Construction Of The Transformation System Of Rhizoctorzia Solani

Rice sheath blight,which is caused by Rhizoctonia solani K(?)hn,is considered to be the most destructive disease of rice worldwide..But so far,people still have known less about the pathogenesis of R.solani,and have not identified the resistance genes in rice varieties.In order to reveal the pathogenic mechanism of rice sheath blight pathogen on molecular level,a genetic transformation system was constructed using protoplasts according to the method of rice blast pathogen(Magnaporthe grisea).Firstly,the protoplasts of R.solani were released from its mycelia by treating with lysing enzyme from Trichoderma harzianum in the solution of 0.8M Nacl(pH 5.8,as osmotic stabilizer), and the conditions of protoplast regeneration and storage were optimized.Secondly,the genetic transformation system of protoplast was constructed by using PEG-medium transformation method and pSM565(contain hygromycin resistance gene)vector DNA was integrated into genome of the fungus.A pair of primer was used to amplify the corresponding region of hygromycin resistance gene by PCR to confirm the successful integration.Two vectors that contain histon enhanced green fluorescent protein(eGFP)for R. solani and M.grisea transformation have been constructed.The histon H3 gene of this two fungus were cloned by PCR according to the gene sequence of M.grisea from Genebank.The PCR product from R.solani was sequenced andanalysed by blastp (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi).The result showed that the gene encode a protein with hight homology to histon H3.Therefore,the histon H3 gene of R.solani was ligated with the C terminal of green fluorescent protein(GFP)gene,and assembled into pSM565 vector.Expression of eGFP in the correct transformants was detected under the fluorescence microscope.