Journal: Scientific Reports
Article Title: ABC transporter mis-splicing associated with resistance to Bt toxin Cry2Ab in laboratory- and field-selected pink bollworm
Figure Lengend Snippet: Mutations affecting PgABCA2 protein in Cry2Ab-resistant pink bollworm from Arizona and India. ( a ) The predicted PgABCA2 protein includes amino (N) and carboxyl (C) termini (pink), two transmembrane domains (TMD1 and TMD2), each consisting of 6 transmembrane regions (TM; orange), three extracellular loops (ECL; green), two intracellular loops (ICL; blue), and two nucleotide-binding domains (NBD; purple). Mutations affecting transcripts of resistant pink bollworm: Circles show premature stop codons from India (red), Arizona (yellow), or both (red and yellow). Triangles show in-frame indels from India (red) or Arizona (yellow). Numbers indicate the affected amino acids. ( b ) Full-length and partial PgABCA2 cDNAs were obtained by direct PCR sequencing, DNA sequencing of cDNA clones, and/or PacBio ® DNA sequencing from susceptible (APHIS-S) and resistant, laboratory-selected (Bt4-R2) pink bollworm from Arizona, USA and India field-selected resistant populations (AM, CK, GAP, KT, and RK). The linear schematic (top) shows the predicted translated domain structure of the 5,187-bp full-length PgABCA2 coding sequence. The predicted protein includes amino- and carboxyl-termini (pink), transmembrane regions TM1-TM12 (orange), intracellular loops ICL1-ICL5 (blue), and extracellular loops ECL1-ECL6 (green). The domain structure connected to the exons that encode the respective domains is shown by dotted gray lines. Each predicted domain is numbered, with ECLs on top and ICLs numbered on bottom of protein schematic. Putative exons 1–31 are numbered, with grey exons indicating regions determined by direct PCR sequencing. Exons colored in light blue were further verified by sequencing cDNA clones (either by Sanger or PacBio ® sequencing). Red bars indicate disruption sites within the full-length coding sequence and the red triangles indicate the location of premature stop codons shown to scale based on the linear schematic of the translated domain structure. Unique cDNA variants are indicated as a, b, c, etc.
Article Snippet: The symmetric barcode-tailed PCR primers were designed based on the PacBio® multiplex PCR primer guidelines ( http://www.2einteractive.com/pacbio/Shared-Protocol-PacBio-Barcodes-for-SMRT-Sequencing.pdf ).
Techniques: Binding Assay, Polymerase Chain Reaction, Sequencing, DNA Sequencing, Clone Assay