The Elementary Investigation Of The Origin Of 20-kb DNA In Crystalline Inclusion From Bacillus Thuringiensis

The recombinant Bacillus thuringiensis strain of HD73(pHC39)produces two different kinds of crystalline inclusions with formsof bipyramidal and cubodidal. They are encoded by cry1Ac and cry2Aagenes respectively. In this study, crystalline inclusions fromrecombinant Bt strain HD73(pHC39) were solubilized selectively andloaded on Ion-exchange Chromatography or Size-exclusionChromatography. Extracted two kinds of 20-kb DNA fragments (20-kbDNAs) associated with Cry1Ac and Cry2Aa protein. Then the cry1Ac geneand the 16s rRNA encoding gene RRA which located on 75 kb plasmidharbored by HD73 and chromosome of Bacillus thuringiensis subspecis.Kurstaki respectively were chosen to be genetic markers to detectchromosome and resident plasmid-specific DNA fragments on 20-kb DNAin crystals, for investigating the origin of 20 kb DNAs in thebipyramidal and cuboidal crystals.1. Construction of recombinant Bt strain HD73(pHC39)Making use of the 20-kb DNA fragments associated with bipyramidalcrystals of Bt 4.0718 as templates, acquired the whole sequence ofcry2Aa gene by using a pair of specific primers of cry2Aa by PCR.The recombinant plasmid pHC39 was obtained through linking the 3.9kb whole sequence with E. coli-B, thuringiensis shuttle vector pHT304after digestion with the same restriction endonuclease, then pHC39was transformed into the HD73 competent cells by electroporation.cry2Aa gene expressed strongly in recombinant HD73(pHC39) strain,typical cuboidal crystalline inclusions were produced, quantitative protein analysis indicated that the amount of Cry2Aa protein wasabove the level of 64.2% of total cellular protein.2. Selective solubilization and Chromatography of solubilizedcrystalline inclusions from HD73(pHC39)Crystalline inclusions from recombinant Bt strain HD73(pHC39)were solubilized selectively under different pH conditions.Experimental results showed that both Cry1Ac and Cry2Aa proteins wereassociated with 20-kb DNA. The Cry1Ac protoxin-20 kb DNAs complexwas Loaded on Ion-exchange chromatography and eluted in NaCO₃/HClbuffer (pH9.5) with increasing gradient of 0-1 mol NaCl. The Cry2Aaprotoxin-20 kb DNA complex was Loaded on Size-exclusionchromatography and eluted in NaCO₃/NaOH buffer(pH11.5). After gettingrid of disturbing DNA component, the 20-kb DNA were extracted fromthe relative purified protein-20-kb DNA fragments complex.3. The original analysis of the 20-kb DNA associated with Cry1and Cry2 protein in HD73(pHC39)In order to analyze the source of the 20-kb DNA, cry1Ac gene whichis located only on the 75 kb resident plasmid harbored by native HD73strain and a 16S rRNA gene RRA which is present only on the chromosomeof HD73 and HD73(pHC39) were used as genetic markers to analyze theorigins of 20-kb DNA fragments by PCR. Two kinds of 20-kb DNA fragmentswere both positive for RRA and cry1Ac genes. It provided evidence that the 20-kb DNA fragments came from chromosome and plasmidharbored by the host strain.4. Identification of chromosomally, located genes on 20-kb DNAfragments15 pairs of primers were designed according to informationpublished on relative database to analyse the section tendency ofchromosomally located genes taking part in 20-kb DNA fragments ofHD73 and provided selective tags for clones.