| Aiming at biocontrol of important agricultural insect pests such as Plutella xylostella, Spodotera exigua and Chilo suppresalis, the commercialized biocontrol bacteria Pseudomonas flurescens P303, Bacillus subtilis B916, a natural isolates B. subtilis B918 from cotton leaves and Bt C002 with high insecticidal toxicity were selected and the construction of versatile plasmid vectors, cloning of Bt cry genes and plasmid replicon, transposition of mini-TnlO transposon and development of genetically modified P. flurescens and B. subtilis were systematically studied. The major research results are summarized as follows.1 Novel E. coli and E. coli-Pseudomonas shuttle plasmid vectors pSpca-/ac and pSpcPoc-/flc were constructed. After continuous culturing for 5 Oh without antibiotic selecting press, the stability of P. flurescens P303 carrying pSpcPa-lac was 100% stability. No deletion, rearrangement and a -complementary function altering could be detected in pSpcPa -lac. Research proved that plasmid pSpcPot -lac and pJMS6a -lac are well compatible with a 99.94% co-existing stability per generation in P303o The biosafety and expressing in different bacteria of spectinomycin resistant gene, the Multi-Cloning-Sites (MCS) and the a-complementation function of plasmid pBlueScript KS(-) contributed to two versatile plasmids, which could be useful and easily manipulating for gene cloning, sequencing and isolation of bacterial plasmid replicon and research on replicating mechanism,2 With plasmid pSpcPa-/ac and B. subtilis 168 research system, a 6909 bps DNA fragment carrying a novel plasmid replicon was isolated from Bt C002 and registered in GenBank as AY1260180 Sequence analysis showed that there are at least three ORF (Open Reading Frame) in the cloned DNA, i.e. ORF145> ORF190 and ORF200 respectively. ORF 190 is a novel protein which only has less than 40% identities with Integrase/recombinase family from bacteria; Both ORF 145 and ORF200 have 97% identities with the N-and C-conserve core sequence of Bt Replicating related protein Rep9741, but with different molecular weighto After continuous culturing for 55.5h at 37℃ without antibiotic selecting press, the stability of plasmid carrying cloned replicon in B. subtilis 168 was 98%. This work laid a foundation for further construction of stable vectors system and genetically modified Bt strains.3 Recombinant shuttle plasmids pZY311 and pZY313 were constructed by inserting full length Bt crylAb gene in the MCS of pSpcP a-lac. Plasmids pZY311 and pZY313 with pJBT2008 a derivative of pJMS6cc-/ac carried crylA and cry2Aa were co-transferred into P. flurescens P303 respectively, the engineered strains with three Bt cry genes were designed as BioP6 and BioPS . After continuous and successive diluting culture for 72h, the plasmids compatible stabilities in BioP6 and BioPS were 83.5%, 89.1% and 80.0%, 84.8%, respectively.4 Results of lab bioassay showed that BioP6 and BioP202(P303 with pJBT2008) have the same insecticidal activities against P. xylostella, while BioPS is much more toxic than BioP202. In year 2000 and 2001, BioPS was tested in the fields for biocontrol of P. xylostella and the larvae decrease rate ( LDR ) after treating 96h using its 10 times diluting ferment were 98.3% and 94.8%, and the LDR for 100 times diluting ferment was 84.8% which is equal to commercialized Bt product (86.1%). No remnants and diffuse could be detected in the soil samples from tested field in year 2000. BioPS has been granted to release in field trial. It is just the characteristic of this work that using the compatibility between plasmids from different computable groups to construct genetically modified bacteria carrying three different Bt cry genes, which is quite different from the strategy combining different modified bacteria carrying one cry gene, e.g. the Cellcap system. It greatly promoted the commercializing proceeding of inter-genus recombinant bacteria by transferring Bt cry genes.5 Bt crylCaS and cry2Ab3 gene were cloned from the strain C002...
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