RECOMBINANT PLASMID DNA PTV 241, METHOD OF CONSTRUCTION OF RECOMBINANT PLASMID DNA PTV 241, METHOD OF CONSTRUCTION OF PLASMID CONTAINING GENE CRY IIIA IN TRANSPOSON TN 917 CAT, STRAIN OF BACTERIUM BACILLUS THURINGIENSIS SUBSPECIES THURINGIENSIS EXHIBITING ACTIVITY AGAINST COLORADO POTATO BEETLE

摘要

FIELD: biotechnology, genetic engineering. SUBSTANCE: invention relates to recombinant plasmid DNA pTV 241 providing an expression of gene cry IIIA encoding delta-endotoxin in Bacillus thuringiensis subsp. tenebrionis that has a complete sequence of vector plasmid pTV 24, complete sequence of Bam HI-Bgl II-fragment of DNA of Bacillus thuringiensis subsp. tenebrionis (size is 2.9 thousand nucleotide pairs), intact gene cry IIIA encoding delta-endotoxin in Bacillus thuringiensis subsp. tenebrionis that is inserted in a sequence of transposon Tn 917 cat. Method of construction of recombinant plasmid DNA pTV 241 involves cleavage of a mixture of plasmid DNA pUC19 and DNA of Bacillus thuringiensis subsp. tenebrionis with restriction endonuclease Hind III. Then cells Escherichia coli JM 103 are treated with a mixture and colorless colonies are selected on Mack-Concki medium with ampicillin. Selected colonies are tested for the presence of DNA sequences showing homology with the corresponding sequences of gene cry IIIA. From cells of one of colony containing the indicated sequences DNA-plasmid with gene cry IIIA is isolated (designated as pBTT 51). Plasmid pBTT 51 is cleaved with restriction endonucleases Bam HI and Bgl II and obtained fragments are mixed with DNA-fragments of plasmid pVT 24 and combined. Fragments are transformed by electroporation of cells Bacillus thuringiensis subsp. kurstaki. Transformants are sown on agarized medium containing chloramphenicol (Cm) and plasmid DNAs are isolated from Cm-resistant cells and the end recombinant plasmid DNA pTV 241 is taken off. Method of construction of plasmid containing gene cry IIIA in transposon Tn 917 cat (size is about 50 thousand nucleotide pairs) involves transfer of recombinant plasmid DNA pTV 241 in cells of strain Bacillus thuringiensis subsp. morrisoni DC and transformed clones showing resistance to Cm are selected. Then these clones are crossed with strain Bacillus cereus that are resistant to rifampicin (Rf). From cellular clones showing resistance to Cm and Rf the end conjugative plasmid is isolated. On the basis of this plasmid strain Bacillus thuringiensis subsp. thuringiensis IPM-37 is obtained and this strain shows activity against Colorado potato beetle and other harmful coleopterous insects. EFFECT: improved method of plasmid construction. 5 cl, 1 dwg, 4 ex