| Botrytis cinerea is an important plant pathogenic fungus,meanwhile,Botrytis cinerea is a important Model species,which plays an important role in helping us to understand the interaction of the fungi with the plant.Dissection of gene functions from the molecular biology level will speed up understanding about the fungal biology of Botrytis cinerea and its pathogenisis molecular mechanism.In addition,it will offer new theoretical knowledge to develop an effective method to control Botrytis cinerea.In our study,the technique of the Agrobacterium tumefaciens-mediated transformation by T-DNA insertion of Botrytis cinerea was established.A library of 320 transformants was constructed with high transformation efficiency of over 75 transformants per 1×106 conidia.After screening through pathogenicity assays on the tomato leaves,we obtained some mutants which reduced virulence evidently compared to the wild-type.Hygromycin B-resistant gene(hph)has integrated into the genome of these mutants since PCR analysis.In our study,we also amplified the DNA flanked sequence which is the integration sites of the T-DNA by using high-efficiency thermal asymmetric interlaced PCR(TAIL-PCR).We blast the obtained DNA sequence with the data bank in the NCBI,and we founded that a large proportion of the gene inserted by T-DNA encode unknown protein.The inserted gene of the mutant No.10 encoded a protein that is similar to the multiprotein-bridging factor.All these transformants were heterokaryons(as is normal in Botrytis cinerea),since PCR with specific primer pairs.Finally,homokaryotic derivatives of the mutant No.7 and No.136 were obtained through the single-spore isolates.Among these transformants,the mutant No.136 is a special transformant,then the mutant No.136 was analysised by Southern blot.The result showed that the mutant No.136 contained only one copy,as a result of the low concentration the DNA,the copy is very vague.Through using RT-PCR,the gene of the mutant No.136 expression was analysised,and proving that the gene integrated has been inactive.The gene integrated of the mutant No.79 encodes a protein which is similar to protein phosphatase 2A regulatory B subunit.Because of the insertion of T-DNA in the mutant No.79,the phenotype of this mutant was different from the wild type strain.Its phenotypes include that slow growth rate,reduced sporulation capacity,decreased pathogenicity on tomato,and sparse mycelium.We cloned this genomic sequence,then through LR reaction,and constructed binary vector which has been transformed into the A.tumefaciens for gene compllementary and function analysis.The mutant No.79 is sensitive to Calcofluor White(800?g/mL),but the wild type and the complemented transformant is resistance to the Calcofluor White on the PDA medium supplemented with 800?g/mL Calcofluor White.When Growing on solid media supplemented with 0.8mg/mL concentrations of cantharidin,effect of cantharidin on colony growth of the mutant No.79 is not different from the the wild type,because of the mutant No.79 is heterokaryons.The mutant No.7 was significantly impaired in vegetative and pathogenic development:they are impaired in growth,morphology,conidiation,and are unable to penetrate unwounded plant tissue.The gene,encoding an unknown protein of Botrytis cinerea,was cloned and characterized.Using BLAST searches,we found this protein is low homologous to that of any filamentous fungi.Finally,we should desiged many experiments that about the mutant No.7 resistance to medicines,and the objective of these experiments was to find out the functional features of the unknown gene.The results of these experiments are that:the growth and colony morphology of the mutant No.7 is similar to the wild type which are exposure to the osmotic stress.In addition,the mutant No.7 are unable to penetrate unwounded plant tissue,and exhibit sensibility to Calcofluor White.Finally,we concluded that the feature of the label gene for the mutant No.7 may be related to the growth,pathogenicty as well as the function of chsIII. |
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