Functional Characterization Of Four Septin Genes And Construction Of Markerless Transgeneic Strains Expressing Insecticidal Protein Vip3Aa1 In Beauveria Bassiana

Beuaveria bassiana is a classic entomopathogenic fungus that has been widely applied for insect pest control in agriculture and forestry. In fungal insecticides based on B. bassiana, active ingredients are formulated conidia which are sensitive to outdoor stresses, sush as high temperature, solar UV irradiation, and applied agrochemicals. This makes it necessary to explore stress-resistant genes for genetic improvement and maintenance of candidate strains. Fungal insecticides with improved virulence or increased stress tolerance gained by genetic engineering often harbor an exogenous herbicide-resistant gene marker, which is the source of an environmental risk. This study sought to elucidate the roles of four septio genes in mediating the fungal development and responses to various chemical and environmental stresses and to probe an approach to constructing markerless transgenic strains that expression an exogenous insecticidal protein in B. bassiana. The results are summarized below.Functional characterization of four septin genes in B. bassiana. Septins are a family of highly conserved GTP-binding proteins and fall in the superfamily of Ras GTPases. The B. bassiana genome harbors four septins, namely BspA/B/C/D which share a sequence identity of more than 79% with the homologues in other 12 filamentous fungi. Single bsp deletions exerted differential effects on the fungal growth, development and conidiation. Conidial germination was accelerated in all the Absp mutants, concurring with delayed septation and reduced septa in germe tubes and hyphae. Their colony growth was suppressed on different carbon/nitrogen sources, particularly on the source carbon source of fructose. The bsp deletions resulted in remarked reductions of conidial yields during normal cultivation in rich SDAY, a standard medium for fungal entomopathogens. Conidial yields on days 4-7 were decreaed by 80-89% in AbspA,49-67% in AbspB, 60-76% in AbspC and 10-39% in AbspD. In addition, some of the deletion mutants became more sensitive to the cell wall stressor Congo red but more tolerant to two oxidants and two osmotic agents during colony growth and conidial germination. Their conididial theromotolerance and UV-B resistance were also differentially changed. However, similar virulence to Galleria mellonella larvae was oberserved in all the deletion mutants and their control strains (wild-type and complementary mutants). These results indicate prominent roles of four septins in the fungal germinmation, hyphal septation and asexual development and their significance for the fungal adaptation to diverse habitats of various insect pests.Construction of markerless B. bassiana transgenic strains with uridine auxotrophy. Function loss of orotidine 5-phosphate decarboxylase (Ura3) has been shown to cause uridine auxotrophy in B. bassiana because it catalyzes uridine biosynthesis. The enzyme-coding gene ura3 was thus used as an endogenous safe marker to replace environmentally risky herbicide-resistant bar marker for construction of markerless transgenic strains expressing the insect midgut-specific insecticidal protein Vip3Aal in B. bassiana Aura3. This was achieved by the use of a dual plasmid vectoring both vip3Aa1 and uras3, which was supposed to restore uridine biosynthesis. As revealed by PCR analysis, the two genes were expressed in most of putative transformants (BbHUV). Further quantitative real-time PCR analysis demonstrated higher transcript levels of vip3Aal in all PCR-verified transformants than in a transformant previous constructed to express the same target gene. The expression levels of the target protein in three selected transformants were also higher than that in the previous transformant based on enzyme linked immunosorbent assay (ELISA). These results highlight a feasibility to construct markerless transgenic strains using ura3 as an endogenous uridine auxotrophy marker.