Construction Of Bacillus Thuringiensis Genetic Modified Strain With Resistance To Plant Disease And Wide Spectrum Of Action

The promoter of gene aiiA and vip83 was replaced with the promoter of gene cry3Aa by overlapping PCR, the resulting genes were inserted into shuttle vector and transformed to Bacillus thuringiensis high-toxicity cell engineering strain YBT-833-2-1 and high-toxicity wild-type strain YBT-1520 respectively, finally two engineering strains were constructed, one has high toxicity to Lepidopteran insects and strong restraint to plant soft rot disease caused by Erwinia bacteria, another has wide spectrum of toxicity to Lepidopteran insects. The form of parasporal crystals and S-layer stucture of Bacillus strain CTC were observed, it was proved that the S-layer proteins of strain CTC could form parasporal crystals. The major results are described as following:1. Construction of Bt engineering strain which has high toxicity to Lepidopteran insects and strong restraint to plant soft rot disease caused by Erwinia bacteriaiV-acyl-homoserine lactones (AHLs), as a kind of autoinducers, are widely conserved signal molecules present in quorum-sensing systems of bacterial pathogenys. this molecules may be degraded by AiiA protein present in B. thuringienis. so that the disease caused by bacterial pathogen were also attenuated. The promoter of gene aiiA was replaced with cry3Aa promoter which promotes the transcription independing the sporulation, to generate the gene pro3A-ahA. This gene was inserted into shuttle vector and transformed to cell engineering strain YBT-833-2-1, yielding the engineering strain BMB821A. Strain BMB821A exhibited stronger ability of AHLs inactivation and more effective restraint to the plant's soft rot disease caused by Erwinia bacteria than those of the parental strain YBT-833-2-1. The LC50 value of strain BMB821A was 3.426uL/mL. showing no obvious differentiation of toxicity to Spodoptera exigua comparing with parental strainYBT-833-2-1, whose LC50 was 2.912uL/mL. This work is the first report of constructing the Bt engineering strain which has high toxicity to Lepidopteran insects and strong restraint to plant disease in the world.2. Construction of Bt engineering strain which has wide spectrum of action Vegetative insecticidal protein (Vip), is a kind of water-soluble protein that starts toexpress at the vegetative period of Bt. It was confirmed that Vip has specially high toxicity to Agrotis ipsilon and Spodoptera exigua. The promoter of gene vip83 was replaced with the cry3Aa promoter, resulting gene pro3A-vip. The expression level and toxicity to Plutella xylostella resulting from expressed Vip protein of gene pro3A-vip were both higher than those of gene vip83. The gene pro3A-vip was inserted into shuttle vector and transformed to wild-type strain YBT-1520, yielding strain BMB218V. The Vipproduced by strain BMB218V was also water-soluble. The toxicity to Spodoptera exigua of strain BMB218V was 11 times higher than that of strain YBT-1520, showing addition of Vip could increase the toxicity to Spodoptera exigua remarkably. The toxicity to Heliothis armigera of strain BMB218V maintained same level comparing with strain YBT-1520. The core-fragment of toxicity of cry 1C was replaced with the ORF ofvip83 to generate the fusion gene vip-1C, this gene expressed 90kD protein in B. thuringiensis acrystalliferous strain BMB171. This work is firstly reported that constructing Bt engineering strain which has wide spectrum of action using pro3A-vip gene. 3. The S-layer protein of Bacillus strain CTC could form parasporal crystal inside cellThe N-terminal amino acid sequence of crystal protein together with the DNA sequence of crystal protein gene etc of strain CTC, were 92% and 87.5% homology to those of S-layer protein of Bacillus anthracis, respectively. It was postulated that strain CTC has S-layer. In this work, it was showed that strain CTC formed S-layer which located on the outer cell wall. The sheet-like structure of S-layer and the parasporal crystals inside the cell were observed during the sporulation period. After the gene etc was transformed to the acrystalliferous B. thuringiens...