The Construction Of Highly Efficient Transformation Library Of Magnaporthe Oryzae

Magnaporthe oryzae is a well-known ascomycete that causes rice blast.A better understanding of the molecular basis of this disease is beneficial for controlling rice blast.As a model fungal pathogen,Cloning of functional genes from M.oryzae will be important to understand other fungus-plant interactions.Construction of genomic library is an important method to clone a gene and study on its function.A high quality of the genomic library and an efficient method for transformation are the basics for cloning the genes by phenotypic complementation.Cloning genes by phenotypic complement is an elemental method.In this study,a cosmid library of M. oryzae was successfully constructed,the DNA fragments and the vector of pBHT2 were available for the construction of genomic library,meanwhile some preliminary exploration was performed to enhance transformation efficiency,mainly the electroporation.This study lays a foundation for cloning gene by phenotypic complementation.All results of the study were summarized as follows:1)Establishment of Genomic DNA Library of M.oryzae with Cosmid VectorIn this research,SuperCos1 was used as a vector to construct the genomic library. High quality genomic DNA was extracted from Magnaporthe oryzae by the method of SDS and proteinase K.The DNA fragments which length about 40 kb were recovered after partially digested with Sau3Al.Then it was ligated to SuperCos1 and packed followed the instruction manual.After transfection with XL1-Blue MR bacteria,4.0×10~4 cosmid clones were obtained that equivalent to 10~5 clones per microgram genomic DNA.The clones were stored with 15%glycerol in the temperature condition of -70℃.The analysis of 50 random recombinants demonstrated that 98%of recombinants were inserted with the foreign DNA fragment which was about 40.5kb length.The volume of the library reached up to 31.75 compared with the size 40Mb of M oryzae genome.According to the formula N=1n (1-p)/1n(1-f)from Clarke and Carbon,the probability of any DNA sequence of M oryzae that can be deteced from the library is higher than 99.99%theoretically.The amplification stock library titer was around 4.6×10~8 cfu/ml,even after three times thawing,the titer still reached to 4.0×10~8 cfu/ml.The results show that the genomic library possesses a good quality which guarantees the screening and identification of functional gene for further studies.2)Preparation of the gene fragments and the vector for the construction of the ATMT libraryIn this research,pBHT2 was used as a vector to construct the genomic library. High quality genomic DNA was extracted from Magnaporthe oryzae by the method of SDS and proteinase K.The 8-15 kb region was recovered after partially digested with Sau3Al.The DNA fragments were obtained to the construction of the Genomic library by gel recovery.Vector pBHT2 was completely digested with BamH1 and treated with CIAP.3)Exploration of efficient transformation of MoryzaeAnother main purpose of the present study was focused on efficient transformation methods in M oryzae.Conditions on electroporation,including electric shock buffer,the voltage of electroporation,protoplast preparation for electroporation. The results showed that electroporation can not bring on an obvious enhancement of transformation efficiency.