Construction of genetic analysis system in Nocardia brasiliensis

1.接合系统：从4株巴西乳杆菌中分离得到的各种营养缺陷型突变体之间没有发现任何接合重组菌株。巴西乳杆菌对溶菌酶的敏感性：巴西乳杆菌对溶菌酶表现出较高的抗性。但在32℃与甘氨酸孵育后，菌株对溶菌酶敏感。菌丝体的分离：将32℃与甘氨酸孵育得到的菌丝体进行裂解。7株中有5株含有pNB1、2、3和4质粒。原生质体制备与再生：从菌丝体中制备原生质体。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没有观察到再生。宿主菌的选育：从IFM0236菌株中分离到温度敏感突变株，从IFM15菌株中分离到类胡萝卜素不产生突变株。后者具有较高的原生质体产量(109/50ml)。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1min，可降低原生质体的再生率。到1/2以下。有必要研究其他转化方法。载体载体：选择类胡萝卜素基因(CAR)作为载体的标记基因。首先，将链霉菌CAR基因克隆到setonii链霉菌中。用限制性内切酶酶切巴西新月浑子的染色体DNA与白纹伊蚊的CAR DNA杂交，得到了较强的条带。将得到的DNA片段与巴西新月球藻的DNA进行连接。

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.