Cloning And Expression Study Of Insecticidal Crystal Protein Genes From Bacillus Thuringiensis 4.0718 Strain

Bacillus thuringiensis (Bt) 4.0718 strain (Number in China Center for Type Culture Collection:200016) is highly toxic to Lepidoptera.The Plasmids were extracted from this strain by Triton X~100 gentle method and the number P1HP4 plasmid were reclaimed respectively by kit. The cryl gene type of the number P1-P4 plasmids was screened by universial primers Unl(d) and Unl(r). 14 kinds of crylAc gene from Ge'nBank were aligned by Jellyfish.software and the consensus regions were found. According to the consensus sequences ,A pair of primers were designed to amplified a 4.2kb element including the dual overlapping promoter and the whole terminor. The amplified 4.2kb element was confirmed to the crylAc gene by cryl sub-genetype PCR-RFLP analysis system .After the TA cloning to vector pUCm-T and subcloning to the Bt-E. coli shuttle vector pHT304, the crylAc gene was expressed in E. coli DH5 a and the CrylAc protoxin was accumulated. In the last , the aery stall if erous mutant was transformed by electroporation. The results are summaried as follows:1. Identification of cryl genetype of plasmidsThe number P1-P4 plasmids were reclaimed respectively from the agarose electrophoresis gel, and the number P1-P4 plasmids respectively as template were amplified by universial primers Unl (d) and Unl (r) of cryl genetype. The number P1-P3 plasmids were amplified to produce the 277bp target element and indicated the cryl genes were existed in those plasmids. According to the PCR-RFLP analysissystem, a 23kb plasmid was appraised by this system. A 1.6kb target element was amplified , and the Restriction fragment length polymorphism map of this element include 1117bp 801bp 518bp and 322bp segments. Comparing with the standard RFLP map of cryl model gene,this 23kb plasmid was testified to comprise crylAa gene and crylAc gene.2. CI on i ng of cryl Ac geneBecause of the high toxic to Lepidopteriaof CrylAc protein, this 昰ene had been taken into- account widely in the regions of construction of insect-resistant engineering strains and the insect-resistant crops. In this study, A alignment of 14 kinds of crylAc gene from GenBank was performed and some high consensus regions were found. According to those datas, A pair of primers from the consensus regions, PR-OF-1 and PR-OF-2, were contrived to amplified a 4.2kb fragment. This element includes the open reading frame of crylAc gene upstream promoter and downstream terminor. PCR-RFLP analysis system proved this element containing the crylAc gene.The 4. 2kb segment from PCR amplification had a A tail in its 3' terminal, and can linked with T tail of 3' terminal of T vector pUCm-T. Two transformants were obtained , pTC9 in forwarddirection and pTCll in backdirection. Cleaved assay indicated the 4.2kb segment was included in recombinant plasmids of two transformants.The 4.2kb element from pTC9 was reclaimed from agarose electrophoresis gel after cleaved by BamH I and Sal I Restriction Endonucleases. Subcloning to Bt-E. coli vector pHT304 was preformed and the transformant EHC34 was screened by PCR-RFLP analysis system.3. Expression of recombinant plasmid pHC34 in E.coli DH5aThe expressed proteins were extracted by a improved protein extraction method from the engineering strains EHC34. All of the proteins were separated by SDS-PAGE and the proteins electeophoresis map was compared with the another proteins electrophoresis map of the E.coli DH5 a (pHT304) control. From this compared results, a 130KD protein was expressed obviously in the transformant EHC34.