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Legume Genomics and Genetics (online), 2011, Vol. 2, No.2, 6-13
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7
The study of
ADH
gene was initiated in corn (
Zea
mays
).
ZmADH1
gene and
ZmADH2
gene were cloned
successfully from corn (Gerlach et al., 1982). The 82%
homology of the deoxyribonucleic acid (DNA) was
between two genes whereas 87% homology in the
sequences of amino acids, which indicated that both of
the genes possible derive from the common ancestor
although the location of the genes in the genome are at
different chromosomes.
ZmADH1
was located at chro-
mosome 1 and
ZmADH2
was located at chromosome 4.
So far, there are some reports on
ADH1
and
ADH2
genes cloned in grape (
Vitis vinifera
), potato (
Solanum
tuberosum
), rice (
Oryza sativa
) and wheat (
Triticum
monococcum
). However, only one
ADH
gene,
AtADH1
,
was reported in Arabidopsis, which located at chromo-
some 1, whose sequences of nucleotides in genome
distributed from 28 980 288 to 28 982 311. While,
there was no any annotation about
AtADH2
. In
Lotus
,
a model plant of legume, the
LcADH1
(Gen- Bank ID:
AAO72531.1) had been identified from tetraploid
species,
Lotus corniculatus
, whereas there was no any
report about the gene,
LjADH1
or
LjADH2
cloned
from
Lotus japonicus,
a popular diploid species so far.
Lotus plant belongs to the genus
Lotus
L. in subfamily
of
Papillionoideae
of Leguminosae family.
Lotus
japonicus,
a diploid species has been instead of tetra-
ploid species of
Lotus corniculatus
used for genetic
study because of small genome size of about 470 Mb,
diploid genome with 6 haploid chromosomes (2n=12)
and a short life cycle of about 2 to 3 months. With the
completion of whole genome sequenced,
Lotus japo-
nicus
has become a convenient model plant for the
studies of genome and genetics in legumes, particu-
larly in reference to rhizobial and arbuscular myco-
rrhizal symbiosis since in the early 1990s (Handberg
and Stougaard, 1992). Based on the conservative char-
acteristics of
ADH
genes, we used MG20, a
Lotus
japonicus
germplasm originated from Miyako Island
of Japan (Kawaguchi, 2000), as experimental mate-
rials to clone homologous
ADH
gene from
Lotus japo-
nicus
cDNA, to express the gene in
E. coli
and yeast
and to characterize the enzyme in this study.
1 Results and analysis
1.1 Cloning and sequence analysis of
LjADH1
In this research we designed a pair of primers based
on the information of
LcADH1
to amplify the
ADH
analog using cDNA from
Lotus japonicus
MG20 as
template. The analog was cloned from MG20 germ-
plasm and then sequenced, which was 1 143 bp in len-
gth encoding 380 amino acids. The further sequence
alignment indicated that the cloned sequence had 99%
homology to an unannotated
Lotus japonicus
sequ-
ence deposited in the GenBank (GenBank ID: CAG
30579.1). We conferred the sequence as
ADH1
of
Lo-
tus japonicus
named as
LjADH1
(GenBank Accession
No.: JN165714).
Multiple sequence alignment was conducted among
putative LjADH1 protein and other ADH proteins
from
Zea mays
,
Triticum monococcum
,
Oryza sativa
,
Arabidopsis thaliana
,
Lotus corniculatus
,
Aegilops
speltoides
,
Vitis vinifera
,
Solanum tuberosum
and
Miscanthus transmorrisonensis
, respectively (Figure 1),
the results showed that the cloned protein sequence
had more than 95% homologous with other ADH
sequences except MtADH (80%). The analysis indi-
cated that the cloned sequence has common ADH
structures of plant ADH including zinc-binding sites,
ADH_N domain and NADB_Rossmann conservative
functional domain. Phylogenetic analysis further
demonstrated that
ADH
gene from
Lotus japonicus
shared the same evolutionary branch with
AtADH
of
Arabidopsis
thaliana
(Figure 2).
1.2 Construction of prokaryotic expression vector
In order to express
the LjADH1
gene in
E. coli
, We
employed the prokaryotic expression vector pQE30,
which has a 6 histidine tag, little influence on targeted
proteins and easy to be purified (Wang et al., 2010;
Liu et al., 2010; 2011), to make the heterologous ex-
pression construct pQE30-LjADH1. The construct
was validated by digestion of
Bam
H
Ⅰ
and
Sac
Ⅰ
to
produce 3.4 Kb and 1.1 Kb fragments by agarose gel
electrophoresis (Figure 3), those fragments in length,
were in accord with the size of pQE30 and
LjADH1
sequence.
1.3 Expression of fusion protein and concentration
determination
We transformed pQE30-LjADH1 into
E. coli
M15
strains to express LjADH1 fusion protein in
E. coli
and optimized the conditions of fusion protein expre-
ssion. The result showed that fusion proteins were ex-
pressed by inducing of IPTG at 0.1 mmol/L of IPTG
concentration, with the optimum induction time 3 h to
4 h and induction temperature at 30 (Figure 4). The
℃
concentration of purified fusion protein 6×His-LjADH1