树状多节孢原生质体的制备和再生  

THE FORMATION AND REGENERATION OF NODULISPORIUM SYLVIFORME PROTOPLAST

作　　者：孙剑秋[1] 于寒颖[1] 张鹏[1] 张彦丰[1] 凌洪博[1] 解玉红[1] 周东坡[1] 平文祥[2] 

机构地区：[1]齐齐哈尔大学生命科学与工程学院,齐齐哈尔161006 [2]黑龙江大学生物工程系,哈尔滨150080

出　　处：《应用与环境生物学报》2001年第4期375-381,共7页Chinese Journal of Applied and Environmental Biology

基　　金：黑龙江省自然科学基金资助项目 (93 C 1 1 );黑龙江省"九五"重大攻关项目 (G98C2 0 1 6 1 )

摘　　要：研究了酶系组成、酶解时间、酶解温度、渗透压稳定剂、pH值、培养基成分、培养方式、预处理、保护剂等因素对紫杉醇产生菌树状多节孢原生质体制备和再生的影响 .结果表明 :将树状多节孢在PDA液体培养基中静止培养 2～3d ,离心收集菌体 ,用 pH 5 .5～ 6 .0的 0 .7mol/L的NaCl配制成的含有 3%纤维素酶、2 %蜗牛酶和 1%溶菌酶的复合酶 ,30℃恒温酶解 9h ,原生质体制备率最高。但是考虑到原生质体的再生 ,以酶解 7h左右为宜 .获得的原生质体经过纯化 ,用 0 .7mol/LNaCl配制的PDA再生培养基进行双层平板培养法再生 ,原生质体的再生率最高 .本研究为以后通过原生质体诱变、转化、融合构建紫杉醇工程菌株奠定了基础 .图 3表 9参The effects of some factors on the formation and regeneration of the taxol-producing fungus Nodulisporium sylviforme protoplasts were discussed in details, including enzyme systems, digesting time and temperature, osmotic pressure stabilizer, pH value, culture medium, culture method, pretreation and protective agent. Nodulisporium sylviforme was cultured in PDA liquid medium for 2～3 d, then the mycelia were gathered by centrifugal method and digested by using compound enzyme consisting of 0.7mol/L NaCl, 3% cellulase, 2% snailase and 1% lysozyme for 9 h at the condition of 30℃ and pH 5.5～6.0. The highest production rate of protoplast was obtained. However, taking account of the regeneration of protoplast, it was suitable to be digested for about 7 h. The protoplasts produced by digesting were purified and regenerated through double layers culture method in PDA medium with 0.7mol/L NaCl. The highest regeneration rate could be obtained. This study has laid the foundation to construct the engineering strain producing taxol by protoplast mutagenesis, transformation and fusion. Fig 3, Tab 9, Ref 16

关 键 词：树状多节孢 原生质体 制备 再生 紫杉醇 微生物发酵 

分 类 号：Q949.320.2[生物学—植物学] Q944