| Lecanicillium lecanii is an important biocontrol fungus,which can parasite variety of aphids,whiteflies,thrips and other greenhouse pests.When spraying its conidial suspension in the field,a good efficacy indicates that Lecanicillium lecanii is a potential biological fungus.However,the effectiveness of L.lecanii is influence by a variety of factors,especially low virulence and slow kill speed.In this study,an exogenous toxin gene omega-actitoxin-Ar1a(ω-ACTX-Ar1a)was chosen,which is a specific insecticides and does not have any side toxin-effect to vertebrates.Furthermore,this exogenous toxin gene ω-ACTX-Ar1 a was introduced into the wild-type strain of L.lecanii via agrobacterium-mediated method,and the virulence of the transformants was improved.The main findings of this study are as follows:1.The isolation and identification of the dominant parasites of the sucking pests.Single spore isolation were used to separate the infection whitefly,entomogenous fungi was Aschersonia placenta,which identified by morphological and molecular biology method.Compared with the biological characteristics of Aschersonia placenta and Lecanicillium lecanii CA-14,which was the dominant entomogenous fungi of sucking pests.Finally,the CA-14 of Lecanicillium lecanii was choosed to gene transformation and effectiveness evaluation.2.Selection of an exogenous spider toxin gene and its synthesis.Through a thorough literature investigation,the insect-related toxin gene informations were selected in GenBank,compared with large number of comparisons,and the omega-ACTX-Ar1 a gene of Hadronyche versuta was select.The gene encoding region was synthesized in a professional company and ligated into the vector plasmid pUC57,and the recombinant plasmid was transformed into E.coli.The PCR and resistance markers were used to select.The recombinant plasmids were extracted from the selected positive clone and used as the template to PCR amplification in order to obtain large number of toxin gene fragments.3.Construction of expression vector of the toxin gene ω-ACTX-Ar1 a.The promoter PtrpC was amplified from the plasmid pBHt2,and the promoter PgdpA and the terminator TtrpC were amplified from the plasmid pAN7-1.Two recombinant fragments PtrpC-ω-ACTX-Ar1 a and PgdpA-ω-ACTX-Ar1a-TtrpC were constructed by connecting the target gene with different promoters and terminator,which were conducted by the overlapping and falling PCR.After the analysis of the sequence and restriction sites of the recombinant fragment and the vector pBht2,KpnI and HindIII were used as the restriction endonucleases.The recombinant fragment the plasmid pBht2 were digested with the restriction endonuclease,and then the digested fragments were ligated by the T4 DNA ligase to construct expression vectors of the recombinant plasmid Pω and PωT.4.Agrobacterium-mediated method via the transformation of L.lecanii.The Agrobacterium tumefaciens strain AGL-1 was prepared,and the recombinant plasmid Pω and PωT were introduced into AGL-1.The tansformed A.tumefaciens containing the recombinant plasmid Pω and PωT were co-cultured with conidia of the wild-type L.lecanii strain CA-14,and then the target gene was introduced into the genome of L.lecanii induced by acetosyringone.Total 100 different transformants were randomly selected in the medium containing hygromycin B,screened and analyzed for genetic stability.The results showed that 66 of the 100 transformants contained the target gene and the transformation efficiency was 66%.All of the 66 transformants growed normally in PDA medium without hygromycin B.The transcripts of 66 transformants were analyzed by RT-PCR.Among the 66 transformants,20 transformants were successfully expressed at the RNA level.Moreover the relative quantification method was used to confirm the gene copy number.The single copy gene NIF3 was selected as the internal reference gene.Finally 8 single-copy and 5 multi-copy transformants were found in the 20 transformants successfully expressed at the RNA level.5.Evalutation of virulence and efficacy of the different recombinant strains against aphids.The wingless Lipaphis erysimi were tested by being dipped in sterile water(containing 0.1% Tween 80)and conidal suspension of the recombinant transformant and the original strain of L.lecanii.The results showed that the lethal time of Lipaphis erysimi infected by the recombinant transformants was shortened in different degrees.The LT50 of the recombinant transformant Pω-40 and PωT-2 were 4.615 and 4.517 d respectively,and they were shortened by 2.209 d and 2.307 d respectively compared to the 6.824 d of the original strain.The results indicated that the introduction of the exogenous spider toxin gene significantly sped up killing aphids by L.lecanii. |
