Basic HTML Version
Legume Genomics and Genetics (online), 2011, Vol. 2, No.2, 6-13
http://lgg.sophiapublisher.com
Research Article Open Access
Cloning and Expression of An Alcohol Dehydrogenase from
Lotus japonicus
and
Characterization of LjADH1
Tuo Zeng
1,2,3
, Shenkui Liu
2
, Ruye Luo
1,3
, Pengtao Gong
2,3
, Degang Zhao
1
, Xuanjun Fang
1,2,3
1. Guizhou Key Lab of Agro-Bioengineering, Guizhou University, Guiyang, 550025, China
2. Alkali Soil Natural Environmental Science Center (ASNESC), Northeast Forestry University, Harbin, 150040, China
3. Hainan Key Lab of Crop Molecular Breeding, Hainan Institute of Tropical Agricultural Resources (HITAR), Sanya, 572025, China
Corresponding author email:
;
Authors
Legume Genomics and Genetics 2011, Vol.2 No.2 DOI:10.5376/lgg.2011.02.0002
Received: 03 Apr., 2011
Accepted: 09 Jun., 2011
Published: 28 Jun., 2011
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Preferred citation r this article as:
fo
Zeng et al., 2011,
Cloning and Expression of An Alcohol Dehydrogenase from
Lotus japonicus
and
Characterization of LjADH1, Legume Genomics and
Genetics (online), Vol.2 No.2 pp.6-13 (doi:10.5376/lgg.2011.02.0002)
Abstract
Alcohol dehydrogenase (ADH), usually using NAD
+
and NADP
+
as coenzymes, widely exists in many organisms, It
plays extremely important roles in growth, development and stress resistance in plants. In this research, we used diploid
Lotus
japonicus
MG20 (originated from Miyako Island of Japan) as plant material to identify the characteristics of alcohol dehydrogenase
gene. Based on the conservative sequences of
ADH
, The
ADH
homologous gene was cloned from
Lotus japonicus
MG20
cDNA,
whose full length gene was 1 143 bp in length encoding 380 amino acids. Homologous analysis showed that the amino acid sequence
of the cloned gene was highly homologous with plant zinc-binding ADH family proteins. We have this gene named as
LjADH1
and
ligated the gene into the prokaryotic vector pQE30 and yeast expression vector pYES2 to make the recombinant vectors of
pQE30-LjADH1 and pYES2-LjADH1 respectively and then were transformed into
E. coli
M15 and yeast
INVScl
. Under the
optimum conditions of expression, the His-tag fusion proteins were highly expressed with 1.12 mg/mL in
E. coli
and 48.2 U/mg
ADH activity examined by the method of Vallee and Hoch. We found that
LjADH1
over expressed in prokaryotic cells can increase
the recombinant strains’ tolerance to H
2
O
2
stress, while
LjADH1
expressed in recombinant yeast can promote growth of the
recombinant yeast under the stress of H
2
O
2
and some heavy metal salts such as CuCl
2
and CdCl
2
except for NiCl
2
. In this research we
have preliminary clues that
LjADH1
is a member of zinc-binding ADH family proteins in plant and that has some functions for
resistance to abiotic stresses.
Keywords
Lotus japonicus
; Alcohol dehydrogenase;
LjADH1
(GenBank Accession No.: JN165714); Prokayotic expression; Yeast
expression; Abiotic stress
Background
Alcohol dehydrogenases (ADH, EC1.1.1.1) are a
group of dehydrogenase enzymes that have extensive
zymolytes and catalyze the interconversion between
alcohols and aldehydes. With the reduction of nicoti-
namide adenine dinucleotide (NAD
+
to NADH), ADH
facilitate the dehydrogenation of primary alcohols or
secondary alcohols, aldehydes and ketones to lose
their hydrogen, In this reaction, the NAD
+
gains dehy-
drogen and become NADH.
ADH widely occurs in all kinds of organs in plant,
which involves in development, maturation and sen-
escence of fruits
, as well as in
the resistant responses to stresses. ADH can be indu-
ced to express by dehydration, cold (Dolferus et al., 1994)
and oxygen deficit (Newman and Vantoai, 1992), as well
as ADH has some links to Ca
2+
signal transduction
(Hwa-Jee and Ferl, 1999). Most of ADH in plants
have the structures with the length of amino acid se-
quence from 370 to 380, which evenly consist of three
folding structures, helix, folding and curling. There is
little difference of secondary structure in ADH among
the plant species, particularly in the parts of conser-
vative function domains. Even though there is a few
differences in amino acid sequences among indivi-
duals, the difference in folding status of peptide chain
is usually very small, which indicates highly conservative
secondary structures should maintain the functional
stabilities. Meanwhile, the highly conservation of ADH
sequences in all kinds of species indicated that the
ADH play an important role in plant growth, so that
any slight mutation in ADH will result in death of plant.
6