Isolation and cloning of Bacillus thuringiensis var Kurstaki HD73 toxin gene and construction of a chimaeric gene for expression in plants.

ABSTRACT

Necessity for the production of transgenic crop plants of India, capable of expression of insecticidal Bt protein in plant to combat lepidopteran pest damage has been strongly felt. Bacillus thuringiensis Kurstaki HD73 crystal protein coded by CryIA(c)73 gene has been found to be sufficiently effective against the major pests of jute and chickpea. An attempt to isolate the gene and make its use in a chimaeric gene construct for expression in plant was carried out. The plasmid CryIA(c)73 gene was cloned and tailored at the 3' end. The expression of the truncated gene was monitored in the minicell systems of E. coli. The entomocidal property was found to be fully retained by the gene product. Deletion of the nucleotides at the 5' end was carried out and chimaeric gene construct of cryIA(c)73 was made in such a way that it was fused in frame with GUS gene under the control of the caMV 35S promoter with Nos polyadenylated terminus. Such a chimaeric gene construct was used as the passenger of a Ti plasmid derived plant vector with kanamycin gene (NPTII) as the additional plant marker. Transformation through infection of tobacco and mustard plant cells in culture was carried out. Plants regenerated from the transformed cells showed the presence of GUS gene indicating the expression of the cloned fused gene. Also, Northern analysis established the presence of cryIA(c)73 gene transcripts in the transgenic plants.