高山被孢霉高产ARA的原生质体融合子筛选

利用原生质体融合技术,对两株单一优良性状差异显著的高山被孢霉菌株F24和G12进行原生质体融合,再采用双灭活或流式细胞分选法筛选优良性状相结合的高产菌株,对双亲菌株原生质体的制备、融合和再生的相关条件进行了研究,并对双灭活和流式细胞分选的具体方法进行了研究.经研究发现,采用培养16 h的高山被孢霉菌丝于混合酶液(蜗牛酶15.0 mg·m L~(-1)、纤维素酶6.0 mg·m L~(-1)、溶菌酶0.5 mg·m L~(-1)和几丁质酶0.2 mg·m L~(-1))中30℃酶解4 h为原生质体制备的最佳条件.并探究得出两种融合子筛选方法的最佳方案,成功筛选出融合子,进一步再生培养验证ARA产量,最终挑选出一株ARA产量达6.32 g·L~(-1)的高产菌株.以前尚未发现此方法在高山被孢霉上的应用,为高山被孢霉育种提供了一种有效的手段.

Screening of Protoplast Fusion of Mortierella Alpina with Highyield Arachidonic Acid

In this study,F24 and G12,two Mortierella alpina strains which have obvious different characters were fused for the purpose of selecting advantages combining ARA high yield strains by using protoplast fusion technique combined with two efficient fusants screening methods. The parental protoplasts preparation,fusion,regeneration conditions and methods were studied. And the specific methods of double inactivation or flow cytometry were studied. Through the study,hydrolyzing 16 h-cultured mycelium in the mixture( 15 mg/m L glusulase,6 mg/m L cellulose solution,0. 5 mg/m L lysozyme and 0. 2 mg/m L chitinase) for 4 h at 30 ℃ is the best condition. Exploring the best conditions of the two fusant screening methods successfully screen out of the fusants. Finally,By further regeneration culture to verify ARA yield,a high-yield strain was obtained which ARA's production was 6. 32 g/L. This method has not been used in the Mortierella alpina before. It provides an effective method for screening the high-yield ARA strains.