Obtaining And Screening Of Insertion Mutant From Setosphaeria Turcica By Mutagenesis Of Restriction Enzyme-Mediated Integration

Northern corn leaf blight, caused by Setosphaeria turcica which is an important filamentous plant pathogenic fungi, is one of the important leaf diseases in corn and can lead to destructive damage in the epidemic years. It is necessary to understand the pathogenesis mechanism of fungi for Fungi's prevention. It's well-known that it is a key of the mechanism of the pathogenesis in pathogenic fungi to identify the pathogenicity-related genes. At present, REMI has been widely used in animal, plant pathogenic fungi and other fungi research. For the past 10 years, we has cloned successfully some disease-related genes of pathogens by REMI. Markers and cloning of pathogenic filamentous fungi related gene are being used more and more by restriction enzyme-mediated integration.On the basis of establishing the transformation system in the laboratory, we found a series of factors such as culturing time of Setosphaeria turcica, the type, time and temperature of cell wall-degrading enzyme enzyme, regeneration culture medium and so on, the factors of which influence release and regeneration of protoplast. The best conditions of protoplast preparation of Setosphaeria turcica is that pycnidiospores of Setosphaeria turcica were cultured 20 h in the Fries, choosing the collapse of one percent, solution of the wall of one percent and a snail's pace of one percent, hydrolysising 4-5 h in the 30℃, hydrolysis process used NaCl buffer as steady infiltration agent, The result showed protoplast concentration can reach 10~6/mL above. The best transformation efficiency is 15 min of ice bath. The proportion of protoplast and joining plasmid affected transformation efficiency greatly, such as adding the vectors of lower conversion decreased transformation efficiency, and the higher affected plasmids' copies in the protoplast.Using REMI transformation techniques, we obtained 310 REMI transformants of Setosphaeria turcica 01-23. To examine the stablility of transformants, these transformants were randomly selected to check the hygromycin-resistance stability for 5 asexual generations on PDA containing 50μg/mL hygromycin B. The result showed that all tested transformants could grow on PDA containing 50μg/mL hygromycin B, but the wild isolate 01-23 can not survive on it. So the result showed that these transformants were stable in genetic. We amplified the genome DNA from these mutants by PCR used the hph gene sequence as primers. These amplified fragments were the same sequence as the hph gene which suggested that the hph gene had been inserted into these genomes. Through Southern blotting tested the copies of the positive transformations of PCR, we found that M142 mutant strain is a single copy transformation. Analysising pathogenicity of M142, found that its pathogenicity became weak and spores morphology changed obviously.