| Three kinds of works were described in this thesis with Bacillus thuringiensis as major material.1. Autoinducer inactivity protein Aii in B. thuringiensis strains and its effect on pathogenicity of plant-pathogen bacteriaAutoinducer, especially AHL molecule, is a kind of cell-to-cell communication molecule that has extensive distribution in gram-negative plant pathogen bacteria. It has direct or indirect relationship with the pathogenicity. A chemical or bacterium that inhibits the activity of autoinducer will affect the pathogenicity.In this project, based on a great quantity of screening experiments, B. thuringiensis and B.cereus strains' culture was found containing a kind of protein which inhibits autoinducer activity. This protein was named Aii and its encoding gene was named aii. Detection of distribution of aii gene and Aii protein in Bacillus species and B.thuringiensis subspecies indicated that except for B.thuringiensis and B.cereus strains, B.subtilis and B.sphaericus strains had lower Aii activity, but B.megalerium strain had no activity at all. PCR amplification results indicated that aii gene could be found in B.thuringiensis, B.cereus, B.subtilis and B.sphaericus strains, but not in B.megaterium. According to the Southern hybridization results, 43 Bacillus strains were divided into several groups, 22 strains were chosen randomly from all these groups for sequencing of aii gene. It was found that the nucleotide acid sequence of aii genes showed 85.4%-100% homology and amino acid sequence of Aii proteins showed 8 8.1%-100% homology.Further study showed that Aii protein was a kind of non-secreted protein, and aii gene was located on the chromosome of B.thuringiensis or B.cereus. Expression of aii gene expanded from vegetative stage to early stage of sporalation. Aii protein not only inhibited the activity of various purified AHL molecules but also inhibited the activity of AHL molecules in bacterial culture. Using Erwinia carotovora strain SCG1 as pathogen, the effect of B. thuringiensis strain 4D1 culture was detected on the rot soft disease. The results indicated that 4D1 culture inhibited the pathogenicity of SCG1 in 24 hours when the cell density of SCG1 was lower than ODeoonm=0.55, but when the incubation time after infection was longer than 24 hours, the effect decreased. However, 4D1 culture showed a little of inhibitory activity when the cell density of SCG1 was higher than OD600nm=0.55. All these results suggested that the cause of 4D1 culture inhibiting thepathogenicity of SCG1 is the decreased concentration of AHL molecule in SCG1 cells, when presenting Aii protein in 4D1 cells, and then not inducing the expression of virulence genes of SCG 1 at low concentration of AHL molecules.A new strategy in controlling plant pathogen bacteria based on inhibiting the activity of autoinducer molecules could be developed. Construction of transgenic plants which express Aii protein or development of new B.lhitringiensis products which produce high yield of Aii protein will be very useful in plant disease control.2. Expression of gfp gene in B.thuringiensis strainGreen fluorescent protein gene is a very useful report gene which could be detected easily. It can be used in the detection of B. thuringiensis environmental release and study of the insecticidal mechanism.In this study, two kinds of specific promoter of B.thuringiensis: cry 3A promoter and Btl-Btll promoter were chosen to construct fusion genes to drive the expression of gfp gene in B.thuringiensis strain. Nucleotide acid sequence analysis indicates that two construts were correct. Recombinant plasmids pGFPExpA that containing cry3Apro-gfp fusion gene and pGFPExpB that containing Btl-BtHpro-gfp fusion gene were transformed into E.coli and B.thuringiensis plasmidless strains, respectively. The Btl-Btll promoter was found to drive gfp gene expression strong green fluorescence in not only B.thuringiensis but also E.coli strain. However, cry3A promoter could not drive gfp gene expression in E.coli, and the expression i...
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