Screening And Analysis Of Insecticidal Activity Of Protein From Bacillus Thuringiensis Against Mythimna Separata(walker) And Ostrinia Furnacalis(guenée)

In recent years, maize’s acreage is the largest and whose cultivation is the most extensive in all crops. Because of global warming and single cropping patterns, plant diseases and insect pests have been increased year by year. Therefore, it is the most importantwork to prevent and control the maize pests. Furthermore, since the chemical control has had many negative effects, biological control is the best way to solve the current problem. Parasporal crystal protein from Bacillus thuringiensis is the main insecticide ingredient. Numerous studies have found that most of the crystal proteins have specific insecticidal activity against Lepidoptera, Diptera, Coleoptera, Hymenoptera, Orthoptera, Mallophaga variety of insects, as well as some nematodes, mites and protozoa. Such that it is widely used in biological control of agriculture in all the world.The insecticidal proteins of Bacillus thuringiensis have insecticidal specificity for a variety of insects, it is beneficial to control pests reasonnally by screening out Bt protein having insecticidal activity to specific insect.In this study, Firstly we optimized the expression conditions of protein in Escherichia coli, we designed two induced temperature, 18℃ and 30℃, three IPTG final concentration, 0.1 mmol/L, 0.5 mmol/L, 1.0 mmol/L. Through repeated study, we found the highest expression level in soluble fraction for Cry1 Bb was 18℃ and 1.0 mmol/L IPTG in final concetration, and the highest in insoluble fraction was 30℃ and the final concentration of 0.1 mmol/L IPTG; The highest in soluble fraction for Cry1 Be was 30℃ and the final concentration of 0.1 mmol/L IPTG, and the highest in insoluble fraction was 30℃,the final concentration of 0.5 mmol/L IPTG; the highest in soluble fraction for Cry9 Ee was 30℃ and the final concentration of 0.5 mmol/L IPTG, and the highest in insoluble fraction was 18℃,the final concentration of 0.1 mmol/L IPTG.The bioassay against Mythimna separata(Walker) of Lepidoptera main maize pests, indicatting that Cry1 Ac, Cry1 Ab, Cry2 Ab, Cry1 Be and Cry1 Bb protein had pesticidal activity against Mythimna separata(Walker), whose LC50 was 5.09, 17.71, 26.75, 27.42 and 43.93 μg/g, respectively; Cry9 Aa, Cry9 Eb, Cry9 Ee protein inhibitted the growth of Mythimna separata(Walker), whose weight inhibition rates were 78.4%, 79%, 86.9%, respectivelyin the concentration of 10 μg/g, and the weight inhibition rates were 92.8%, 95.7%, 96.75% in the concentration a 100 μg/g. Cry1 Ba, Cry1 Ca and Vip3 Aa protein had no significant activity against to Mythimna separata(Walker).The bioassay against Ostrinia furnacalis(Guenée) of the Lepidoptera Pyralidae, indicating Cry1 Bb and Cry1 Be had insecticidal activity to Ostrinia furnacalis(Guenée), whose LC50 was 0.46 g/g and 24.01 g/g, respectively.The new gene ng1 was constructed with expression vector, protein expression and functional verification of protein. Since the p EB-ng1 expression vector whose expression levels was very low under various conditions, so we carried out to replace the vector construct p ET21b-ng1 expression vector, and gained higher expression in the E. coli Rosetta. In addition, we determined biological activity of the protein against Plutella xylostella(Linnaeus), Colaphellus bowringi(Baly), Spodoptera exigua(Hübner), Helicoverpa armigera(Hübner), Mythimna separata(Walker) and Ostrinia furnacalis(Guenée). Through the above test insect screening, we found Ng1 protein against to Plutella xylostella(Linnaeus) and Ostrinia furnacalis(Guenée). The LC50 was 3.168 μg/mL against Plutella xylostella(Linnaeus), and 31.257 μg/g against Ostrinia furnacalis(Guenée). but it had no significant toxic effect on Colaphellus bowringi(Baly), Spodoptera exigua(Hübner) and Helicoverpa armigera(Hübner). It has a certain growth inhibition at a concentration of 320 μg/g against Mythimna separata(Walker).