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Fig. 7. Schematic representation of intron-containing hairpin constructs designed for genetic transformation. Plasmid construction information: The promoter region of taRAFTIN1a (?1,716 bp from ATG) was retrieved by PCR with OL3861 and OL3862; that of taRAFTIN1b (?2,092 bp from ATG) with OL3863 and OL3862; and the osRAFTIN1 promoter (?1,309 bp from ATG) with OL3142 and OL3143. HindIII-BamHI digested amplicons were cloned into pAMW477 (1) in which the TAA1a coding region had been replaced with a GUS gene from plasmid pRD420 (2) to generate Promoter:GUS chimeras for taRAFTIN1a (pAMW484), taRAFTIN1b (pAMW483), and osRAFTIN1 (pAMW486). Intron hairpin RNA intermediate clone pAMW487 containing taRAFTIN1a promoter, the complementary sequence of osRAFTIN1 cDNA (159 nt from start codon to nucleotide 415), its corresponding sense genomic DNA sequence with additional 114 nt of upstream genomic sequence including an 82-nt intron, and a 35S terminator (named taRAFTIN1a::hp-osRAFTIN1) was constructed by coligation of a BamHI-SpeI restricted PCR fragment of osRAFTIN1 cDNA amplified with OL3888 and OL3889 and an XbaI-EcoRI restricted PCR fragment of osRAFTIN1 genomic DNA with OL3886 and OL3887 into BamHI-EcoRI sites of plasmid pAMW484. Similar strategy was used to make clone pAMW488 containing taRAFTIN1b::hp-osRAFTIN1, clone pAMW489 containing osRAFTIN1::hp-osRAFTIN1 and pAMW503 (35S::hp-osRAFTIN1). Clone pAMW491 containing taRAFTIN1b promoter, complementary sequence of taRAFTIN1a cDNA (181 nt from start codon to nucleotide 544), its corresponding sense genomic sequence with additional 148 nt of upstream genomic sequence including a 99-nt intron), and a 35S terminator (named taRAFTIN1b::hp-taRAFTIN1a) was created by co-ligation of a BamHI-SpeI restricted PCR fragment of taRAFTIN1a cDNA amplified with OL3892 and OL3893 and an XbaI-EcoRI restricted PCR fragment of taRAFTIN1a genomic DNA with OL3890 and OL3891 into BamHI-EcoRI sites of plasmid pAMW485. Similar strategy was used to generate subclones pAMW492 containing osRAFTIN1::hp-taRAFTIN1a and pAMW504 containing 35S::hp-taRAFTIN1a. Subclone pAMW502 containing taRAFTIN1a::hp-taRAFTIN1a was constructed by insertion of the small fragment from BamHI-KpnI double digested pAMW491 into the corresponding sites of pAMW484. The small fragment of clones pAMW487, pAMW488, pAMW 489, pAMW491, pAMW492, pAMW502, pAMW503, and pAMW504 double-digested with HindIII-KpnI was ligated with the small HindIII-KpnI fragment (35S::hph cassette) of plasmid pBShph, and plasmid pHS723 (3) predigested with HindIII and BamHI to obtain intron hairpin RNA transformation vectors pAMW495, pAMW496, pAMW497, pAMW498, pAMW500, pAMW506, pAMW507, and pAMW508, respectively.
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