The Characterisics Of Crystal Protein Gene Cry26Aa And Cry28Aa Expression In Bacillus Thuringiensis

Bacillus thuringiensis subsp. finitimus produced at least two parasporal inclusions. One inclusion was formed within the exosporium and remained with the spore after mother cell lysis. A second inclusion formed somewhat later exterior to the exosporium. Bt 020 isolated by our lab also has this phenomenon. And these two strains have the same constitutions of plasmid and parasporal crystal protein based on PFGE and SDS-PAGE.Strains of finitimus not only have the distinguished location of parasporal crystal protein, but also the unique spectrum of susceptible larvae. This may partially attribute to packaging an inclusion within the hydrophobic exosporium so as to keep the inclusion in the water where target larvae maybe actively feeding. Or spore has some synergic effects to the insecticidal protein by releasing some pathogenic factors during the process of germination. So it must have some important and pratical value to construct a "bio-capsule" insecticide by using spore-associated proteins, such as increasing the exposure resistance of insecticidal protein under the UV light or drought. In addition, understanding the origin of cry genes and how their regulation became integrated with sporulation is also the hot spot of research.According to the crystal protein gene sequence in finitimus, cry26 and cry28 were cloned from strain 020 and transformed into the crystal negative Bt strain BMB171 by shuttle vectors, alone or together. Crystals can be observed both inside and outside of the exosporium when co-expression of these two genes. But when they expressed alone, cry26 gene leaded inclusion body formation within or outside of the exosporium, whereas cry28 gene only induced crystal formation outside of exosporium. All of the proteins could not stably exist.On the other hand, the free parasporal protein degraded easily as well. PCRamplification suggested that strain 020 has slh fragment of s-layer gene and csaAB operon.Along with the previous result that strain CTC of finitimus could form parasporal crystals. by s-layer protein, a putative s-layer protein may also exist in strain 020 and has somecooperative effect to spore-associated crystal formation.In above, it suggests cooperation between cry26 and cry28 gene could lead crystal forming inside of the exosporium. But other help proteins maybe also involved in enclosure.