Agrobacterium Tumefaciens-mediated Transformation And T-DNA Integration Mutation Of R.Solani AG1-IA And Rice Sheath Blight Resistance Materials Screening

Rice sheath blight, one of the world abroad rice diseases, together with rice blast and bacterial leaf blight are called three most devastating diseases in China. At present, with short pole breeding strategy and more nitrogenous fertilizer applied, the disease is one of main factors for decreasing yield of rice. And billions kilogram rice lost every year, especially in high temperature and humid condition,10%to30%yield of rice would be lost, during the worst period more than50%yield of rice would be lost. Rice sheath blight has already become the most serious disease in south rice growing region.Rhizoctonia solani AG-1IA is one pathogenic fungus of Rice sheath blight. The fungus is widespread all over the world and has a wide range of host. It can infect two hundred kinds of plants. It is pathogenic to important crops such as rice, wheat, maize, potato cotton, peanut, and other important economical crops, Rsolani AG-1IA is hard to generate conidiospores in natural condition, can not produce asexual spore. Furthermore, too many nuclei exist in one single cell. At the present, the genomic research of R.solani AG-1IA is lagged. This article using Agrobacterium-tumefaciens-mediated transformation to gain international standard R.solani AG-1IA transformants, detecting pathogenicity of transformants to explore the pathogenicity gene of R.solani AG-1IA and analyse function. In order to explain the pathopoiesis further. At the same time, because of short of R.solani resistance materials in China, cultivating rice core germplasm repository material to screen R.solani AG-1IA resistance plant. Main research (content below) includes two aspects:1. Rsolani AG-1IA transforamnts gained and analysing(1) R.solani AG-1IA morphology and hygromycin resistance testInoculated R.solani AG-1IA in PDA medium for three days, and took some hypha on glass slide. Then dyed them with gossypol and observed dichotomous branching and hypha anastomosing. Dyed by DAPI, two to five nucleus will be found. R.solani AG-1IA be cultivated seven days, seven days later take hypha to microscope. Sclerotium characteristic showed short rods. Inoculated activated R.solani AG-1IA into PDA medium with concentration of hygromycin are0mg/L,5mg/L,10mg/L,15mg/L,20mg/L,25mg/L separately. When the concentration of hygromycin is20mg/L, hypha can not grow.(2) Preparing and regeneration protoplast of R. solani AG-1IAUsing enzyme-acting, the optimal way to harvest1.67×109protoplasts per gram from R. solaniAG-1 IA,12h aged mycelium in0.6M MgSO4stabilizer (pH5.2) combining cellulase(10mg/m1)and driselase (10mg/ml)needs to be dealt with at35℃for3h. The most suitable condition is16-hour-aged mycelium in0.6M MgSO4buffer (pH5.2) combinating cellulase and driselase at35℃for2.5hours. Under that condition, the regeneration rate is the highest.(3) Agrobacterium tumefaciens-mediated transformation of protoplast and myceliumPreparing concentration of acetosyringone is40mg/L, pre-inducing for5hours, OD of Agrobacterium is0.5-0.8, concentration ratio of Agrobacterium and protoplast is100:1. With inducing medium, making co-culture temperature24℃, screening by1%half solid medium with hygromycin, and36hours later, transformation efficiency is highest. Mycelium transformation, while OD of Agrobacterium is0.8and co-culture temperature is28℃, transformation efficiency is the highest after48hours co-culture.(4) Transformants pathogenic research and mycelium no fusion analysisThrough Agrobacterium tumefaciens-mediated transformation of protoplast and mycelium, screening by hygromycin and molecular level,120transformants have been attained. Take the second upper leave of rice9311, inoculated by mutation of T-DNA insertion, cultured in man-make climate culture box (28℃,80%humid), and cultivated for24hours. Compared with wild type R. solani AG-1IA, mutation observed and analysed, the result is60low pathogenic mutation and27no fusion mutation, and3partly fusion mutation would be gained.(5) Single oligonucletide nested PCR28DNA sequences have been gained by single oligonucletide nested PCR. Sequences align successfully in the genome of R.solani AG-1IA,2are no fusion mutation,1is sequence of low pathogenicity and no fusion mutation, and1sequence of low pathogenicity and partly fusion mutation. Homology rate of these sequence and AG-1IA-08162, AG-1IA-08822, AG-1IA-08161.AG-1IA-03672in the genomic is100%. But at present the function of three gene is unknown, only one is known as G protein regulating factor.2. Rice sheath blight resistance material screening and creating(1) Rice sheath blight resistance material screeningCultivated185species of rice core germplasm repository material and5species9311mutation, and took the second upper leave to inoculate R.solani AG-1IA. Cultured in man-made climate culture box (28℃,80%humid) for24hours. Compared with9311,on the basis of area scab of rice leaves, screened16rice sheath blight resistance materials and62rice sheath blight susceptibility materials. (2) Mutating rice9311by chemical mutagen to create rice sheath blight resistance materialsWith chemical mutagen (Ethyl methylsulfonate), the concentration of phosphate buffer is0.05mol/L and0.4%-0.5%EMS. Set up four treatment conditions, like A, B, C, D and disposed seed of rice9311. Sowed seeds in Hainan and gain self-hybridization mutation seeds. Then sowed the mutation seeds in Wenjiang experimental field. Took the second upper leave inoculate R.solani AG-1IA. It turned out that rice sheath blight resistance has strengthened after disposed by treatment conditions A and B, while resistance had no difference under conditions C and D.