紫杉醇产生菌Nodulisporium sylviforme原生质体诱变研究  

Study on the Mutagenesis of Taxol-producing Fungus Nodulisporium sylviforme Protoplast

作　　者：赵凯[1] 周东坡[1] 平文祥[1] 邹积宏[1] 马玺[1] 

机构地区：[1]黑龙江大学生命科学学院,哈尔滨150080

出　　处：《中国生物工程杂志》2004年第9期63-68,共6页China Biotechnology

基　　金：黑龙江省"十五"重大攻关资助项目 (GA0 2C10 1);哈尔滨市重点资助项目 ( 0 114 2 112 6)

摘　　要：对酶系组成、pH、酶解温度和酶解时间等影响树状多节孢原生质体制备和再生的因素和原生质体诱变进行了研究。结果表明 ,原生质体制备和再生的最佳条件为 :用pH5 5～ 6 0的0 7mol LNaCl配制由 3%溶壁酶 + 3%蜗牛酶 + 1 %的溶菌酶 + 3%纤维素酶组成的复合酶系 ( 1ml酶液 2 5 0mg湿菌体 ) ,在 30℃恒温水浴条件下酶解 6h ;然后 ,将获得的原生质体过滤洗涤后 ,在含0 7mol LNaCl的PDA再生培养基上 ,采用双层平板培养法再生制备到的原生质体。树状多节孢紫杉醇产生菌原生质体诱变的最佳条件为 :30w紫外灯、距离 30cm、照射 5 0s;UV + 0 6%LiCl复合诱变、照射时间 40s,诱变菌株经初筛和复筛 ,选出了两株高产紫杉醇的原生质体诱变菌株———UV40 -19和UL50 -6,其产量从出发菌株紫杉醇的产量 ( 31 4 0 7μg L)分别提高至 376 38μg L和392 63μg L。The effects of some factors that include enzyme systems, enzymolysis temperature, enzymolysis time, pH value on the preparation and regeneration of the taxol-producing fungus Nodulisporium sylviforme protoplasts, and mutagenesis of protoplasts in the experiment were discussed. The results showed that the optimal conditions of the preparation and regeneration of protoplast were 3% lywallzyme+3% snailase+1% lysozyme+3%cellulase which were made in 0.7mol/L NaCl solution with a pH 5.5～6.0, and enzymolysed in a water bath(30℃) for 6h. The prepared protoplasts were regenerated by using bilayer plate culturing method. The optimal condition of mutagenesis of protoplasts was achieved when the protoplasts of Nodulisporium sylviforme that contained 0.6%LiCl in the medium were irradiated by a 30w UV lamp in a 30cm distance for 40s.The mutants UV 40-19 and UL 50-6 were obtained by the primary and secondary screen, which increased their yield of taxol from 314.07μg/L to 376.38μg/L and 392.63μg/L, respectively.;

关 键 词：紫杉醇产生菌 树状多节孢 原生质体制备 诱变 酶解时间 酶解温度 酶液 菌株 株高 高产 

分 类 号：Q939.5[生物学—微生物学] S646[农业科学—蔬菜学]