Identification of a Bt toxin Cry1Ab resistance gene in the silkworm, Bombyx mori
Bombyx mori was used as a model for studying the genetics of Bt toxin resistance in this research. Bt toxin is a family of polypeptides produced by Bacillus thuringiensis, a Gram-positive bacterium, during sporulation. The Bt toxin is hypothesized to kill several lepidopteran species by forming pores in the midgut cell membrane and causing cell death due to homeostatic imbalance. Its increasing use as a pesticide brings the risk of developing resistant strains through genetic mutation and environmental selection. Mapping and isolating the mutant genes that confer resistance in different lepidopteran species can provide information needed to understand relationships between species and possibly the mechanism of the toxin resistance. Previous genetic studies in this and other laboratories using a standard leaf smear bioassay with neonate progeny from F1 crosses between resistant and sensitive strains and backcrosses between F1 females and resistant males showed that resistance to toxin Cry1Ab was recessive in several strains of B. mori; further, complementation tests among resistant strains of different geographic origin indicated that the majority of the resistance was caused by a single Bt-r mutation. Based on a map using restriction fragment length polymorphisms that localized resistance to chromosome 15 (W. Hara, Y. Kim and K. Miyamoto, personal communication), primers were designed to flank the intron regions within the DNA sequence for PCR amplification of known and annotated genes of B. mori chromosome 15 including Boceropsin (GenBank Acc. No. AB064496), Bmm101 (GenBank Acc. No. AU235098), CM22 (GenBank Acc. No. BY914068), and 9 other regions in order to detect polymorphisms in progeny DNA. Using these and other anchor loci, the location of a major Bt-r mutation was confirmed on chromosome 15 in female informative backcrosses using resistant Japanese strains 106 and 214, and sensitive European strain 555, and localized it between Boceropsin and Bmm101 (∼1.67Mbp range), based on 101 (strain 106) and 88 (strain 214) progeny surviving a discriminating dose of toxin in male informative backcrosses. With additional sequencing data evidence and gene model prediction (S. Atsumi, Y. Kim, and K. Miyamoto, personal communication), the Cry1Ab resistance was confirmed to result from the same mutation in several B. mori strains. The mutation I identified in this study also suggested it may be related to an ATP-binding cassette (ABCC2) transporter which was recently proposed as a resistance mechanism which is different from two known ones (Gahan et al. 2010). The genetic mapping method used here for detecting and identifying Bt toxin resistance confirmed a new genetic basis of the resistance mechanism by using B. mori as the model organism and molecular markers that can be helpful for mapping the same gene for other species in the future. ^
Biology, Molecular|Biology, Genetics
"Identification of a Bt toxin Cry1Ab resistance gene in the silkworm, Bombyx mori"
Dissertations and Master's Theses (Campus Access).