Prokaryotic Expression Of Cry1aa-cpti Fusion Protein And Its Litura Insecticidal Activity Toward Prodenia Litura Fabricius

Prodenia litura Fabricius seriously affect the agricultural and forestry production as overeating pest, a large number of chemical pesticides are used which cause environmental pollution, serious harm human and animal health, besides easy to aspirin resistant.However, bio-insecticides are high cost, insecticides slow, poor stability. So find an bio-insecticides which faster, better stability, lower cost, use bio-pesticides first, then use chemical pesticide control strategies, give full play to biological insecticides and chemical pesticides to their respective advantages, reduce the quantity of chemical pesticide, may be a better way to integrated control the Prodenia litura Fabricius.Bacillus thuringiensis (BT) is currently the most widely used microbial pesticides.The gene encoding insecticidal crystal protein (Insecticidal Crystal Proteins, ICPs) named as cry genes, crylAa gene encoding protein Cry1Aa which has insecticidal activity against lepidopteran, the protein in the practical application is poor stability, slow and affected by environmental-impact.Cowpea trypsin inhibitor (cowpea trypsininhibitor, CpTI)come from the edible parts of cowpea, with a broad spectrum insect resistance and insect tolerance to its easy features, has been used as an important candidate gene in insect-resistant plant genetic engineering,such as tobacco,rice,cotton,tomatoes and other crops.In the classification CpTI belong to Bowmun-Birk family, a large number of medical studies have shown that proteins of the family are no harm to human and animals.crylAa and cpti genes based on advantages of the two genes are fused expressed, using in vitro recombinant DNA techniques, a large number of fusion protein were expressed, then study the insecticidal activity against Spodoptera litura.Firstly use the PBI121 plasmid as template, PCR amplificated the crylAa-cpti gene. Then connected crylAa-cpti gene with the pMD18-T Simeple cloning vector, sequencing results showed that the gene was successfully connected with pMD18-T Simeple cloning vector. Expression vector was constructed by two steps, firstly used NdeⅠand SalⅠenzyme to digest pET-30a vector, then used NdeⅠand SalⅠenzyme to digest pMD18T Simeple vector. Connect the obtained gene fragments with NdeⅠ/SalⅠdigested pET30a vector to construct the expression vector, transformed to BL21(DE3).IPTG was used to induced the recombinant engineering bacteria to express with Cry1 Aa-CpTI fusion protein inclusion body, and then used ultrasonic crushing, inclusion bodies washing buffer washed,then inclusion bodies were purified.Specific band of expression products showed in 80.73kDa by SDS-PAGE.The temperature (27,29,31,32,35,37,39℃),induction time (1,2,3,4,5,6 h) and IPTG concentration (0.4,0.6,0.8,1.0,1.2 mmol/L) were optimized to determine the optimal conditions, the results showed that induction of expression at 37℃,1mmol/L IPTG induced 3h,the amount of fusion protein was highest. Cry1 Aa-CpTI fusion protein stability analysis shows that at 37℃, the fusion protein was not degradaed within 4 days,5 days began to degrade.Indoor activity assay showed that, CrylAa-CpTI fusion protein on the 2nd instar larvae of S. litura have antifeedant effect, 1mg/mL Cry1Aa-CpTI fusion protein, when insecticide-treated time is 24h, antifeedant effect was 83.8%;insecticide-treated time is 48h, the average mortality rate 20%; insecticide-treated time is 72h,appeared the phenomenon of death, with an average mortality rate was 70%.Emamectin benzoate (emamectin benzoate), and chlorpyrifos are commonly used in the field as litura pesticides, LC50 were determined by feeding mixed with toxic emamectin benzoate and chlorpyrifos on the 2nd instar larvae of S. litura:handle 24h,48h and 72h-hour LC50:insecticide-treated time was 24h, LC50 emamectin benzoate was 4.1363μg/L, LC50 of chlorpyrifos was 273.4257μg/L; handle 48h, LC50 of emamectin benzoate was 3.2784μg /L, LC50 of chlorpyrifos was 130.4301μg/L; handle 72h,LC50 of emamectin benzoate was 0.7279μg/L, LC50 of chlorpyrifos was 115.1665μg/L.Fusion protein with Cry1 Aa-CpTI for 2 instar larvae of S. litura 24h, and then measured emamectin and indoor chlorpyrifos toxicity. Handle 24h, LC50 of emamectin benzoate was 0.8257μg/L, decreased 80.01%, chlorpyrifos 88.3617μg/L, decreased 67.68%; handle 48h, LC50 of emamectin benzoate was 0.4083μg/L, decreased 87.55%, chlorpyrifos 45.0294μg/ L, decreased 65.48%; handle 72h, LC50 of emamectin benzoate was 0.2242μg/L, decreased 69.20%, chlorpyrifos 31.6220μg/L, decreased 72.54%. Handle 24h,48h and 72h, LC50 of emamectin lower than the average alone 78.92%,68.57% of chlorpyrifos on average lower.Litura indoor pesticides test showed that:Cry1Aa-CpTI fusion protein has strong antifeedant effect to Prodenia litura Fabricius, such as firstly used the fusion protein and Cry1Aa-CpTI to treat with 2nd instar Larvae, The average use of emamectin benzoate and chlorpyrifos pesticides was reduced by 78.92% and 68.57%.when used alone can achieve the same effect. Therefore, this study firstly use prokaryotic expression of the fusion protein Cry1 Aa-CpTI to develop a new biological pesticides, the developed formulations can be used in conjunction with chemical insecticides, firstly applicated the biological pesticides, further use chemical insecticides to Spodoptera litura insects; the strategy can significantly reduce the use of chemical pesticides, while reducing the resistance, to achieve double effect of environmental protection and prevention.