| Two main parts were included in the present thesis.Part I. Cloning and expression cryIgenes of Bacillus thuringiensisin different host strains.1. Expression of cry genes located in native plasmid in different flagella serotype strainsTo study cloning and expression of ICPs genes, an EcoR I-F fragment of crylA (a) gene from pESI was inserted into pSELECT-1 with T7 RNA polymerase promoter in vitro. The plasmids of Bacillus thuring fensisYBT-803 and YBT-791 were analyzed by southern hybridization using an RNA probe of EcoRI-F fragment and by PCR identification with cryl mixture primers. The results showed that YBT-803 contained cryIA (a), cryIA (b) and cryIA (c) genes, and YBT-791 contained crylA (b), crylA æ‹¢} and cryll genes. These genes were located in >38MDa plasmids. The 38MDa plasmid containing crylA (b) and cryII genes from YBT-791 was purified and transformed by electroporation into the acrystalliferous mutants 4D10 (serotype H3ab) and Bti IPS 78/11 (serotype H14). The twodifferent serotype transformants, MBL-1 and MBL-2 harboring cryl A (b) and cryII were obtained, respectively. SDS-PAGE analysis showed that the transformants could express 130kDa and 65kDa proteins. Three kinds of parasporal crystals of bipyramidal, cubical and embedded shapes were observed under an electronmicroscope. The cubic crystals were more numerous than those in the original strain. Moreover, most of the cubic crystals existed as embedded crystals, which were not observed in the original strain. Bioassay of third instar plutella xylostella larvae showed that insecticidal activity of MBL-1 and MBL-2 were 2.6 and 1.7 times higher than that of the original strain, respectively. It was interesting to note that MBL-1 and MBL-2 contained the same gene type, but insecticidal activity of MBL-1 was higher than that of MBL-2 only to difference in hostserotypes. This suggested that there was a match relationship between the c/y gene type and serotype of the host.2. Cloning and expression of err/gene of Bacillus duringKvm9YBT-803 strain. Bacillus tiwringiensis strain YBT-803 was toxic to plutelh xyloStella larvae.The fragment of plasmids digested with Pst I/Bam HI were analyed by southern blot using c/7/gene RNA probe. Two positive fragments of 7.9kb and 4.4kb were ligated to a shuttle vector pXI 61 and transformed into E. coli1G\. The cloned strains, EL-1 and EL-2 harboring recombinant plasmids were obtained. PCR analysis showed that the 7.9kb fragment contained a typical cry/A (h) gene but the gene-type of the 4.4kb fiagment was not identified scince it possessed characteristics of both crylA (h) and cry IA fa). In order to learn more information about the 4.4kb fiagment, the 653 bp fiagment at 5'-terminal was sequenced. However, the sequence indicated no homology that of crylA (b) and crylA (a). SDS-PAGE showed that EL-1 and EL-2 expressed full-length 130kDa protein in E. coll However, the recombinant plasmid from EL-1 and EL-2, was transformedrespectively by electi operation into acrystalliferous mutant Bti IPS 78/ll(Serotype H14) and resulted in BL-I and BL-2. The expression products of BL-1 and Bl-2 were DOkDa and 65kDa, respectively. Therefore, the 4.4kb fragment from pBL-2 was ligated to a shuttle vector pHT304 with Bt replicon and transformed into acrystalliferous mutant 4D10(Serotype HM,) and resulted in a cloned strain BL-3. This strain could express only 130kDa protein. These results suggested that the 65kDa protein was a degraded product, Bioassay result showed that insecticidal activity of BL-3 was 3-fold higher than that of BL-2 against 3rd instar plutelh xylostella.3. Expression of crylA (b) gene in pseudom on a s fluoreseent pfx-18In order to study expression characteristics of ICPs gene in Pseudom onas fluorescent, a new simple transformation method of Pf was established. The transformation frequency achieved lO^cells/ug DNA when Pseudom onas fluorescent, was grown to 0.55 at ODr,oo, and incubated for 2 minutes at 42 XH prior to treatment with 25mmol/L CaCl2.
|
PDF Full Text Request