Abstract
Most crop production practices rely on the use of chemical insecticides to combat crop diseases, caused by microbes and insect pests, and increase production yields. However, the misuse of chemical insecticides has resulted in the outbreaks of insect pests as well as environmental pollution. Furthermore, the controlled insect pests develop resistance to the insecticide rendering their subsequent applications ineffective. In addition, the use of chemical insecticides for insect pest control makes it difficult to grow crops in a sustainable and environmentally sound manner. Therefore, the protection of plant crops against insect pest damage without damaging and polluting the environment is essential. Candidate alternative is the use of proteins encoded by the toxin complex (tc) genes which are insecticidal toxins that will serve as microbial based insecticides as well as deployment of transgenic crops. Toxin complex (tc) is a tripartite high molecular weight complex and is known to be insecticidal towards insect species belonging to Coleoptera, Hymenoptera and Lepidoptera orders. This group of toxins may be used as alternatives to the toxins derived from the extensively used Bacillus thuringiensis for deployment of transgenic plants as well as usage as microbial based insecticides. The present study aimed at finding relatively low-cost and environmentally friendlier options to control and manage insect pest damage and their subsequent diseases. A codon-optimised insecticidal gene (tccZ) identified in Pantoea ananatis strain MHSD5 (a bacterial endophyte previously isolated from Pellaea calomelanos) was used to transform Escherichia coli BL21 (DE3) to allow expression of the tccZ gene and to test its larvicidal activity against Tenebrio molitor larvae. Furthermore, the bacterial cell suspension of Pantoea ananatis strain MHSD5 was used to assay its larvicidal activity against T. molitor and Galleria mellonella. Herein, we report the success of cloning of tccZ gene into pET SUMO vector and ultimately the transformation into E. coli BL21(DE3). However, despite conducting a time course of expression to determine optimal conditions for expression of tccZ protein, we were unable to detect tccZ protein expression and therefore could not purify and characterize tccZ expressed protein. Escherichia coli BL21(DE3) transformed with tccZ gene exhibited larvicidal activity against insect larvae. The injectable toxicity of the E. coli cells means that the tccZ protein may have potential to be developed as a biopesticide as well being used in deployment of transgenic plants. Keywords: tccZ gene, toxin complex, Pantoea ananatis strain MHSD5, pET SUMO, E. coli BL21(DE3), bacterial endophyte, Tenebrio molitor, Galleria mellonella, insect pest control.
M.Sc. (Biotechnology)