(A) Gene structure of the KCBP gene. Black rectangles indicate exons, gray rectangles indicate the 5′ and 3′ untranslated regions, and thick lines indicate introns. The arrow indicates the location of the T-DNA insertion in the kcbp-1/zwiA mutant. (B) PCR-based identification of the T-DNA insertion. LP and RP indicate a pair of KCBP specific primers. BP indicates the T-DNA border primer. (C) RT-PCR-based identification of the kcbp-1/zwiA allele. Red arrows indicate the position and the direction of primers, which are listed in the Supplementary file 1.
* Sequencing analysis of the PCR product amplified by the LBb1.3/RP primer pair revealed that the T-DNA insertion is in the third exon and located at the position of 957 bp downstream the ATP start codon of the KCBP gene and introduces a premature stop codon 61 bp downstream the insertion site by frame shift. Thus, the kcbp-1/zwiA mutant is expected to produce a short peptide of 284 amino acids containing the first 263 amino acids of KCBP at a lower level. The sequence of the PCR product is pasted below:
Note: The 325-bp fragment, bolded, is the sequence of 633–957 bp downstream the ATG start codon of the KCBP gene; and the remaining 186-bp fragment is the T-DNA sequence. The 21 bp, underlined and bolded, is the sequence of the primer 031704-RP; and the 19 bp, underlined, is the reverse complement sequence of the primer LBb1.3.