| The cry2Aa8, ciy2Aa9 genes were cloned by using of partial digestion of plasmid DNA, Southern blotting, and PCR-RFLP techniques. The engineering Bt strains BiotlI2O, BiotIIl76 and Biot11732 containing cry2Aa8 gene were constructed. Also the insecticidal activity of BiotIIl76 was tested in this study. 1. Cloning of cry2Aa Gene from B-G-8 and Bt66 Strains Plasmid DNA of B-G-8 was digested partially with Sau3AI and 4-7kb fragments of the DNA were collected and inserted into cloning vector pUCP19 treated by Baml-IL This enabled construction of recombinant plasmid library. Based on PCR results, many recombinants were obtained. A 4.5kb fragment containing cry2Aa gene was cloned. After subctoned and sequenced, this gene was found to be composed of 1902bps. The amino acid sequence of ICP was deduced from the nucleotide sequence of ciy2Aa gene and the MW was 70.853kDa. This cry gene had been registered in EMBL/GenBank (Accession number AJ2732 18) and designated as cry2Aa9 by International Nomenclature Committee of Bt- 8 - endotoxin Gene. Identified Bt66 Strain harbored crylEa and cry2Aa genes. Southern hybridization of plasmid DNA of Bt66 strain was carried out. Hindu digested fragments from Bt66 plasmid DNA were hybridized with cty2Aa DNA probe. The results showed one positive band of 5.0kb. The 5.0kb fragment was inserted into the HindIII site of cloning vector pBluescriptSK(+), and then transformed into E. coli JM 107. PCR and enzyme digestion results demonstrated that the positive transformant clones contained the 5.0kb fragment of the gene. By subcloning, a 4.0kb BamHI fragment was obtained. Nucleotide acid sequence as well as deduced amino acid analysis suggested that this gene was composed of 1902bps and one of the bases had been changed compared with known cry2Aa gene (from A to G) and subsequently resulted in one amino acid change (from Ser to Gly). 633 amino acids were deduced from its nucleotide sequence and its MW was 70.821 kDa. Also, this cry2Aa gene was registered in EMBLIGenBank (Accession number 252262) and named as a new gene called ciy2Aa8. 2. Expression of cry2Aa Gene and Construction of Engineering Strains Expression vectors pGWI 105 (Bt-E. coli-Pf) and pZG3 15 (Bt-E. calf) were constructed by cry2Aa8/cry2Aa9 inserted into pGM 1105 and pHT3 15 respectively. The transformant BiotII2O was obtained from introduction of pGWI 105 into the acrystalliferous mutant strain 2 BE2O by electroporation. By the above method, pGW1 105 was transfered into natural isolates UVI7 and HD-73 respectively, then Bt genetic engineering strains of BiotlIl76 and Biot11732 were constructed. The expression of Cry2Aa crystal proteins in BiotlIl76 and Biot11732 was characterized by SDS-PAGE and optical microscope. The results, an intence protein band of Cry2Aa8 7OkDa in SDS-PAGE and formation of cubic crystal, indicated that cry2Aa8 could express normally and stably in UV 17 and HD-73. The expression was due to ORF2 protein contained in pGWI 105 that was known to promote Cry2Aa crystal formation during sporulation. On the other hand, the situation was greatly different in BiotlI2O. Transformation of introduction pGW1 105 into Pseudomonas fluorescens strain P303 had been proved successfully by PCR-RFLP. The growth of Bt strain UVI7 and 1-10-73 was not affected on pGWI 105 being introduced into it. 3. Toxicity of Engineering Strain BiotII 176 to Plutella xylostella Insecticidal activity of engineering strain BiotII 176 holding two distinct cry-type genes... |
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