Prokaryotic Expression Of Bt Insecticidal Proteins And Their Toxicities To Bradysia Difformis

In this study,8 wild Bacillus thuringiensis(Bt)strains were screened from Purple mountain,and Bt strain 23-5 with good insecticidal activity against Bradysia difformis was obtained.At the same time,the strain was identified by morphological identification,physiological and biochemical identification,16 S r DNA identification and identification of insecticidal protein genotypes.The prokaryotic expression of insecticidal protein contained in the strain was subjected.These results will provide a theoretical basis for the development and application of Bacillus thuringiensis microbial pesticides.The specific research results were as follows:1.Insecticidal activity of 8 Bt strains against Bradysia difformis was tested by stomach poison method.The results showed the corrected mortality rate of 23-5 was highest,reached 84.48%,and the lowest was 43.11%.The inhibitory effect test on mushroom hyphae showed the effect on the growth of Pleurotus ostreatus,Auricularia auricula,Agrocybe aegerita and Agaricus bisporus.8 Bt strains inhibited the growth rate of four edible fungi hyphae in different degrees,but Bt strains did not affect the overall growth of edible fungi,except Auricularia auricular.Therefore,the following tests were performed on the Bt strain 23-5.2.The identification test of 23-5 was carried out by morphological identification,physiological and biochemical identification and 16 S r DNA identification,and the type of insecticidal proteins contained in strain 23-5 was determined by PCR-RFLP identification method.The results showed that the strain contained crystal protein,the physiological and biochemical identification and 16 S r DNA identification also showed that the strain was Bacillus thuringiensis.PCR-RFLP identification showed that the strain contained the insecticidal proteins genotype combination cry4/10 + cry11 + cyt1 + cyt2.3.Primer design of each insecticidal protein genes showed that the lengths of cry4 B,cry10,cry11,cyt1 and cyt2 genes were 1470 bp,2043 bp,1941 bp,750 bp,and 792 pb,respectively.Cloning vector and expression vector of each genes was constructed separately for PCR verification and double enzyme digestion verification.The bioinformatics analysis of the insecticidal protein genes indicated that the Cry4 Ba,Cry10Aa,Cry11 Aa and Cyt2 Ba proteins were hydrophilic proteins,and the Cyt1 Aa protein was a hydrophobic protein,we also obtained its amino acid composition,physicochemical properties and conserved domains,respectively.4.Each insecticidal protein expression vector genes was expressed and purified.After induction by IPTG,12% SDS-PAGE results indicated that the target proteins Cry4 Ba,Cry10Aa and Cry11 Aa were mainly present in the precipitate which in the inclusion body form.They required a refolding method to re-dissolve the target protein,then carried out the Ni column affinity purification.The target protein Cyt2 Ba was mainly present in the supernatant and expressed in a soluble form,which can directly carry out the Ni column affinity purification;the expression of the target protein Cyt1 Aa was not obvious.The sizes of the fusion proteins Cry4 B,Cry10,Cry11 and Cyt2 were 70 KD,84KD,88 k D and 70 k D,respectively.5.The target proteins of each engineering strain were separately extracted.The protein concentration was determined by the Brandford method,then the toxicity test of each target proteins was measured.The protein Cyt2 Ba had the highest insecticidal activity and the corrected mortality rate was 70.18 %.The protein Cry4 Ba,Cry10Aa and Cry11 Aa had no significant difference in the insecticidal activity of the mosquito,the corrected mortality rates were 33.33-35.09 %.