Construction Of Genetic Transformation System For Nomuraea Rileyi

There are some important pests in the insect family Noctuidae (Insecta: Lepidoptera). Nomuraea rileyi (Farl.) Samson,an important entomogenous fungus, is a specific pathogen of noctuid insects, and as an important mortality factor, it sometimes causes epizootics in noctuid populations, resulting in substantial suppression to the outbreak of the pests. As a fungal biocontrol agent, it inevitably experiences a latent period for infection development in host hemocoel and kill insects much more slowly than chemical insecticides. In addition, it has high environment requirements. These disadvantages have often discouraged the use of its fungal formulations in insect pest control. However, genetically modified technology can improve its virulence and environment resistence to meet market need. Insect chitinase is a major hydrolyzing enzyme which is produced during fungal infection. Chitinase can degradate the materials with chitin that play the role of physical or chemical barriers for insects and reduce the defence capacity of the host, helping infection process happen. Scorpion venom has strong and specific toxicity to insect. With high specificity and smaller coding gene, scorpion venom is an ideal material for study of protein engineering, neurobiology, immunology and ion channels. In the present study, two foreign genes, bbchit1, a Beauveria bassiana chitinase gene, and aait, an insect-specific neurotoxin peptide gene from North African scorpion, Androctonus australis, were transferred into Nr03 and Nr09, two wild-type strains of Nomuraea rileyi, to construct transgenic strains, respectively.The plasmid pbarGPE1-aait containing selection marker and target gene was constructed firstly in this experiment. The plasmid was used as intermediate vector to amplify a gene fragment with strong promoter, terminator and aait gene. The GpdA-aait-TrpC gene fragment was inserted into pRNA-hygro-U6.1 plasmid and constructed a new plasmid, pRNA-hygro-U6.1-aait, containing the hygro gene and aait gene. The optimal inhibitory concentrations were selected by drug resistance assays with conidia. The best inhibitory concentration for hygromycin was 80μg/ml, and the best inhibitory concentration for benomyl was 2μg/ml. With blastospore transformation method, Nr03 was transformed into Nr03-A, a monovalent transgenic strain containing scorpion venom gene aait, while Nr09 was transformed into Nr09-B, another monovalent strain containing Beauveria bassiana chitinase gene bbchitl by use of a vector pbenGPS3-bbchit1. The result of bioassay showed that virulence of the two transgenic strains on the larvae of the Chinese cotton boll worm, Helicoverpa armigera, was significantly higher than that of the respective wild-type strain. LC₂₀ of Nr03-A was reduced by 13.46 times and LC₂₀ was shortened by 44.4% as compared to that of Nr03 while LC₂₀ of Nr09-B was reduced by 10.85 times and LC₂₀ was shortened by 42.2% as compared to that of Nr09, indicating that the overexpressed transgenic aait and bbchitl both can significantly increase the virulence of the transformants.