Study On Agrobacterium Tumefaciens-mediated And Peg-mediated Transformation Of Valsa Mali

In our country, Valsa canker of apple has characters of wide distribution and serious pathogenesis, so apple growers called the disease "cancer".It has restricted the further study of the regularity of Valsa mali var. mali (Vmm), caused inefficient, blind and passive disease control work, and caused serious yield and economic loss, because of the little knowledge of the pathogenesis of this disease. Therefore, How to use molecular biology to reveal the molecular pathogenic mechanism, pathogenic mechanism and pathogenic process gene regulatory mechanismin of Vmm will clear the essence of Vmm and host interactions, and promoting the control theory and practice research process of Vmm at the premise of ineffective resistant varieties.So, We adopted the methods of ATMT and PEG mediated transformation to transformate Vmm, and we established genetic transformation system of Vmm take advantage of PEG and Agrobacterium-mediated transformation initially. The plasmid pBIG2RHPH2-GFP-GUS carrying hph gene was used and Vmm 03-8 isolate was used as the host strain.1. According to the system of ATMT of G. graminis(Zhuling 2010). we established optimal conditions for transformation of Vmm as follow: A. tumefaciens cultured to OD600=0.30 in induced medium with 400μM AS for 6 hours, the concentration of the Conidium of Vmm is 106 per mL, transferring the glass paper to Co-IM, incubating 400μL induced A. tumefaciens cells and Vmm 3 days at 25℃.2. Until now, according to the system of ATMT of Vmm, we had obtained 28 transformants, and the transformant efficiency was 6 transformants per 106 condium of Vmm; We found that the selectable marker gene hph was integrated effectively into the genome of Vmm according to the detection of PCR and Southern boltting analysis. After 5 subculturing on PDA, all the transformants still grew, By the observation of the growth characteristic of transformants, we found that the colony color and spore production situation had changed.3. We still used the methods of PEG to transformate the proplast of Vmm. At 50mg/mL Driselase+10mg/mL Lysing enzymes concentration, the mycelium of Vmm that cultured in YEPD medium for 48 h was hydrolyzed in 10 mL enzymes liquid / 0.5 g wet mycelium for 2 h. The protoplast yield was 4×10~7 CFU/mg. 4. Therefore, by the system of PEG-mediated transformation, we had obtained 218 transformants, and the transformant efficiency was 44 per g DNA. Analysis of the transformants by PCR and Southern blotting showed that the selectable marker gene hph was integrated effectively into the genome of Valsa mali var. mali. After 5 subculturing on PDA, 87.5% transformants could grow.According to what we have disscussed above, the stability test of transformants suggested that the foreign gene hph was stable in heredity.At the same time, these two transformation systems developed in this paper are valuable and important tool for the further study of the pathogenic gene of Valsa mali.