. The latter mutant gave good protoplast yield (10^9/50ml culture).7. Effect of PEG on the regeneration of protoplast: Treatment with 20-25% PEG 1000 or 4000 for 1 min decreased the regeneration freg. to under 1/2. It would be nedessary to examine other transformation methods.8. Vector plasmid: As a marker gene of vector, carotenoidd gene (car) was selected. First, car of streptomycetes was cloned in streptomyces setonii. Hybridisation between restriction enzyme dijested chromosomal DNA of N. brasiliensis and car DNA of S. setonii gave strong bands. The DNA fragment would have car of N. brasiliensis will be ligated with plasmid DNA of N. brasiliensis.