Gene Cloning and Characterization - Genetics, Genomics and Breeding of Crop Plants - page 191

meristems and floral organs revealed a complex expression pattern
(Michelotti et al. 2007). HtKNOT1 may play a dual role being required to
maintain the meristem initials as well as initiating differentiation and/or
conferring new cell identity.
The sequence information from a group 1 late embryogenesis-abundant
(Lea) cDNA clone of sunflower was used to isolate the Ha ds10 G1 gene from
genomic DNA (Prieto-Dapena et al. 1999). This gene contained an intron at
a conserved position with a size of 1,024 bp. Transcription from the Ha ds10
G1 promoter was strictly seed-specific and originated from at least two close
initiation sites. Almoguera et al. (2002) were the first to report cloning and
functional characterization of a heat shock transcription factor that was
specially expressed during embryogenesis in the absence of environmental
stress in sunflower. To isolate trans -acting factors involved in the
developmental activation of small heat shock protein (sHSP) gene promoters
the yeast one-hybrid cloning approach was used (Li and Herskowitz 1993).
A cDNA library specific for sunflower embryos containing 830,000
individual transformants, all with an average cDNA insert of 1.3 kb, was
established. For one-hybrid screening the reporter yeast strain was
transformed with DNA prepared from the embryo cDNA library, after
amplification of 1,660,000 primary clones. Five million yeast transformants
were plated on SD-His-Leu + 15 mM 3-aminotriazole. Twenty-four putative
positive yeast clones were selected. Four of the cDNAs encoded the same
heat shock transcription factor, which was named HaHSFA9 (Almoguera et
al. 2002) because it was very similar to AtHSFA9 described previously (Nover
et al. 2001). In sunflower, HaHSFA9 proved to be a transcription factor
involved in the developmental activation of the two small heat shock protein
genes Ha hsp17.6 G1 and Ha hsp17.7 G4 . The activation is strictly restricted
to the embryo (Almoguera et al. 2002). Using the Ha hsp17.6 G1 promoter as
bait (Díaz-Martín et al. 2005), the same yeast one-hybrid technique was
used to clone the corresponding transcription factor, named sunflower
drought-responsive element binding factor 2 (HaDREB2). Functional
analysis of the interaction of the two transcription factors HaHSFA9 and
HaADREB2 demonstrated that both transcription factors synergistically
trans-activate the Ha hsp17.6 G1 promoter in bombarded sunflower embryos
(Díaz-Martín et al. 2005).
Carotenoid biosynthesis takes place in the plastid, but all known
enzymes in the pathway are nuclear-encoded and post-translationally
imported into the organelle (reviewed in Sandmann 2001). For cloning
the cDNA of the sunflower phytoene synthase gene ( HaPsy ),
amino acid sequences of previously cloned Psy genes were aligned
(Salvini et al. 2005). Regions with the highest conservation were identified
and two degenerate oligonucleotide primers were designed:
P-A, 5'-TTCCKGGGASTTTGRGYTTGTTG-3' (forward) and P-B,