title: Screening of cry-type promoters with strong activity and application in Cry protein encapsulation in a sigK mutant.
abstract: To optimize the expression of cry genes in a Bacillus thuringiensis sigK mutant failing in crystal releasing, the transcriptional activity of the cry promoters cry1A, cry3A, cry4A, and cry8E was compared using lacZ gene fusions. A beta-galactosidase assay indicated that the cry8E promoter showed the highest transcriptional activity. A novel Escherichia coli-B. thuringiensis shuttle vector pHT315-8E21b was constructed for cry gene expression using the cry8E promoter and the multiple cloning sites from vector pET21b, based on vector pHT315. SDS-PAGE analysis showed that the expression of the cry1Ac gene directed by the cry8E promoter was increased by approximately 2.4-fold over the expression directed by the cry3A promoter. The cry1Ba gene was expressed in the sigK mutant with the constructed vector pHT315-8E21b. Normal bipyramidal crystals encapsulated in mother cell were observed by transmission electron microscopy (TEM). The encapsulated Cry1Ba protein expressed in the sigK mutant showed activity against Ostrinia furnacalis and Plutella xylostella similar to that of the released Cry1Ba protein expressed in the acrystalliferous strain HD73 and can be protected from inactivation by UV light. All these results suggest that the cry8E promoter can be an efficient transcriptional element for cry gene expression in sigK mutants and can be utilized for the construction of a genetically engineered strain.
authors.affiliation: State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193, China.
authors.forename: Changmei
authors.initials: C
authors.lastname: Zhou
authors.forename: Qingyun
authors.initials: Q
authors.lastname: Zheng
authors.forename: Qi
authors.initials: Q
authors.lastname: Peng
authors.forename: Lixin
authors.initials: L
authors.lastname: Du
authors.forename: Changlong
authors.initials: C
authors.lastname: Shu
authors.forename: Jie
authors.initials: J
authors.lastname: Zhang
authors.forename: Fuping
authors.initials: F
authors.lastname: Song
mesh_terms: D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics / Q000378:metabolism*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D004731:Endotoxins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D006460:Hemolysin Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D007915:Lepidoptera / Q000187:drug effects; D046529:Microscopy, Electron, Transmission; D011401:Promoter Regions, Genetic / Q000235:genetics*
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis