The Effects Of Helper Protein On The Expression Of Insecticidal Crystal Protein In Bacillus Thuringiensis

The effects of the 20kDa helper protein (P20) from Bacillus thuringiensis subsp. israelensis were examined on the expression and crystal formation of insecticidal crystal protein CrylAc, and towards the end to construct engineered strains to be both environment-friendly and highly efficient. The major results are described as following:In the presence of P20, the expression of CrylAclO was 3.5 times of the control without P20, the formed crystals were in an average size of 1.85u.m long and 0.85um wide, shown as a typical bipyramid with 3-time volume of the control crystal. Consequently, the transformant harboring P20 gene and cry 1 Ac 10 exhibited 2.5-time toxicity of the control transformant against neonate larvae of Heliothis unnigera. Further study revealed that serious degradation of CrylAc happened during the whole stage of sporulation, while the 20kDa protein could prevent neonate peptides only instead of the full protoxins from degradationTwo plasmids with the ability of site-specific recombination were obtained, one is pBMB1808 carrying P20 gene and crylAclO between two specific resolution sites (res), the other is pBMB1820 harboring only P20 gene. Also, a Bt replicon was included between these two "res" sites. Other genes such as tnpl. antibiotic resistance genes and an E.coli replicon etc were positioned outside of the two "res" sites. Therefore, a site-specific recombination would happen between two "res" sites in B. thuringiensis, and enable the transformant to get rid of the unwanted DNA fragments. Thus, these two plasmids would be useful in the construction of an environment-friendly engineered strain with high productivity of ICPs.Three engineered Bt strains BMB21882, BMB83383 and BMB15358 were obtained by electro-poration of pBMB1808 into three natural isolates with a high productivity of ICPs and high toxicity against H. armigera and Plutella. xylostella. Quantification results revealed that two times of CrylA protoxin produced in the transformants relative to their receipts. And larger bipyramidal crystals were resulted in a size of 2.40 u.m long and 1.05am wide, with 2-3 time volumes of their receipts' crystals. In contrast, the transformants' spores became shorter or spherical instead of long rods. Further study showed that continuous accumulation of CrylA protoxins during the whole sporulation stage was obtained in the engineered strains, while in natural strains a serious hydrolysis of 130kDa protoxin occurred before crystal formation.Despite of the high expression of ICPs in these transformants, bioassay results failed to show the expected toxicity against their target insects, especially in the case of neonate larvae. However, in vitro activation of crystals in H. armigera midgut juice showed that much more toxic peptides were generated by transformants than the receipts. Further study suggested that the lowered toxicity might be resulted from the poor solubility of crystals in vivo.Unfortunately, the obtained transformants failed to eliminate the antibiotic resistance gene harbored in pBMB1808. due to both the prompt excision in most transformants before antibiotic selection, and DNA modification on pBMB1808 in the obtained transformants. Therefore, more efforts needed to obtain an ideal engineered strain.