Characterization Of Important Functional Genes From Bacillus Thuringiensis

Abstract Insect resistance has been a key problem in the use of Bacillus thuringiemis. Great efforts are presently being undertaken by many researchers in different universities or institutes to look for new B. thuringiensis cry genes. PCR-RFLP has been successfully used for rapid detection and characterization of toxin genes of B. thuringiensis. The identification systems of cry1-cry4, cry7, cry9 and cry 10 have been established by other researchers. In the present study, cry5, cry6, cry8 and cry11 identification systems were established and cry1-cry11-type genes of 36 B. thuringiensis strains (including 6 standard stains and 30 Wuyi Mountain isolates) had been determined by the PCR-RFLP systems. B. thuringiensis strains harboring cry1 represented 88.9% of the strains while 61.1% and 2.8% of the strains contained cry2 and cry11 genes, respectively. None strains harboring cry3, cry4, cry5, cry6, cry7, cry8, cry9 or cry10 were detected. Twenty-two different cry gene profiles (cry1Aa, cry1Ab, cry1Ac, cry1Ad, cry1Ba, cry1Cb, cry1Db, cry1Ea, cry1Eb, cry1Fa, cry1Fb, cry1Ga, cry1Gb, cry1Ka, cry1La, cry1Ia, crylIb, cry2Ab, cry2Ac, cry2Ad, cry2Ae and cry11Ad) were detected in the analysis. The first generation of transgenic cotton produces a single toxin, CrylAc. The evolution of resistance to both B. thuringiensis subspecies and individual toxins has been demonstrated in the laboratory for several kinds of insects and found in field population of Plutella xylostella. Because Cry2Ab and CrylAc share limited identity, strong cross-resistance between transgenic plants producing these toxins is impossible. Second-generation transgenic cotton that produces B. thuringiensis toxin Cry2Ab alone or in combination with CrylAc has been developed abroad. To date, there are 7 cry2Ab genes and 1 cry2Ad gene in the Bacillus thuringiensis toxin nomenclature database (http://www.lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/). In this study, two cry2-type genes, designated cry2Ab8 and cry2Ad2 by the database, had been cloned and sequenced from B. thuringiensis Wuyi Mountain isolates WB2 and WB10, respectively. The sequence analysis revealed both of them have an open reading frame of 1902 bp, encoding a protein of 633 amino acid residues. A computer based homology search program of NCBI revealed that the deduced amino sequence of cry2Ab8 shared maximum 99.5% identity with the known Cry2Ab toxins. When Cry2Ab8 was compared with those of other Cry2Ab proteins, 3 common substitutions, Ala411Val, Ile629Leu and Ser630Pro, were found. The alignment of the deduced amino sequence of cry2Ad2 with...