Cloning And Expression Of LuxS Gene From Bacillus Thuringiensis And Its Effects On Plant Disease Resistance

Quorum sensing is the regulation of gene expression in response to fluctuations in cell-population density and allows populations of bacteria to synchronize group behaviors. Quorum-sensing systems can be divided into two paradigmatic classes: LuxI/LuxR-type quorum-sensing systems in Gram-negative bacteria which produce acyl-homoserine lactone (AHL) autoinducers, and oligopeptide/two-component-type quorum-sensing ciruits in Gram-positive bacteria which produce autoinducing peptide (AIP) autoinducers. In contrast to the two paradigmatic quorum-sensing systems, Vibrio harveyi produces and responds to an AHL-type autoinducer termed AI-1 and a novel signal molecule named AI-2. AHL and AIP autoinducers are species-specific signaling molecules for intraspecies communications. AI-2 serves as a universal signal molecular for interspecies communications. The structure of AI-2 has recently been elucidated and its biosynthesis depends on LuxS protein. Further studies of bacterial quorum sensing systems will give new insights into novel mechanisms of interspecies cellular communications, with the possibility of finding new ways of attempting to defend bacterial diseases.This thesis was aimed to study the cloning and expression of the luxS from Bacillus thuringiensis and the effects of the AI-2 signal molecular biosynthesis on plant disease resistance. To determine whether B. thuringiensis produces AI-2, the V. harveyi luminescence bioassay was used. Culture supernatants of Bt strains induced luminescence in V. harveyi reporter strain BB170, which can only response to AI-2 signal moleculars, indicating that the Bt strains have the luxS gene.By aligning the nucleotide sequences of the B.anthracis and B.cereus luxS genes, a pair of primers were designed and an 474-bp fragment was amplified from Bt strains by PCR. Sequence analysis of the fragment revealed that the luxS gene from Bt had nucleotide homologs similarities of 96.8% and 96.6% withB. anthracis and B. cereus luxS genes, respectively, and amino acid similarities of 98.7% for both. Similar result was also clearly confirmed in the phylogenetic dendrogram. When expressed in Escherichia coli DH5a cells, the cloned fragment restored AI-2 activity to these cells.These data indicated that the fragment is a functional luxS ortholog in B. thuringiensis necessary for AI-2 expression.To test the expression of the gene in plant, the cloned luxS gene from B. thuringiensis was constructed into pCAMBIA 1305.2 vector. The resulting plasmid was mobilized into Agrobacterium tumefaciens strain and used for tobacco transformation. Using the V. harveyi luminescence bioassay, expression of the luxS gene was detected in transformed tobacco. Induced defence responses were observed after inoculated with plant viruses.