Cloning, Expression Of Novel CRY Gene From Bacillus Thuringiensis And Exploation Of Insecticidal Function

The control of insect pests for agriculture is always important problem in China. A large number of chemical pesticides was used to solve the problem. However, the abuse of chemical pesticide, had great harm to human health and the environment. At the same time, the pests had resistant, we had to use more chemical pesticide, increasing the cost of prevention and treatment, pollution control. To solve these problems, biological control was the one of the effective ways. In the past 60 years, because it was safe to the environment human and animal, and it's residue-free, Bacillus thuringiensis (referred to as Bt), as a good bio-pesticides, it played an important role in pest control, it has developed into the largest global production, the application area of Bt was the most extensive microbial pesticides.Bt was a spore forming Gram-positive bacteria, and its parasporal crystal protein is toxic to a variety of insect pests. Different Bt proteins with different agricultural pests have different biological activity, the insecticidal spectrum is specificity, that required us to discovery of new genes and explores a new activity.First of all, we used PCR-RFLP identification of Bt strains S6, found that there may had cry1A, cry1E, cry1K gene.we designed full-length genotype cry1Ah primers to amplification by S6 strain plasmid. Cloned cry1Ax gene and expressed Cry1Ax proteins in E. coli., tested biological activity with diamondback moth (Plutella xylostella (Linnaeus)) and the Asian corn borer (Ostrinia furnacalis (Guenée)). It had high toxicity, LC50 were 0.6990μg ml-1, 5.0939μg mg-1. with cotton bollworm(Helicoverpa armigera (Hubner)), it had significant weight inhibition, LWC50 were 17.4908μg ml-1.The bioactivity of protoxins of Cry1Ba3 and Cry1Ia8 expressed in Bt and E. coli respectively, were performed against the larvae of P xylostella (L.) strain with high resistance to Cry1Ac toxin. The bioassay result demonstrated that Cry1Ba3 and Cry1Ia8 were high toxicity against resistant larvae of P. xylostella (L.). The 50% lethal concentrations (LC50s) were 0.2175,0.6706μg ml-1 separately; Toxicity of Cry1Ba3 was three folds as much as that of Cry1Ia8. The mixture of Cry1Ba3 and Cry1Ia8 exhibited intense insecticidal activity with 0.4375μg ml-1 of 50% lethal concentration. The calculated synergistic factor suggested that it neither was obvious synergism, nor antagonism between Cry1Ba3 and Cry1Ia8. Statistical analysis from the bioassays of resistant and susceptible strains indicated that the two protoxins didn't have cross resistance with Cry1Ac.On the use of PCR-RFLP can not be identified, the strain SC7-D1, two-step PCR method was used, cloning out a new gene fragment, the total length was 1414bp, translated into protein and BLAST, the results show that it had a typical three-domain in cry-type genes, Cry8Ka2 was the highest similarity with it, 46 percent, it may be a new holotype gene.We used a variety of Bt protein in the laboratory, to test biological activity with Clostera anachoreta and Lymantria dispar. Lymantria dispar: Cry2Ab 0.39ppm mortality 16.7%, 1.563ppm mortality was 20%, Cry1Ah 1.563ppm at the highest levels of mortality for 60%, Cry1Ba 25ppm highest mortality was 56.7%, Cry2Ad 0.39ppm of the mortality rate was 43.3%. Clostera anachoreta: 100ppm concentration, Cry1Ia, Cry2Ab, Cry1Ah, the mortality rate was 20%, 40%, 80%.Purified protein Cry2Ab4, and used the method of cutting SDS-PAGE, Cry2Ab4 antibodies were prepared to study the mechanism of insecticidal Cry2Ab4.