赤霉素产生菌原生质体制备、纯化与再生工艺的研究  

Study on production,purification and regeneration process for protoplast of gibberellin-producing strain

作　　者：张文宣 赵南 朱江萍 

机构地区：[1]江西新瑞丰生化有限公司,新干331307

出　　处：《中国抗生素杂志》2012年第4期276-279,297,共5页Chinese Journal of Antibiotics

摘　　要：目的对赤霉素产生菌原生质体进行制备、纯化、再生工艺改进与完善。方法本文通过微孔滤膜作载体,在含甘氨酸的菌丝生长培养基固体平板上,培养取得幼嫩菌丝体,并对原生质体制备时的复合破壁酶液组分配比、酶解时间,原生质体纯化再生工艺进行了改进和完善。结果实验证明:①在幼嫩菌丝体的制备过程中,采用微孔滤膜平板法培养幼嫩菌丝体,易于控制菌龄,生长在微孔滤膜上的菌丝体不混杂有多余的培养基成份,不必经洗涤,可直接酶解,简化了实验步骤;②在原生质体制备过程中,采用以纤维素酶2%+蜗牛酶1%+果胶酶0.4%作为复合破壁酶液的组成,可使原生质体的释放量从105升至106;③在原生质体纯化过程中,采用先慢速离心沉淀,后洗涤滤除菌丝断片的操作方法,可使无壁娇嫩易碎的原生质体可依附菌丝断片作缓冲载体一起沉淀,有利于纯化收集到完整的原生质体,提高原生质体的活性再生;④在原生质体再生过程中,选用含有聚胨、酵母膏成份的RM再生培养基,可使原生质体的再生率达到16%以上。结论原生质体制备、纯化、再生工艺的改进与完善,确保了诱变育种基因突变后的遗传个体绝大部分都是纯合体,避免了高产性状经传代后水平回复的现象,为赤霉素理化诱变育种工作的顺利开展创造了良好条件。Considering the gibberellin-producing strains are multinucleate mycelium without spores, it is necessary to make the GA mycelium a number of individual free protoplasts by means of enzymolysis for the cell wall before protoplast physiochemical induced mutation of gibberellin-producing strain. Employing the microporous membrane as a carrier, tender mycelium can be cultivated in the solid plate containing mycelium growth medium of glycine, and optimizing the parameters such as distribution ratio of the composite enzyme for cell-wall breaking, enzymolysis time and purification and regeneration process for the protoplast. The experiments showed that： （1）In the process of producing the young mycelium, the age of strains are easy to be controlled and the experiments procedures are simplified by employing the microporous membrane as a carrier because of that there is no need to scrub the mycelium without redundant culture medium so that the mycelium can be enzymolysis directly. （2）In the process of producing the protoplast, the release amount of protoplast can be increased from 105 to 106 by employing the composite enzyme for cell-wall breaking made from 2% cellulose, 1% snailase and 0.4% pectinase. （3）In the process of purifying the protoplast, the delicate and fragile protoplast without cell wall can be precipitated with the mycelial fragments as the buffer carrier by means of low-speed centrifugation precipitate first and wash out mycelial fragments second, it is conducive to purify and collect the integral protoplast and enhance the activity of protoplast regeneration.（4）In the process of regenerating the protoplast, the RM regenerated culture medium containing poly peptone and yeast extract was selected, then the regeneration activity was significantly enhanced up to 16% at least. It ensures that the genetic breeding mutations are mostly individuals homozygous and the phenomenon which flat response of the high yield character after subculture is avoided on the basis of the improvement and maturity

关 键 词：赤霉菌菌丝体 原生质体 复合破壁酶 诱变育种 

分 类 号：Q933[生物学—微生物学]