Establishment And Optimization Of A System For Transformation Of Rice Sheath Blight And Its Protoplast Via Agrobacterium Tumefaciens

Rice(Oryza sativa L.) is one of the most important food crop in the world,whose yields is largest among China grain crops,up to109t.More than one third of people in the world use it as main food source.Rice sheath blight,bacterial leaf blight and rice blast were three main diseases of cultivated rice throughout the world,resulting severe effect on rice yields.Reserches on bacterial leaf blight and rice blast have been carried out in depth for long time while very few was conducted to investigate pathogenic mechanism of Rice sheath blight especially interaction between rice and Rhizoctonia solani Kvhn is unclear and no virulence gene was reported until now.In this paper,an dual plasmid pCAMBIAl300sGFP that carrying green fluorescent protein (GFP) gene as a tool to tract interaction between plant and pathogens,hygromycin phosphotransferase gene as a selection marker was constructed to transform Rhizoctonia solani mycelial and its protoplasts via Agrobacterium tumefaciens.A system have been established and optimizated to produce proplasts. The optimal way of harvesting1.67×10protoplasts per gram from Rhizoctonia solani is to deal with12-hour-aged mycelium in0.6M MgSO4stabilizer (pH5.2) combining cellulase and driselase at35℃for3h. Meanwhile, we attain the most suitable condition about protoplast regeneration, the condition is to deal with16-hour-aged mycelium in0.6M MgSO4stabilizer (pH5.2) combining cellulase and driselase at35℃for2.5h, and incubation with26degree centigrade in the regeneration medium.Over6000mutants that will provide plentiful materials for our research have been gained via transformation of protaplast with Agrobacterium tumefaciens.In order to assure to improve the probability of obtaining mutants,a transformation system was established and optimizated. The optimal yields of transformants was reached via Agrobacterium tumefaciens with Agrobacterium tumefaciens/protaplasts as100:l,with AS(200μmol/) added during induction stage (10h)and co-transformation stage(36h) at24℃.Transformation via Agrobacterium tumefaciens was compared to REMI and PEG-mediated transformation,it was more stable and efficiency.Forward genetic urged us to analysis phenotype and character of mutants related to genes which provide us many references to gene functional analysis,such as colony morphology,hypha morphology in microscope,phsiology and pathogenicity.Some mutant flanking sequence have been cloned via Tail-PCR using primers designed from board sequence of dual plasmid.In addition, green fluorescent protein(GFP) was applied to observe the pathogenic process of Rhizoctonia solani under fluorescent microscope which lay the foundation to study the pathogenic mechanism of Rhizoctonia solani.