稻瘟病菌原生质体制备条件优化实验研究

Optimizing process of protoplast preparation in Magnaporthe oryzae

原生质体的制备一直是现代分子生物学研究的重要内容。以稻瘟病菌作为供试菌株,优化了其原生质体的制备条件。优化的原生质体制备条件包括:使用新鲜的菌丝进行接种;加入玻璃珠一起培养,通过振荡使菌丝体破碎分离以获得更多的生长点;菌悬液继续加入新鲜培养液进行培养;原生质体沉淀时选择3 000r/min离心10min,保证最大限度地收集原生质体。实验证明,通过这4个条件优化后,原生质体的产量有了极大的提高,每毫升酶液可制备10 8～109个原生质体。

Protoplast preparation is an important study in modern molecular biology.Several conditions in preparing the protoplasts of Magnaporthe oryzae were optimized.The optimized conditions included using fresh mycelia of Magnaporthe oryzae strains to inoculate adding glass beads（10 mm in diameter） in liquid medium while incubating,and shaking vigorously every 4 h to encourage uniform growth of mycelium.The cultures were then transferred to petri dishes with fresh liquid medium and incubated at 30℃ for one day.Protoplasts were depostion by centrifugating at 3 000 r/min for 10 min,this speed could decrease the loss of protoplasts mostly.After these four conditions optimized,protoplasts were easily obtained at about 108~109 mL-1.