Study On Correlation In Relation To Nucleotide Sequences Between 20-kb DNA In Insecticidal Crystals And Plasmids As Well As Chromosome From Bacillus Thuringiensis

Through cloning and expression of cry1Ac gene on 20-kb DNA in bipyramidal crystals from Bacillus thuringiensis strain 4.0718, this study testified that 20-kb DNA contained the coding sequences of crystal proteins. In order to explore the origin of the 20-kb DNA fragment, the study detected distribution of cry-type genes on plasmid and chromosome of Bacillus thuringiensis strain 4.0718 by means of Southern blotting. The existence of the same cry-type genes on plasmid and chromosome, as that on 20-kb DNA in bipyramidal crystals from Bt strain 4.0718, rendered direct evidences to the previous speculation that 20-kb DNA on crystals from B.thuringiensis was derived from plasmids or chromosome.1. Cloning and expression of cry1Ac gene on 20-kb DNA in bipyramidal crystals from Bt strain 4.0718A pair of primers were designed to amplify 4.2-kb fragment containing the promoter, the coding region and the terminator of crylAc gene on 20-kb DNA. The PCR product was subcloned into pMD18-T vector through T/A ligation. The resultant recombinant pTAc4 was digested with BamHI and salI . Then the 4.2-kb digested fragment was cloned into Bt-E.coli shuttle vector pHT304, producing pHAc4. The plasmid pHAc4 was transformed into acrystalliferous strain XZM-101 by electroporation. The expression of cry1Ac gene in transformant XZM-101/pHAc4 was monitored by AFM observation and SDS-PAGE analysis. Typical bipyramidal crystals was observed under AFM and 130-kD protein band on SDS-PAGE gels was found. Bioassay results showed transformant containing pHAc4 was toxic against plutella xylostella larvae.2. Localization of cry genes in Bt strain 4.0718Chromosome of Bt strain 4.0718 which was digested with NotI in advance was subjected to pulsed-field gel electrophoreses(PFGE). The digested chromosome and plasmids of Bt strain 4.0718 were transferred to hybond-lsT nylon membranes respectively, and were blotted with the DIG-labeled probe that was derived from 1.6-kb PCR fragment from cry1Ac gene. The results of southern blotting showed that both plasmid and chromosome of Bt strain 4.0718 harbored cry-type genes.3. Identification of cry-type genes on chromosome from Bt strain 4.0718PFGE was used to separate chromosome from other DNA elements of Bt strain 4.0718. Then chromosomal DNA was recovered from gel slices by electroelution. Two pairs of universal primers Unl(d)/Unl(r) and Un2(d)/Un2(r) were used to detect cry-type genes on chromosome after the recovered chromosomal DNA was digested with SalI. Two PCR products with predicted sizes of 277bp and 689bp were obtained. For RFLP analysis of cry1 and cry2 genes, two pairs of specific primers were used for PCR amplification. 1600-bp PCR product corresponding to cryl genes amplified with k5un2/k3un2 was digested with PstI and XbaI. 1200-bp PCR product corresponding to cry2 genes amplified with S5un2/S3un2 was digested with Hindi and MspI. The restriction fragment length polymorphism of the PCR products showed that the chromosome of Bt strain 4.0718 contained cry1Aa, cry1Ac, cry2Aa, cry2Ab genes.