Gene Transformation And Bioassay Of Lecanicillium Lecanii(Zimmerman) Viegas For Increasing Virulence To Aphis

Lecanicillium lecanii, an entomopathogenic fungus with a wide range of host insect species, ishighly pathogenic to aphid，aleyrodid, scales and thrips, but its killing time needs to be shorten.Scorpion venom, a group of polypeptides consisting of60-70amino acids, is highly toxic to insect pests.This research tried to clone the BmKIT gene encoding the protein which leads to quick shrinking andanaesthetization of insect pests from Buthus martensii Karsch, and then introduce it into a wild strainof L. lecanii by Agrobacterium-mediated transformation after connecting with TrpC, a strong fungalpromoter, to get a genetically engineered L. lecanii strain which may kill insect pests quickly. The majorresearch results obtained in this study are as follows:1. Cloning of the scorpion venom gene: the BmKIT gene encoding scorpion venom was cloned bycutting telson of B. martensii, extracting total RNA from the telson, synthesizing the first chain ofcDNA by reverse transcription using the total RNA as template, designing the primers based on DNAsequence reported by previous researchers, and then PCR amplification. DNA sequence analysisindicated that it was highly homologous with that reported in previous researchers, and it was213bp inlength.2. Construction of expression vector: author ligated TrpC with BmKIT and then got TrpC-BmKITgene, a fragment with580bp as indicated by electrophoresis, after digesting BmKIT gene and TrpC genegotten from pBHt2plasmid with SacI/BamHI and HindIII/SacI, respectively. The TrpC-BmKIT genewas transferred into competent cells of E. coli after it was inserted into T-DNA region of plasmidpBHt2.3. Transformatiom: the pBHt2carried TrpC-BmKIT was introduced into Agrobacterium AGL-1,which was then used to transform L. lecanii CA14.4. Identification of transformants: the percentage of negative transformants was8%as indicated byPCR analysis of the DNA extracted with CTAB from50putative transformants. RT-PCR analysis ofpositive transformants showed that BmKIT could be transcripted regularly.93.5%of the transformantscould grew well in PDA plates amended with hygromycin B at concentration after being successivelysubcultured in PDA plates for20generations, indicating that most of the transformants were stablegenetically.5. Assay for biological activity of the transformants: transformant T-1, T-2and T-3carrying BmKITwere tested for their toxicity to Lipaphis erysimi by dip-molding method. Results indicated that thetransformants were highly virulent to the pests in comparison with their parental strain CA14. Mortalityof the3transformants were46.7%-48.9%, which is more higher than their parental strain CA14, at the4^thday after inoculation. Mortality of the3transformants were100%, and76.7%for their parentalstrain CA14, at the6thday after inoculation. And most of the dead insect bodies gave rise to significantwhite fungal mold.