产色链霉菌SY20-2与龟裂链霉菌SY20-4的种间原生质体融合

Protoplast Fusion of Streptomyces Chromogenes SY20-2 and Streptomyces Rimosus SY20-4

利用卡那霉素（250 μg/mL）、链霉素（1 000 μg/mL）和罗红霉素（150 μg/mL）标记亲本菌株SY20-2、SY20-4,进行种间原生质体融合.试验结果表明,培养基中加入0.5%～1%甘氨酸可以提高菌丝体对溶菌酶的敏感度,有利于原生质体的释放.溶菌酶不仅影响原生质体的形成,而且影响原生质体的再生率,因此在35℃水浴条件下,SY20-2选择酶浓度2 mg/mL,作用时间30 min;SY20-4选择酶浓度4 mg/mL,作用时间40 min,最适于原生质体的再生.以45%PEG（分子量1 000）作助融剂,融合频率为0.124%,选出153个融合子,其中3株重组子的稳定性及抑菌活性较好,重组子C对烟草野火病菌的抑菌活性比对照要好.

Streptomyces chromogenes SY20 - 2 and Streptomyces rimosus SY20 - 4 was signed by kanamycin （250μg/ ml）, Streptomycin（ 1000μg/ml）and mxithromycin（ 150μg/ml）. When 0.5 % - 1% glycin were added into the liquid medium, mycelium was much sensitive to lytic enzymes and yields of pmtoplast were high. Lytic enzymes is not only affect release of protoplast but also regeneration of pmtoplast. So an optimized protocol is that Streptomyces chromogenes mycelium were hydrolyzed for 30min with lytic enzymes at 2mg/mL or Streptomyces rimosus mycelium were hydrolyzed for 40 rain with enzymes at 4mg/mL at 35℃. 45% （W/V）PEG（molecnlar weight 1000） as fusogen, protoplast fusion rate is 0. 124% and 153 recombinant progenies were obtained. Among them 3 recombinant progenies with high inhibition activities on the Pseudomonas syringae pv. tobaci were obtained.