The Research Of Agrobacterium Tumefaciens-mediated PacC Gene Knockout System For Penicillium Italicum

Penicillium italicum caused large batch of citrus fruits rot during postharvest storage and transportation,which results in huge economic loss.In order to control the disease effectively,it is necessary to explore the pathogenesis involved.A lot of research have shown that when P.italicum infected citrus fruits,they secreted large amounts of acids to altered the environmental pH,damage the defensive system of host.P.italicum inducing environmental pH through signal transduction pathway of PAL and activate transcription factor PacC via signal transduction thus making response to environmental pH value.By means of genetic conversion technology mediated by Agrobacterium,and the△pacC mutants was obtained by transformation.It is possible to investigate the function of gene pacC of P.italicum and accordingly to make further exploration on pathogenesis of P.italicum.The main research results are as follows:1.Construction of pacC gene knockout vector:Based on fusion PCR,this study design primers with complementary ends.By extension of the PCR product overlapping strand,fused the upstream sequence of target gene pacC,hygromycin B resistance gene and downstream sequence of target gene pacC merge into a fragment.After the recombinant fragment was obtained,the recombinant plasmid ACC3300 was successfully constructed by ligating the recombinant fragment into vector pCAMBI3300 by digestion with PMD19-T as the intermediate vector.2.Establishment and optimization of P.italicum transformation system mediated by A.tumefaciensIn this study,by exploring the influence factors on transformation system mediated by A.tumefaciens,for instance,spores suspension liquid concentration,species of A.tumefacien,co-culture time,co-culture temperature,the addition of Acetosyringone(AS)pre-culture stage,the type of carrier membrane and the concentration of antibiotics,the optimum conditions obtained for the transformation were as follows:the concentration of P.italicum conidia is 1x10~5-1x10~6cfu/mL,mixed with Agrobacterium which was containing knockout vector and pre-induced by AS,then they are coated on Coculture-ingducing(Co-IM)medium with cellophane sheets.After incubation at 25℃for 36 h,remove the cellophane sheets into PDA medium that containing 50μg/mL hygromycin B as well as 200μg/mL cephalosporin,can obtain single colony transformants in large quantity,they are superior transformation system.Transformants selected randomly successively subculturing on PDA medium without antibiotics for five rounds,removed it to selective medium containing hygromycin B,it grown stably and contained hygromycin B gene by verification of PCR,which reveals that it is steady and high efficient genetic transformation system.3.Two △pacC mutant strains were obtainedThrough the mediation and transduction of Agrobacterium tumefaciens,pacC knockout vector that obtained recombinant fragment integrated in P.italicum genome,has got 346 transformants.The transformants were extracted and verified by PCR.Two of these transformants did not contain the pacC gene and contained the hygromycin B resistance gene.It suggests that two△pacC mutant strains have been successfully obtained which is the basis of follow-up research that pacC plays roles and functions in P.italicum infecting citrus.