Identification And Characterization Of A T-DNA Insertion Mutant, Mu10, From Beauveria Bassiana

Entomopathogenic fungi are important biocontrol agents against agricultural pest and vectors of human pathogens.Compared to other biotic agents,fungal insecticide actively invade the target pest insect and easily cause insect epidemic in the narture.In addition,the target insect hardly develop resistance to the fungal agents.Thus,the fungal agents have been intensively investigated to control pest insect.However,littler is known about the molecular mechanisms involved in fungal development and pathogenicity,which to some extent impeded the application of mycoinsecticide.It is necessary to elucidate the mechanisms of for development of mycoinsecticede.Beauveria bassiana,the important entomopathogenic fungus,kills a variety of coleoptera and plant-sucking bugs,has been universally used as a biological agent to control agricultural pest insects.A T-DNA insertion collections of Beauveria bassiana was constructed by Agrobacterium tumefaciens-mediated transformation.A T-DNA insertion mutant,Mu10,with significant reduce in virulence anginst Myzus persicae and evident difference in colony morphology was identified.The mainly results are as follows:1.Identification of the T-DNA insertion mutant related to virulenceA large number of random T-DNA insertion transformants of B.bassiana were obtained by Agrobacterium tumefaciens-mediated transformation.A T-DNA insertion mutant Mu10 presenting deficience in virulence was screened through bioassay.After inoculation of conidia suspension at a concentration of 5×10⁷ conidia/ml,the accumulative mortality of Myzus persicae caused by the mutant Mu10 was decreased by approximately 80%compared to that treated with the wild-type strain.2.Analysis of biological characteristics in mutant Mu10On rich nutrient media（SDAY）,growth of the mutant was slower than that of wild type strain under normal condition.Moreover,growth of the mutant was heavily inhibited under the hyperosmotic stress condition（containing 1 mol/L NaCl）.On the minimal media（CZM）,no significant difference in growth was observed between the wild-type and Mu10 under normal condition or hyperosmofic stress condition.At temperature of 32℃,the mutant Mu10 showed a obvious reduction in growth on SDAY media compared to the wild-type strain,while no significant difference of growth on CZP media was observed beteen the two strains.When grown on SDAY media,the conidial yield of Mu10 was significantly decreased by 58.70%and 80.53%under normal conidition and hyperosmotic stress conditions（containing 1 mol/L NaCl） compared to the wild-type strain,respectively. Meanwhile,the biomass of the mutant was reduced by 22.62%and 13.71%in SDY liquid media under normal condition and hyperosmotic stress,respectively.When cultured on CZM,no significant difference was observed in conidial yield and biomass between Mu10 and the wild-type strain under normal condition or hyperosmotic stress. Conidia germination was seen to be 35.33%（SDAY）and 36.23%（CZM） less in Mu10 as compared to wide type at 16 h after inoculated.When grown under nomal conditions（1/4 SDY） and hyperosmotic stress conditions（containing 0.6 mol/L NaCl）,both glycerol and arabitol contents in mycelia of the mutant were a bit lower than those of the wild-type strain,but no significant difference in glycerol and arabitol accumulation was found between the two strains.The content of trehalose showed a significant decrease in mutant Mu10 when exposed to hyperosmotic stress conditions,but there was no difference between the mutant and the wild type under the normal conditions.3.Isolation of T-DNA tagging sequenceTo abtain T-DNA insertion flanking sequence,chromosome walking was carried out by YADE method and around 1.9 kb was cloned.BLAST result showed The tagging sequence showed no homologous to any sequence in GenBank using BLAST rearches, suggesting that the sequence is an unknown gene or an unencoding region.4.Construction of homologous recombination verctorpPZPtk8.10 containing a bar cassette as a positive selection maker,and HSVtk cassette（encoding thymidine kinase that converts nucleoside analogs such as 5-Xuoro-2-deoxyuridine[F₂dU]to a compound toxic to fungi） as a negative selection marker on the T-DNA was a master vector.The targeted sequence replacement vector was construced by flaning the 5’ end and 3’ end fragment of the tagging sequence to the 5’ and 3’ end of the bar cassette in vector pPZPtk8.10.The resulting vector was delivered into transform B.bassiana.Targeted sequence replacement mutants can be selected by both the positive（PPT） and negative（F₂dU） selection agents.Two homologous recombination mutants,T5 and T7,was obtained which can be used for further rearches on molecular mechanisms of fungal development and pathogenicity.