Figure 2
Southern analysis of chromosomal DNA of N. attenuata transformed with binary vectors containing antisense-cDNA for N. attenuata hydroperoxide lyase ( hpl ), allene oxide synthase ( aos ), lipoxygenase ( lox ) and digested with Ssp I. A) T-DNA of transformation vector pNATHPL: PCaMV/TCaMV, CaMV 35S promoter/terminator: PNOS/TNOS NOS promoter/terminator, as-hpl, antisense hpl; sat-1, nourseothricin resistance gene; LB/RB, left/right border of T-DNA; SspI, recognition site for restriction enzyme; s, amplicon ampNAT1 (conventional TaqMan® probe); n, amplicon ampNOT2 (Minor-Groove-Binder probe); Southern probe, 260 bp radioactive PCR-labeled probe for Southern blot; vectors pNATAOS, pNATLOX have the same organisation as pNATHPL except that that as-hpl is replaced by as-aos or as-lox, respectively. B) Southern blots of progeny of the hemizygous line A443-1 transformed with pNATHPL, 21 plants were examined (plants 1–24, except 10 and 17; *, DNA of plant Nr. 16 was blotted, but not included into the segregation ratio, because of low concentration); 5 plants without T-DNA, 16 plants with T-DNA: result corresponds to the sensitive/resistant ratio of 1:3 in segregration analysis of a hemizygous line; P (plasmid), 2 ng pNATHPL; WT, wild-type DNA. C) Southern blots for progeny of homozygous lines (2 from each line) transformed with pNATHPL, pNATAOS or pNATLOX; 1 as-hpl line and 2 as-lox lines show two bands on the blot, indicating two insertion sites for the T-DNA; P (plasmid), 2 ng pNATHPL; WT, wild-type DNA.