Gene Cloning and Characterization - Genetics, Genomics and Breeding of Crop Plants - page 211

Knotted-1 like genes constitute a family of genes, whose products are
transcription factors involved in several aspects of plant development. Tioni
et al. (2003) had isolated three knotted-1 like genes in sunflower and analyzed
their expression patterns. One of the genes, HAKN1 , was highly expressed
in leaves and roots, whereas other gene, HaKN2 , showed preferential
expression in stem, especially in fascicular and interfascicular cambium
and phloem (Tioni et al. 2003). The interaction of the homeodomain of the
sunflower HAKN1 transcription factor with DNA was studied by site-
directed mutagenesis, hydroxyl radical footprinting and missing nucleoside
experiments (Tioni et al. 2005). For this, the homeodomain of HAKN1 was
expressed in E. coli as a fusion protein with the maltose binding protein
using the vector pMALc2. The fusion protein was purified by affinity
chromatography in amylose resin and used for DNA-protein interaction
studies. A 24-bp nucleotide (HAKN1 binding site, BS1) containing the
sequence TGT(G/C)ACA was used as the DNA target. Electrophoretic
mobility shift assays were performed with HAKN1 and the oligonucleotide
BS1 and its variants containing changes at single positions. Based on the
analyses, a model for the interaction of HAKN1 with DNA was proposed
(Tioni et al. 2005).
The protein PLIM-1 from sunflower is expressed exclusively in mature,
free pollens (Baltz et al. 1996). The pollen protein as well as some of the
mutant forms created by site-directed mutagenesis were expressed in E. coli
to obtain protein for studies of its interactions with nucleic acids. The PLIM1
protein bound DNA and RNA in vitro to form large complexes, while the
mutant polypeptides lost the ability (Baltz et al. 1996).
Serrano-Vega et al. (2005) studied the substrate specificity of an acyl-
acyl carrier protein (ACP) thioesterase, which determines the fatty acids
available for biosynthesis of storage and membrane lipids in seeds. Through
the heterologous expression of HaFatA1 in E. coli , the acyl-ACP-thioesterase
FatA1 was purified and characterized, showing that sunflower HaFatA1
cDNA encodes a functional enzyme with preference for monounsaturated
acyl-ACPs. The HaFatA1 thioesterase was most efficient (kcat/Km) in
catalyzing oleoyl-ACP, both in vivo and in vitro. Finally, using available
structure prediction models, a 3D model of plant acyl-ACP thioesterases
was proposed. In addition, the model was tested by mutating the residues
proposed to interact with the ACP protein in the FatA thioesterase by site-
directed mutagenesis (Serrano-Vega et al. 2005).
6.5 Conclusions
In the recent years, cloning and characterization of genes from sunflower
have made a huge progress. Although, so far, no gene has been successfully
cloned by map-based cloning attempts, candidate gene approach has proved