水稻纹枯病菌(Rhizoctonia solani)原生质体制备与再生技术及优化

Technology and optimization of preparation and regeneration of protoplasts from rice sheath blight fungus Rhizoctonia solani

水稻纹枯病的病原菌是立枯丝核菌(Rhizoctonia solani Kühn),其病原菌(R.solani Kühn)又称为水稻纹枯病(rice sheath blight)菌,由立枯丝核菌侵染引起的水稻纹枯病是水稻上最重要的病害之一。为建立高效的水稻纹枯病菌原生质体制备技术体系,采用0.7mol/L NaCl作为渗透稳定剂,对Glucanex、溶壁酶(lywallzyme)、纤维素酶(cellulase-R-10)、离析酶(macerozyme-R-10)、蜗牛酶(snailase)、崩溃酶(driselase)和裂解酶(lysing enzyme)等7种不同的胞壁裂解酶降解水稻纹枯病菌GD-118菌株细胞壁的作用进行测定,并对不同酶组合及其酶质量浓度进行优化筛选。结果显示:Glucanex对水稻纹枯病菌细胞壁的降解效果最好,用20mg/mL Glucanex处理1g菌丝4h后,可释放118.5×104个原生质体;用15mg/mL Glucanex和10mg/mL lywallzyme混合酶处理,可高效降解水稻纹枯病菌细胞壁并获得大量原生质体,原生质体产量达3.09×107个/g菌丝,再生率达58%;Glucanex和lywallzyme的混合酶对不同水稻纹枯病菌菌株的细胞壁降解和原生质体释放没有显著差异。可见,上述制备水稻纹枯病菌原生质体的方法具有较好的高效性和适用性,可满足该菌分子遗传研究的需要。

Summary Rice sheath blight caused by Rhizoctonia solani Kuhn is one of the most important diseases on cultivated rice worldwide. Unlike most other fungal pathogens, R. solani forms heterokaryotic vegetative mycelia with multiple nuclei per hyphal cell and is unable to produce haploid asexual spores under normal conditions. These characteristics of R. solani may make it difficult to perform genetic transformation and functional analysis of genes. Production of large amount of protoplasts from this fungus is a prerequisite for the studies of molecular genetics, such as protoplast fusion and fungal transformation. Previously, some lyric enzymes and conditions for releasing R. solani protoplasts have been tested and optimized and several protocols for the preparation and regeneration of protoplasts from R. solani mycelium have been developed by some researchers. However, the efficiency of R. solani protoplasts releasing by these protocols is sometimes unstable due to different strains of R. solani or experimental conditions. Therefore, it is necessary to develop an efficient method for preparing protoplasts of rice sheath blight fungus. The objectives of the present study were to evaluate various cell wall degradation enzymes and their combinations for releasing protoplasts from R. solani myeelium, and to develop an efficient protocol for yielding protoplasts. Using 0.7 mol/L NaCl as stabilizer solution, seven different cell wall degradation enzymes, including Glucanex, lywallzyme, cellulase-R-10, macerozyme-R-10, snailase, driselase and lysing enzyme, and their combinations were evaluated for releasing protoplasts from R. solani GD-118 mycelium which was harvested from potato dextrose liquid medium cultured at 28 ℃for 36 h. The number of released protoplasts was counted by using haemocytometer under microscopy. The optimal concentration of lytic enzymes for the generation of protoplasts was determinated and the conditions to obtain and regenerate protoplasts of the fungus were also optimized. Among the seven tested lyric enzymes, Glucanex was the most suitable enzyme for the digestion of R. solani GD-118 cell wall. The protoplast yield in the treatment with 20 mg/mL Glucanex for 4 h was 23.7 × 104 cell/mL （e. g. 118.5× 104 cells per gram mycelium）. Moreover, the results showed that the optimal mixture enzymes of 15 mg/mL Glucanex and 10 mg/mL lywallzyme were effective in releasing protoplasts with the production of 3.09 × 10^7 protoplasts from per gram R. solani GD-118 mycelium, and the obtained 58% protoplasts could be regenerated. In addition, the combination had similar effects on digesting cell wall of different strains of rice sheath blight fungus. Taken together, the mixture lytic enzymes of 15 mg/mL Glucanex and 10 mg/mL lywallzyme can effectively digest the cell wall of rice sheath blight fungus and produce abundant protoplasts.