Gene Cloning and Characterization - Genetics, Genomics and Breeding of Crop Plants - page 207

Apart from Arabidopsis , tobacco was used to test the function of genes
isolated from sunflower. Chimeric constructs containing the promoter and
upstream sequences of Ha hsp17.6 G1 , a small heat shock protein gene,
reproduced in transgenic tobacco ( Nicotiana tabacum ) its unique seed-specific
expression patterns previously reported in sunflower (Carranco et al. 1999).
Seed-specific overexpression of the transcription factor HaHSFA9 ( H. annuus
heat-stress factor A9) in transgenic tobacco was linked with increased seed
longevity, and with the overaccumulation of a subset of small heat-shock
proteins (sHSPs) in seeds (Prieto-Dapena et al. 2006). In addition, Prieto-
Dapena et al. (2008) could demonstrate that the constitutive overexpression
of the seed-specific HaHSFA9 transcription factor under control of the CaMV
35S promoter is sufficient to confer tolerance to severe dehydration, outside
of the developing seed context, to vegetative tissues of transgenic tobacco.
For studying genes from the fatty acid biosynthesis, expression of these
genes in E. coli proved to be valuable. To characterize the enzyme activities
from the stearoyl-ACP-desaturase and the acyl-ACP thioesterases FatA and
FatB from sunflower seeds Serrano-Vega et al. (2003, 2005) cloned, sequenced
and overexpressed the recombinant genes in E. coli .
6.4.3 Mutant Analyses
6.4.3.1 Mutants in Fatty Acid Synthesis
With regard to fatty acid synthesis, the most well known mutation in
sunflower is the pervenet mutation, obtained by treating a wild type
population (VNIIMK83) with dimethylsulfate that resulted in the first high-
oleic sunflower cultivar (Soldatov 1976). The oleoyl-phosphatidyl choline
desaturase ( FAD2 ) is necessary for the synthesis of linoleic acid from oleic
acid (Ohlrogge and Browse 1995). Three FAD2 genes ( FAD2-1 , FAD2-2 and
FAD2-3 ) have been identified in sunflower (Hongtrakul et al. 1998b; Martinéz-
Rivas et al. 2001). Whereas FAD2-1 is strongly expressed in developing
seeds (Hongtrakul et al. 1998b), FAD2-2 and FAD2-3 are weakly expressed
(Martinéz-Rivas et al. 2001). Isolation and sequencing of the corresponding
FAD2 cDNA clones from the high-oleic variety, designed HA-OL9 FAD2-1 ,
HA-OL9 FAD2-2 , and HA-OL9 FAD2-3 , showed 100% identity to the
corresponding FAD2 sequences from the normal type variety (Martinéz-
Rivas et al. 2001). However, Hongtrakul et al. (1998b) as well as Lacombe
and Bervillé (2001) discovered that FAD2-1 was duplicated and very weakly
expressed in mutants homozygous for the Ol mutation. Schuppert et al.
(2006) further investigated the duplication of the FAD2-1 . The upstream
repeat ( FAD2-1U ) carries a 1.69 kb intron in the 5'-UTR, whereas the
downstream repeat ( FAD2-1D ) is missing the first 1.54 kb of the 5'-UTR and
intron. FAD2-1 maps to LG 14 of the public sunflower map (Schuppert et al.
2006). Novel FAD2-1 alleles were found in exotic low-oleic genotypes.