50mL-管摇床培养暨24孔板法筛选麦角菌突变株  

50mL-TUBE SHAKING CULTURE AND 24 WELL-PLATE ASSAY FOR THE SCREENING OF ERGOT MUTANTS

作　　者：朱平[1] 王全[1] 朱慧新[1] 何惠霞[1] 

机构地区：[1]中国医学科学院中国协和医科大学药物研究所卫生部天然药物生物合成重点实验室,北京100050

出　　处：《微生物学通报》2000年第3期192-194,共3页Microbiology China

摘　　要：麦角菌原生质体用０．３ｍｇ／ｍＬ亚硝基胍（ＮＴＧ）分别处理２０，４０，６０和８０ｍｉｎ。随机挑取ＮＴＧ处理后的原生质体再生单菌落共８０个，分别接种到５０ｍＬ离心管（内装１０ｍＬ液体培养基）中，在２４℃、２２０ｒ／ｍｉｎ摇床上培养１２ｄ。在紫外灯（２５４ｕｍ）下选出４６株荧光相对较强的培养物在２４孔板上作Ｖａｎ Ｕｒｋ蓝色反应分析。挑选９株呈深蓝色反应的菌株作ＨＰＬＣ分析，其结果与２４孔板法有明显的相关性。在麦角隐亭产率上，Ｎｏ．４５最佳（来自ＮＴＧ ８０ｍｉｎ剂量组），Ｎｏ．１８、 Ｎｏ．２６次之（来自ＮＴＧ ４０ｍｉｎ剂量组）。５０ｍＬ－管摇床培养与２４孔板法相结合，有效地提高了筛选速度和成功率。Protoplasts of Claviceps purpurea were treated with N-methyl-N'-nitro-N-nitrosoguanidine (NTG) for 20, 40, 60 and 80min, respectively. Eighty regenerated monocolonies of the NTG treated protoplasts were picked up and inoculated individually in the 50mL-tube containing 10mL of liquid medium, and cultivated on a rotating shaker at 24℃, 220r/min) were assayed on the 24 well-plate for their Van Urk blue color reactions. Nine isolates which gave darker blue color were further analyzed by HPLC. The results of HPLC were correspondent with those of the 24 well-plate assay. Three isolates showed relatively higher yields of ergocryptine and the best one was the No.45, isolated from the 80min dosage group; the other two isolates (No. 18, No.26) belonged to the 40min dosage group. It was demonstrated that 50mL-tube shaking culture combining with the 24 well-plate assay could increase the screening speed and successful chance.

关 键 词：麦角菌 摇床培养 麦角碱类药物 诱变育种 24孔板 

分 类 号：Q93-336[生物学—微生物学]