Gene Cloning and Characterization - Genetics, Genomics and Breeding of Crop Plants - page 198

Homeodomain-leucine zipper (Hd-Zip) proteins constitute a family of
transcription factors found only in plants. For isolation of partial cDNA
clones containing homeobox sequences, a PCR-based strategy was employed
on total DNA from a sunflower stem cDNA library constructed in lambda
gt10 as previously described (Gonzalez and Chan 1993). One of the clones
represented a member of the Hd-Zip family that was named Hahb-4 (Gago et
al. 2002). The full-length cDNA sequence was 674 bp long and contained an
ORF of 177 amino acids. In order to investigate the genomic structure of the
Hahb-4 gene, genomic DNA was amplified with several oligonucleotides
comprising the entire cDNA. A single intron of 101 bp was detected between
nucleotides 381 and 382. To isolate the promoter region of Hahb-4 , inverse
PCR was used on genomic DNA. A 720 bp fragment was isolated that
contained the TATA box 24 bp upstream from the transcription initiation
and two putative ABRE in the -290 and -165 regions (Gago et al. 2002). The
transcription factor Hahb-4 is upregulated by drought and ABA in roots,
stems and leaves (Gago et al. 2002).
Recently, a sunflower cDNA microarray containing about 800 clones
covering major metabolic and signal transduction pathways was used to
study gene expression profiles in leaves and embryos of drought-tolerant
and -sensitive genotypes subjected to water deficit under field conditions.
In total, 409 genes were proven to be differentially expressed among
genotypes, water treatment and organs (Roche et al. 2007).
6.3.4 Disease Resistance
The identification of common domains like nucleotide binding site (NBS),
leucine rich repeats (LRR) and toll interleukin receptor (TIR) in cloned
resistance genes (Mindrinos et al. 1994; Whitham et al. 1994; Lawrence et al.
1995) made candidate gene approach for resistance genes in other species
possible (Meyers et al. 1999; Dangl and Jones 2001; Jones and Dangl 2006).
Conserved amino acid sequence motifs in the NBS domain have been widely
used to isolate and classify NBS-LRR encoding genes (Meyers et al. 1999;
Pan et al. 2000).
To date, 11 genes have been postulated to provide resistance to one or
more races of downy mildew ( Plasmopara halstedii ) in sunflower (Rahim et al.
2002). Apart from map-based cloning attempts, candidate gene approach has
been used to clone the genes corresponding to these resistances. Gentzbittel et
al. (1998) used degenerate primers designed from conserved NBS domains of
N from tobacco (Whitham et al. 1994), RPS from A. thaliana (Mindrinos et al.
1994) and L6 from flax (Lawrence et al. 1995) to clone resistance gene analogs
(RGAs). The amplification products represented a multigene family. One of
the clones was sequenced and mapped close to the Pl 6 gene. Gedil et al.
(2001a) placed Pl 1 and six RGCs of the NBS-LRR type on the HA370 x HA372