禾谷丝核菌(Rhizoctonia cerealis)原生质体制备与再生的研究

Protoplast preparation and regeneration of Rhizoctonia cerealis

禾谷丝核菌(Rhizoctonia cerealis)是引起我国小麦纹枯病的主要致病菌。为了建立高效稳定的禾谷丝核菌遗传转化体系,本试验比较研究了不同细胞壁降解酶、酶液浓度、酶处理温度和时间等因素对禾谷丝核菌原生质体制备的影响,利用正交设计试验优化了原生质体再生条件。结果表明,液体培养6d的菌丝,采用15mg/mL溶壁酶+10mg/mL蜗牛酶组成的混合酶液,30℃下酶解4h,可以获得较高的原生质体释放量,可达到3.0×106个/mL;禾谷丝核菌原生质体再生的最佳条件是以SuTC缓冲液作为渗透压稳定剂悬浮原生质体,采用单层混菌法接种于TB3再生培养基,原生质体再生率可达到58.6%。禾谷丝核菌原生质体制备和原生质体再生条件的优化,为深入研究禾谷丝核菌生长发育的分子遗传学基础和进一步探索小麦纹枯病的致病机理奠定了基础。

Rhizoctonia cerealis is the main pathogen of wheat sharp eyespot in China. In order to obtain high-quality protoplasts for the transformation of Rhizoctonia cerealis, the factors influencing preparation of R. cerealis protoplasts were studied. The results showed that the suitable conditions for protoplast preparation were as fol- lowed., mycelia of 6 d, a mixture of 15 mg/mL lywallzyme and 10 mg/mL snailase, digested at 30 ℃ for 4 h The protoplast yield reached 3.0 × 10^6 cell/mL. The optimal regeneration conditions for R. cerealis protoplasts were as followed： SuTC as osmotic stabilizer, TB3 medium, inoculating by mixing the protoplasts with unsolidified medi- um. The protoplast regeneration rate could reach 58.6%. This study is an important foundation for exploring the molecular genetics of the development and pathogenic mechanism of this pathogen.