Regulatory Mechanisms, Protein Expression and Biological Activity of Photolyase Gene from Spodoptera littoralis Granulovirus Genome.

One of the most important factor that affects the efficient using of baculoviruses as a biopesticide is their sensitivity to UV irradiation. In this study, a photolyase gene (phr) of 1.4 kbp DNA fragment was cloned and characterized from Spodoptera littoralis granulovirus, an Egyptian isolate (SpliGV-EG1). A sequence of 466 amino acid were deduced when the gene was completely sequenced with a predicted molecular mass of ~ 55 kDa. Transcriptional regulation analyses revealed that phr transcripts were detected early at 6-h post-infection (hpi) and remained detectable until 72 hpi, suggesting their transcriptional regulation from a putative early promoter motif. An approximately ~ 55 kDa protein fragment was expressed from phr-induced bacterial culture and detected by SDS-PAGE and western blotting. In addition, direct exposure to UV irradiation resulted in a twofold decrease in SpliGV-EG1 occlusion bodies activation compared with Spodoptera littoralis nucleopolyhedrovirus (SpliNPV) occlusion bodies which decreased with about 129-fold after exposure to UV irradiation based on median lethal concentration value (LC50). The obtained results suggested that the presence of photolyase gene possibly alters the inactivation of SpliGV-EG1-occluded bodies by UV irradiation. These results support the role and application of the photolyase protein to improve the damaged DNA repair mechanism as well as resistance of SpliGV to UV light inactivation.

Characterization, cloning, expression and bioassay of vip3 gene isolated from an Egyptian Bacillus thuringiensis against whiteflies.

Throughout the vegetative life of Bacillus thuringiensis, vegetative insecticidal proteins (Vip) are produced and secreted. In the present study, the vip3 gene isolated from Bacillus thuringiensis, an Egyptian isolate, was successfully amplified (2.4 kbp) and expressed using bacterial expression system. The molecular mass of the expressed protein was verified using SDS-PAGE and western blot analysis. Whiteflies were also screened for susceptibility to the expressed Vip3 protein (LC50). In addition, ST50 was determined to assess the kill speed of the expressed Vip3 protein against whiteflies compared to the whole vegetative proteins. The results showed that the potency of whole B. thuringiensis vegetative proteins against whiteflies was slightly higher than the expressed Vip3 protein with 4.7-fold based on LC50 value. However, the ST50 parameter showed no significant difference between both the B. thuringiensis vegetative proteins and the expressed Vip3 alone. The results showed that the vip3 gene was successfully expressed in an active form which showed high susceptibility to whiteflies based on the virulence parameters LC50 and ST50. To our knowledge, this study showed for the first time the high toxicity of the expressed Vip3 proteins of B. thuringiensis toward whiteflies as a hopeful and promising bio-control agent.