小麦纹枯病菌原生质体制备条件及再生菌株致病性研究

Protoplast Preparation Conditions of Rhizoctonia cerealis and Pathogenicity of Regenerated Strains

以小麦纹枯病菌HD-6为对象,进行原生质体制备与再生研究,并将再生菌株与原始菌株的致病性进行比较分析,为建立小麦纹枯病菌高效遗传转化体系提供依据。选用溶壁酶、崩溃酶、蜗牛酶、纤维素酶4种常见酶分别对小麦纹枯病菌进行酶解,并通过单因素试验分析酶解温度、酶解时间以及溶液pH值对原生质体得率的影响。结果表明,4种酶均能够裂解小麦纹枯病菌细胞壁得到一定数量的原生质体,其中崩溃酶的裂解效果最好,每克菌丝得到的原生质体数可达到2.6×108个。该酶的最佳反应温度是24℃,最适的酶解时间是3h,最适的pH值是6.0。获得的原生质体可以完成再生,并且再生菌株与原始菌株的致病力没有显著差异。

Rhizoctonia cerealis strain HD-6was used for studying the protoplast preparation and regeneration,and the pathogenicity of wild-type and regenerated strains was compared.Four lytic enzymes,lywallzyme,driselase,snailase and cellulose,were used for the protoplast preparation independently.The effects of enzymolysis temperature,enzymolysis time and solution pH on protoplast preparation were investigated according to the single factor tests.Among the four enzymes,the driselase had the best efficiency for protoplast preparation and the protoplast quantity per gram of mycelium reached 2.6×108.The best conditions for the protoplast preparation were that the mycelium of R.cerealis was digested at 24℃in driselase solution（pH6.0）for 3 h.The prepared protoplasts could produce mycelium,which had the similar pathogenicity to that of the wild-type strain.