Study On Expression Of Bt Toxin Protein Gene In Engineered Bacterium And Toxicity Of Bt Toxic Protein To Insect

In recent years significant progress has been made on Bt genetics and molecular biology, especially the Bt toxin protein gene in engineered bacterium. This paper in which Bt toxin protein cloned in engineered bacterium was studied included three parts. In the first part, the expressions of seven Bt toxin protein genes cloned in engineered E. coli were studied. Engineered bacteria were cultured in LB culture medium for 24 hours at 37℃, with the shake speed of 150~200rpm. After that, collected bacterium was frozen and thawed repeatedly, then followed by breaking up with ultrasonic, centrifugation and SDS-acrylamide gel electrophoresis. The results showed that ECE52 has the highest expression level of toxin protein among seven engineered bacteria. ECE128 and ECE125 were next to ECE52. The optimal cultural conditions of ECE52 were explored through culturing the bacterium in test tube, flask and fermentor respectively. The results indicated that ECE52 would grow best during 8~12h cultured at 37℃. Bt toxin protein was isolated from engineered bacterium by gel filtration on Sephacryl S-200 and wished with Na2CO3/NaHCO3 solution. The purified protein showed one band on SDS-acrylamide gel electrophoresis, and the molecular weight of it was 133 KDa. When the purified Bt toxin protein was provided as food, the mortality of the fourth instars larvae of silkworm would reach 100% in 24h.The second part was the study on toxicity of the expressed protein of engineered bacterium. We found that the toxin protein expressed by ECE52possessed toxicity to the fourth instars larvae of silkworm, whereas the comparison bacterium was innocuous. The mortality of silkworm would reach 100% in 72h when fresh leaves treated by 0.125mg/ml expressed-protein were provided as food for only 12h. All tested silkworm would die in 48h or 24h with 0.25mg/ml or 0.5mg/ml expressed-protein. The LC50 value at 24h, 48h, and 72h was 0.101mg/ml, 0.045mg/ml, 0.028mg/ml, respectively. And the LC95 value was 0.325 mg/ml, 0.173 mg/ml, 0.070 mg/ml, respectively. The results of variance analysis showed that both the expressed-protein concentration and the feed time affected prominently the mortality of sikworm. At the same time, the interaction of them also affected prominently the mortality. On the other hand, after feeding the leaves treated with low concentration of expressed-protein continuously, the high death rate would appear. The survivors' weights were lighter than those without provision of expressed-protein, and couldn't become pupas. We could conclude that the toxin protein expressed by crylAa in ECE52 possessed high toxicity to the larvae of silkworm. The mortality was determined by both the expressed-protein concentration and the feed time, and the growth of silkworm would be inhibited if fresh leaves as food for silkworm were treated with low concentration of expressed-protein.In the third part, we studied the effects of some factors, including chitinase, chemist additives, ultrasonic, ultraviolet on toxicity of expressed-protein to the third instars larvae of silkworm. The results showed that all of them had significantly positive effects on ECE52. The more increased the additives' concentration, the more synergism showed. But the mortality of silkworm was zero when fresh leaves treated with the high concentration additives (ZnSO4, EDTA), the rate was 3.3% when high concentration chitinase or formic acid was provided. The results of variance analysis showed that both the protein concentration of the additive and the feed time affected prominently the mortality of silkworm. The interaction affected prominently the mortality too. The synergisms of expressed-protein caused by the three additives were compared though the bio-statistics methods. It indicated that the synergism of expressed-protein caused by EDTA was more obvious than others, and the order of the efficiencies was EDTA> formic acid >ZnSO4. The toxicity ofthe 0.25mg/ml expressed-protein treated by ultrasonic ten minutes was double than the untreated. The toxicity of the expressed-protei...