| The expression and application of gfp in Bacillus thuringiensis were described in this thesis. The research results are summarized as following:1. Preliminary Study on the Expression of gfp Gene in Bacillus thuringiensisThe gene gfpmut3a with specific promoter of B.cereus were cloned into a shuttle plasmid pHT304 between E.coli. and B.thuringiensis. It was then introduced into recipient strains BMB171, CryB, IPS78-11, 4Q7 and HD80-21 by electroporation. The results shown that green fluorescence could only been detected in acrystalliferous strains.2. Studies on the activites of promoters of Bacillus by using gfp geneThree kinds of promoters from Bacillus: P441-12, PCry3A and PBtI-BtII were chosen to drive the expression of gfp gene and were cloned into shuttle plasmids. Three recombinant plasmids of pGFP-304, pGFPExpA and pGFPExpB obtained were transformed into recipient strains of E.coli DH5a and B. thuringiensis BMB171 by electroporation. The results shown that gfp gene driven by Pcry3A could only express in BMB171. The VbtI-btII was found to show strong green fluorescence not only in BMB171 but also in DH5a. The activities of Pcry3A in recipient strains were much weaker than that of P44-12 and PBtI-BtII.3. Microcalorinetric study on recombinant strains of Bacillus thuringiensis containing diversity of promotersFluorescent intensities of BMB171(pGFP-304), BMB171(pGFPExpA) and BMB171(pGFPExpB) were detected by using bioactivity monitor. The results showed that gfp gene could be expressed by three kinds of promoters. Thermogenic curves and the results of metabolism heat output revealed that the heat output of BMB171(pGFP-304) and BMB171 (pGFPExpB) were less than that of BMB171(pGFPExpA).4. Construction of recombinant strains labeled with gfp gene in Bacillus thuringiensisBy SOE technic, a recombinant plasmid pBMBZGC10 with P4412 was obtained by the ligation of gfp-crylAc 10 fusion gene and vector plasmid pAD4412. Another recombinant plasmid pBMBZGC11 with PBtI-BtII was obtained by replacing promoter.And then they were introduced by gene pulser into acrystalliferous strains BMB171 and CryB to obtain the recombinant strains BMB171(pBMBZGC10) , CryB(pBMBZGC10), BMB171(pBMBZGC11) and CryB(pBMBZGCll). The results shown that green fluorescence could only been detected in recombinant strains BMB171(pBMBZGC10) and CryB(pBMBZGC10) by fluorescent microscopy. The expression of the fusion genes about 150 kDa ~ 160 kDa can be detected by SDS-PAGE.5. Analysis of fluorescent detection in recombinant strains of Bacillus jhuringiensis by microscopyFor all fluorescence microscopy work, Zeiss Axioplan Leica Diaplan and Leica M2FIII fluorescence Stereomicroscopes equipped with filter sets were used. Finally contained the following filters: excitation filter 488nm, and a 522(DF32)nm (only on the Zeiss microscope) emission filter. Furthermore, fluorescence intensity of recombinant strains detection by instrument of Bio-assay Reader. The GFP-long pass filter set was composed of: excitation filter 485nm, emission filter 520nm. All the data and pictures were done using professional soft of HT Soft 2.0 and Laser Sharp 2000?6. Studies on the effects of the 20kDa helper protein (P20) in recombinant strains of Bacillus thuringiensisA shuttle plasmid pBMB20-2 containing p20 gene was introduced into BMB171 (pBMBZGC10) and CryB(pBMBZGC10) by electroporation to obtain recombinant strains BMB171(pBMBZGC10-20) and CryB(pBMBZGC 10-20). The results showed that green fluorescence could be detected in them under the fluorescent microscope. Further study revealed that the 20 kDa helper protein can increase expressing of fusion protein gene by fluorescence intensity analysis.7. Studies on the construction of labeled recombinant strain and its cry 1 Ac 10 gene horizontal transfer of Bacillus thuringiensisA recombinant plasmid pBMBZGClO was obtained by the ligation of gfp-crylAc10 fusion gene and vector plasmid pAD4412. It was then introduced by gene pulser into acrystalliferous strain CryB and a recombinant strain CryB(pBMBZG...
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