Gene Cloning and Characterization - Genetics, Genomics and Breeding of Crop Plants - page 190

thaliana ( AtL1L ) and Phaseolus coccineus ( PcLIL ) were used to amplify cDNAs
derived from immature zygotic embryos of H. annuus (Fambrini et al. 2006).
The rapid amplification of cDNA ends (RACE) approach was used to isolate
the 5' and 3' ends of the cDNA. The reconstructed full length cDNA sequence
(840 bp) from sunflower contained 642 bp coding sequence, 39 nucleotides
of a 5'-untranslated region (UTR) and 159 nucleotides of 3'-UTR. The
nucleotide sequence represents the entire region coding for a putative peptide
of 214 amino acids. The sunflower HaL1L gene encodes a heme-activated
protein 3 (HAP3) subunit of the CCAAT box-binding factor. HaL1L transcripts
are accumulated primarily at the early stages of zygotic embryogenesis. In
order to see whether HaL1L transcription also played a role in somatic
embryogenesis, the mutant EMB-2 was investigated. This mutant represents
a somaclonal variant, which had been previously induced in vitro from leaf
explants of the tetraploid ( 2n = 4 x = 68) interspecific hybrid Helianthus annuus
x H. tuberosus (Fambrini et al. 2000). EMB-2 produces ectopic embryo and
shoot-like structures, arranged along the veins in clusters. The epiphyllous
proliferation of ectopic embryos on EMB-2 leaves was associated with HaL1L
mRNA accumulation (Fambrini et al. 2006). In conclusion, transcription of
the HaL1L gene is maintained both in zygotic and somatic embryogenesis.
HaL1L could be involved in switching the somatic cell fate towards
embryogenic competence (Fambrini et al. 2006).
Knotted1 -like homoeobox ( KNOX ) genes are expressed in specific
patterns in the plant meristems and play an important role in maintaining
meristematic cell identity (Michelotti et al. 2007). For cloning of the H. tuberosus
knotted1 -like gene ( HtKNOT1 ), total RNA from H. tuberosus was used with
the Superscript Preamplification kit (Invitrogen) to produce cDNA
(Chiappetta et al. 2006). Reverse transcription was carried out with
Superscript II retrotranscriptase in the presence of the adapter primer (AP,
5'-GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTT-3') provided with
the kit. The cDNA was then obtained by PCR with the following degenerate
primers: P-1, 5'-CCDGMDYTRGAYCARTTCATGG-3' (forward) and P-4,
5'-ATRAACCARTTGTTKATYTGYTTC-3' (reverse). These primers were
selected in a region with the highest conservation among members of the
class I knox genes: KNAT1 and KNAT2 from Arabidopsis (Lincoln et al. 1994),
SBH1 from soybean (Ma et al. 1994) and LET6 from tomato (Chen et al. 1997).
The PCR reaction yielded a PCR product of 450 bp, which was cloned into
the pDrive Cloning Vector (Qiagen). The RACE approach was used to isolate
the 5'- and 3'-ends of the HtKNOT1 cDNA. The full length cDNA sequence
(1,398 bp, DDBJ/EMBL/GenBank database accession No. AJ519674)
contained 1,089 bp CDS, 54-nucleotides of 5'-untranslated region (UTR),
and 255-nucleotides of 3'-UTR. The HtKNOT1 gene encodes a predicted
protein of 362 amino acids with a calculated molecular weight of 40.2 kDa.
Expression analyses of HtKNOT1 in stems, inflorescence meristems, floral