Basic HTML Version
Triticeae Genomics and Genetics 2012, Vol.3, No.1, 1
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6
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2
has been suggested to be mainly due to the differential
expression of stress-responsive genes (Joshi et al.,
1997). Many genes respond experimentally to water
stress, however, their precise functions either in
tolerance or sensitivity often remain unclear (Ludlow
and Muchow, 1990; Smith and Griffiths, 1993). It is
thus critical to study functions of stress-induced genes
to understand the mechanisms involved in stress
tolerance in plants. Correlating phenotypic adaptations
with molecular characters should enable us to evaluate
the role of dehydrins during adaptation (Guo et al.,
2009). Therefore, the possible relationship between
diverse molecular trait of DHN6 and phylogenesis
was investigated in two-, four- and six-rowed barley
lines in this research.
1 Results and Analysis
1.1 Cloning and sequence analyses of
Dhn
6
Taken the barley genomic DNA of BZ-26, BZ-16,
BZ-12, respectively as the template, we amplified the
target genes. The results of agarose gel electrophoresis
validation showed that the specific fragments between
1 500 bp and 2 000 bp in length can be obtained, when
the annealing temperature was 55
℃
(Figure 1).
Identification of the recombinant of BZ-12 was further
digested with
EcoR
Ⅰ
and
Hind
Ⅲ
(Figure 2). Sequence
analyses found that the length in BZ-26, BZ-16 and
BZ-12 was 1 657 bp, 1 705 bp and 1 767 bp,
respectively, and the sequence alignment displayed that
the homology among them reached 93.08%. Moreover,
the
Dhn
6 sequence in two-rowed barley (BZ-26) was
the shortest, apart from absence 42 bp nucleotide
sequences at +583 bp , as well as at +787 bp lacking
TGGTGC and at +908 bp lacking GGTGTC starting
from ATG. In addition, another 47 nucleotide sites
appeared mutation. Compared with BZ-26, BZ-16 was
surplus 48 bp nucleotides at +583 bp and +787 bp,
additionally, there were 11 base sites appeared mutation.
However, the surplus of 63 bp appeared at +791 bp in
six-rowed barley (BZ-12). Using the online software
Blastn to retrieve the GenBank database, the results
revealed that the
Dhn
6 gene sequence in this paper
could not be exactly matched with any sequence that
has been submitted, though the sequence homology
was very high, which indicated that
Dhn
6 could be
applied to identify species.
Figure 1 PCR result of BZ-12
Note: M: DL5000 DNA marker; 1: 55
℃
; 2: 60
℃
Figure 2 Identification of the recombinants by PCR and
restriction digestion of BZ-12
Note: M: DL5000 DNA marker; 1: PCR product; 2: Digestion
with
EcoR
Ⅰ
; 3: Digestion with
EcoR
Ⅰ
and
Hind
Ⅲ
1.2 Characteristics of DHN6 amino acid sequences
Analyses of open reading frame (ORF) with ORF
finder showed the ORF in BZ-26 was shortest about 1
458 bp in length, encoding 486 amino acid residues,
and that of BZ-16 and BZ-12 was about 1 506 bp, 502
amino acid residues and 1 569 bp and 523 amino acid
residues, respectively. The amino acid sequence
analyses displayed that the deduced protein (Y2SK2
type) was composed of the highly conversed motifs
including in Y-sequence (2), S-sequence (1) and
K-sequence (2) in three typical rowed barleys (Figure 3).
Comparing amino acid sequences of DHN6 of
different rowed barley with that of hull-less barley
(GenBank accession No. AF043091), we found 21
mutant sites in the deduced protein in those three