Construction Of A High-efficiency Genetic Operating System For Purple Spore

Paecilomyces lilacinus is currently in the commercialization of nematode biocontrol fungi.In this study,the new species Purpureocillium lavendulum was used as the starting strain.Firstly,the ku80 gene was knocked out and recovered by the method of homologous recombination technique.Then,the morphological and virulence of the ku80 knockout strain and the complemented strain were determined.After determining that the ku80 knockout strain had the same nematicidal effect as the wild type strain,the gene brlA was knocked out in the wild type strain,ku80 knockout strain and ku80 complemented strain,respectively,to compare the recombination rates.This study laid the foundation for efficient gene knockout of P.lavendulum.The main results of this study were described as follows:1.P.lavendulum ku80 gene knockout and complementationThe plasmid pPK2-neo-GFP was digested and the upstream and downstream homologous fragments of ku80 gene were ligated at both ends of the neo gene by In-fusion.Through Agrobacterium tumcfaciens mediated transformation(ATMT)the ku80 gene of P.lavendulum was replaced with gene neo by homologous recombination.Transformants were screened by G418 and the ku80 knockout strain was obtained by PCR screening and confirmed by Southern blot.In addition,the ku80 full-length gene was ligated into the plasmid pPK2-bml-GFP follow bml gene(anti-fungicide benomyl resistance gene).The constructed plasmid was transformed into ku80 knockout strain by ATMT.Transformants were screened by benomyl and the ku80 complemented strain was screened by PCR and confirmed by Southern blot.2.Knockout ku80 did not influence the growth and nematicidal activity of P.lavendulumBy comparison,the results are shown that the colony morphology and growth rate of these three strains showed consistency.At the same time,through the comparison of ku80 knockout,complemented and wild type strain of C.elegans infection effect,determine the ku80 knockout and complement strain of nematodes with virulence are same as wild type strain.Thus,knockout ku80 did not influence the growth and nematicidal activity of P.lavendulum and ku80 knockout strain can be used as background strain for further research.3.Functional verification of ku80 knockout strainUsing the brlA gene as an example,to verify whether knockout ku80 can improve the recombination rate in P.lavendulum.We used the OSCAR technology which was constructed by Paz Z and improved by professor Fang Wei-Guo to construct the brlA knockout plasmid.Using agrobacterium tumcfaciens mediated transformation,the brlA knockout plasmid transformed into P.lavendulum wild-type,ku80 knockout and ku80 complemented strains,respectively.After comparing knockout efficiency of brlA,wetA and abaA(these two plasmids were constructed by others in the research group)in these three strains,we found that the knockout of ku80 gene can improve the recombination efficiency of other genes in this fungus.The main innovation of this paper:For the first time in P.lavendulum to destruct the nonhomologous end joining pathway which lead to gene targeting inefficiencies.The specific operation through knockout of the ku80 gene to disrupt vital NHEJ pathways,it is mainly because of the Ku protein complexes are crucial to NHEJ,so that the probability of homologous recombination will increase.The OSCAR method in constructing the brlA knockout plasmid is firstly used by Paz Z and improved by professor Fang Wei-Guo of Zhejiang University,we use the method of ATMT to transform the P.lavendulum.