Expression of mosquitocidal Cyt1Aa toxin from Bacillus thuringiensis subsp. israelensis in Asticcacaulis excentricus

Asticcacaulis excentricus, who lives in upper-layer waters providing food resource to the mosquito larvae and has been proven to be a successful host to produce the mosquitocidal binary toxins or Cry11Aa toxin from Bacilli (Liu et al., 1996, Nat Biotech 14: 343; Armengol, et al. , 2005, Curr Microbiol 51: 430), was developed to express cyt1Aa gene from Bacillus thuringiensis subsp. israelensis (Bti). Two A. excentricus transformants were constructed with the attempt of producing CytlAa alone and alongside with Cry11Aa, repectively. Detection of expressed Cry11Aa and CytlAa proteins by immunoblot in the recombinant A. excentricus clones showed that either cry11Aa or cyt1Aa was expressed well solely but not simultaneously although both restriction analyses of plasmid DNA and DNA sequencing showed that the transformed plasmid was identical to scheme. To investigate the reason why the recombinant A. excentricus harboring both genes and their ribosome binding site (RBS) sequences expressed only Cry11Aa, the total RNA of A. excentricus cells was extracted and revealed three-band pattern in which all RNA molecule weights are not greater than 16S RNA of Escherichia coli by formamide agarose gel electrophoresis, indicating that different RNA systems within these two Gram-negative strains required distinguishingly organised constructs to express multiple foreign genes. It is hypothesized that an extra promoter upstream of RBS sequence is required to express cyt1Aa in the cry11Aa-cyt1Aa tandom plasmid