Development Of Agrobacterium Tumefacien-Mediated Transformation System For Aspergillus Flavus

The plant pathogenic fungus Aspergillus flavus displays a wide host range such as seeds of maize,peanuts and rice,and pose serious health hazards to humans and domestic animals by producing aflatoxins(AF),the most toxic and carcinogenic secondary metabolites.Understanding of the molecular basis of this disease is beneficial for preventing its colonization in crop seeds.The Agrobacterium tumefaciens-mediated transformation(ATMT)technique was a major breakthrough in transformants,the random and single-site insertion pattern.ATMT has proven to be an efficient tool for gene function analysis.In this study,a transformation system for A.flavus based on Agrobacterium tumefaciens-mediated approach was developed,the main results were as follows:1.Sensitivity of A.flavus to three different antibiotics was tested.Bleomycin was determined as the selection marker in A.flavus transformaion.Growth of 1×106 per milliliter A.flavus spores could be inhibited with 100?g/mL bleomycin.And the optimum concentration of cefotaxime sodium(Cef)on selection medium was 300?g/mL.2.To construct the pDHBle vector suitable for A.tumefaciens mediated transformation of A.flavus,a two-step subcloning strategy was followed.Firsty,PgpdA-ble-TtrpC expression box was constructed on the backbone pAN7-1 by replacement of the hygromycin B phosphotransferase gene(hph),with the bleomycin gene(ble).And the new vector is named pANBle.Secondly,PgpdA-ble-TtrpC expression box was digested and inserted into pDHt vector.3.To investigate the transformation efficiency of A.tumefaciens mediated A.flavus protoplasts compared to spores,the suitable conditions for preparation of A.flavus protoplasts were determined.The optimum condition for preparing protoplasts from A.flavus spores was as follow,cellulase enzyme:Snail enzyme:lywallzyme=1.5%:1.5%: 1.5%,30?water bath 3h.The formation rate of protoplasts was 97.3% and the regenration rate was 89.2%.This work lay the foundation for genetic transformation of A.flavus.3.A.tumfaciens mediated transformation system for A.flavus was developed preliminarily.The co-cultivation time,temperature,concentration of A.tumefaciens and cell materials of A.flavus were investigated in the transformation.The results show that the transformation frequency of A.flavus using ATMT was optimal when 105 conidia were co-cultivated at 22? for 2days.Transformaion for A.flavus protoplasts is in process.4.Part of transformats were validated by PCR amplification.The primers were designed based on the sequence of ble gene.The positive transformants were inoculated on the SM medium supplemented with 100?g/mL bleomycin at 30? for 6 generations.The stable insertion of T-DNA into the transformants was observed by PCR analysis,targeting the ble gene.In this study,we developed an ATMT system for A.flavus preliminarily,using the ble gene as antibiotic selector marker for recombinants.The effectiveness of this method was measured and will be further validated as a genetic molecular tool for A.flavus.