| Bacillus thuringiensis has been widely used for insect control worldwide becauseof the specific larvicidal activity of parasporal crystals produced during sporulation. Itwas reported that the parasporal crystals contained 20-kb heterologous DNA fragmentsintimately associated with protoxins, which might play a key role in the assemblingand stability of parasporal crystals as well as the activation of crystal toxin in themidgut of target larvae. However, the nature of the interaction of the Bt protoxin withDNA, the sequences and origin of 20-kb DNAs remained to be investigated. Thepresent investigation was undertook to determine the sequences and origin of the 20-kbDNAs in the parasporal crystals from native and recombinant Bt strains. The data willbe helpful for further understanding the mechanism of interaction between Bt protoxinand DNA as well as the role of the DNA in the structure and function of Bt crystalprotein.A wild-type Bt strain 4.0718 (CCTCC No. M200016) is highly toxic to the larvaeof Helicoverpa armigera and Plutella xylostella. Bt strain 4.0718 harbored cryl andcry2 genes and could produce bipyramidal and cuboidal crystals. MALDI-TOF massspectrometry identification of protein spots on the 2-DE gel revealed that this strainexpressed Cry1Ac and Cry2Aa proteins. The Cry1 protoxins of Bt strain 4.0718 werefound to be associated with 20-kb DNA, on which cry1Aa, cry1Ac, cry2Aa and cry2Abgenes were detected through PCR-RFLP strategy. Furthermore, cry1Ac and cry2Aagenes on the 20-kb DNAs were cloned into Bt strain Cry-B and well-expressed, suggesting that 20-kb DNAs in the crystals contained functional cry genes.A foreign cry2Aa gene was transferred into Bt subsp, kurstaki HD73, thusproducing recombinant strain HD73(pHC39). The strain could produce bipyramidalcrystal of Cry1Ac protoxin and cuboidal crystal of Cry2Aa protoxin. PCR analysesrevealed that 20-kb DNAs in both bipyramidal and cuboidal crystals harbored 20-kbDNAs from chromosome and large plasmid. RAPD analyses of total DNA of HD73and 20-kb DNAs in the crystal of this swain revealed that only part of genomic DNAwas involved in the formation of protoxin-20-kb DNA complex. |
