Gene Cloning and Characterization - Genetics, Genomics and Breeding of Crop Plants - page 210

to be essential to provide or perceive an appropriate signal for the initiation of
axillary meristems during both vegetative and reproductive phases.
The isolation of pigment-deficient mutants is crucial to identify genes
controlling chloroplast biogenesis (Hennigsen and Stummann 1982; Leister
2003). In H. annuus , pigment deficient mutants have occurred spontaneously
(Rodriguez et al. 1998; Fambrini et al. 2004) or have been induced by in vitro
culture (Barotti et al. 1995). The nuclear mutation xantha1 ( xan1 ) is
characterized by many pleiotropic effects (Fambrini et al. 2007). Homozygous
xan1 / xan1 seedlings under field conditions die after depletion of
cotyledonary reserves and can only be recovered by selfing normal
heterozygous plants ( Xan1 / xan1 ). These mutants show aberrant
development of chloroplasts, deficient pigment content and reduced CO 2
assimilation rate.
6.4.4 Site-directed Mutagenesis and Chimeric Constructs
To understand the developmental regulation of plant sHSP genes, the
promoter and regulatory elements need to be cloned and analyzed
(Almoguera et al. 1998). Promoter and 5'-flanking sequences of Ha hsp17.7
G4 (Coca et al. 1996), a small heat shock gene from sunflower, have been
isolated and shown to confer developmental regulation in zygotic
embryogenesis using chimeric constructs. However, deletion analyses did
not separate heat shock response from developmental regulation. Site-
directed mutagenesis resulting in sequential nucleotide substitutions in the
heat shock element regions of the Ha hsp17.7 G4 promoter was performed
(Almoguera et al. 1998). The substitutions were introduced by PCR
amplification of plasmid DNA using Pfu DNA polymerase and different
olignucleotides containing the desired substitutions. A two-step PCR (Chen
and Przybyla 1994) was used, in which “megaprimers” containing the
mutations were obtained after the first round of amplification, and gel-
purified for utilization in subsequent reaction, which amplified DNA
fragments that were flanked by restriction sites in the plasmid polylinker.
Obtained promoter sequences were combined with gus for expression
analyses in tobacco (Almoguera et al. 1998). Dual regulation of the Ha hsp17.7
G4 promoter during embryogenesis could be demonstrated. Thus, whereas
activation of the chimeric genes during early maturation stages did not
require intact heat shock elements (HSE) in the promoter region, expression
at later desiccation stages was reduced by mutations in both the proximal
(-57 to -89) and distal (-99 to -121) HSE. In contrast, two point mutations in
the proximal HSE that did not severely affect gene expression during zygotic
embryogenesis eliminated the heat shock response of the same chimeric
gene in vegetative organs (Almoguera et al. 1998).