The cloning of <i>ABS2</i>.

Abstract

<p>A. <i>abs2-1D</i> was genetically linked with T-DNA. Total leaf DNAs were extracted from 16 progenies of an <i>abs2-1D</i>/+ heterozygous plant. The DNAs were digested with <i>Hind</i>III and restriction fragments were separated with electrophoresis followed by transfer to a nylon membrane. The blot was probed with <sup>32</sup>P labeled <i>BAR</i> gene sequences. Plants that did not show <i>abs2-1D</i> phenotypes were indicated by arrows. B. Confirmation of a single T-DNA insertion in <i>abs2-1D</i>. Genomic DNAs from <i>abs2-1D</i> plants were digested with indicated restriction enzymes. After electrophoresis and transfer to a nylon membrane, the blot was hybridized with <sup>32</sup>P labeled <i>BAR</i> gene sequences. There is one <i>Eco</i>RI site in the probe sequence so two hybridizing bands were observed. C. Cloning of <i>abs2-1D</i>. In the <i>abs2-1D</i> mutant, activation tagging T-DNA was inserted between At2g36080 and At2g36090. Solid lines represent intergenic regions, while white boxes represent genes in the vicinity of the T-DNA insertion. The right border of the T-DNA was facing At2g36080. D. Semi-quantitative RT-PCR analysis of the expression levels of At2g36080 and At2g36090 in wild-type, <i>abs2-1D/+</i> heterozygous and <i>abs2-1D</i> homozygous mutants. <i>Actin2</i> expression was shown as a control. Total cellular RNAs were extracted from the aerial parts of two-week-old seedlings. 1 µg DNase I treated RNA from each sample was used for cDNA synthesis. RT-PCRs were performed with indicated numbers of cycles.</p

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