Construction of genetic analysis system in Nocardia brasiliensis

1.接合系统：4株N. brasiliensis.2. N. brasiliensis对溶菌酶的反应：N.巴西曲霉对溶菌酶具有高抗性。但在32 ℃下与甘氨酸孵育使菌株对溶菌酶敏感.质粒的分离：裂解在32 ℃下用甘氨酸孵育制备的菌丝体。7株菌中有5株携带质粒（pNB 1、2、3和4）。原生质体的制备和再生：从菌丝体制备原生质体。原生质体的产量因菌株而异（10^6-10^8个/50 ml培养物），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可再生原生质体。在含蔗糖和琥珀酸钠的培养基上均未观察到再生现象。宿主菌的选育：从IFM 0236中分离到温度敏感突变株，从IFM 15中分离到不产类胡萝卜素突变株。后者的原生质体产量较高（10^9个/50 ml培养物）. PEG对原生质体再生的影响：20- 25%PEG 1000或4000处理1 min，再生频率降低。低于1/2。有必要研究其他转换方法。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，在链霉菌中克隆了链霉菌的car基因。用限制性内切酶消化的N. brasiliensis和S. setonii产生强条带。该DNA片段将具有N的car。巴西根瘤菌与N.巴西的。

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.