Page 9 - Legume Genomics and Genetics

Legume Genomics and Genetics (online), 2011, Vol. 2, No.2, 6-13
http://lgg.sophiapublisher.com
11
hol dehydrogenase activity. In respect to stress tole-
rance of fusion proteins over-expressed in the E. coli
and yeast, the recombinant strains grew well than that
of the wild type. While, LjADH1 gene was ligated into
the yeast vector pYES2 to over-express LjADH1 gene
in yeast cells, the result showed that the growth status
of the recombinant yeast harboring pYES2-LjADH1
were much better than that of the wild type with pYES2
under the stresses of 10 mmol/L CuCl2, 100 μmol/L
CdCl2, 150 μmol/L CdCl2 and 3.5 mmol/L H 2O2, res-
pectively, but except for 1.8 mmol/L NiCl2. It was ob-
vious that the LjADH1 protein over-expressed in yeast
might increase the yeast cells’ tolerance to abiotic stre-
sses. 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.
3 Materials and Methods
3.1 Materials used in this research
The Lotus japonicus MG20 seeds were kindly
provided by Dr. Da Luo (Professor of Sun Yat-Sen
University, P. R. China) and were deposited in Hainan
Institute of Tropical Agricultural Resources (HITAR,
China). The seeds were sanitized and germinated in
greenhouse for 24 h at 30℃ prior to planting in
flowerpots (57 cm) with 12 h/12 h day/night cycle at
26 , 80% humidity. Two weeks later, the seedlings℃
were collected and cleaned for ready.
The bacteria and plasmids used in this research in-
cluding E. coli JM109, Yeast strain INVScl (Clontech),
E. coli M15, pMD18-T (TaKaRa), pQE30 (Qiagen),
pYES2 (Clontech) were deposited in the lab of Alkali
Soil Natural Environmental Science Center (ASN
ESC), Northeast Forestry University. Enzymes and
chemicals including restriction enzyme, EX Taq TM,
T4 DNA ligase, kanamycin et al . were purchased from
Takara Company.
3.2 cDNA synthesis, Gene cloning and sequence
analysis
Total RNA from MG20 was extracted using Biozol
Total RNA Extraction kit (Biomiga). The first strand
cDNA of Lotus japonicus MG20 was synthesized by
RT-PCR using RNA (AMV) reverse transcription kit
(Takara) by RT-PCR following the procedures as: 30 ,℃
10min; 42 , 15min; 50 , 15min; 55 , 15min; 60 ,℃ ℃ ℃ ℃
15min; 90 , 10min; 5 , 5min; for 30 cycles.℃ ℃
A pair of primers was designed according to known
LcADH1 gene, forward primer was given as 5'-TAG
CTATGTCGACCACAGCT-3', reverse primer as 5'-
AACTCAGTCCCCAAATAGGG-3'. ADH analogus
gene was amplified from Lotus japonicus MG20 cDNA
under the procedures as follows: pre-denaturation at
95℃ for 3 min, followed by 30 cycles (95℃ 30 sec,
52℃ 30 sec and 72 ℃ 2 min), and ended at 72℃ for
10 min. The PCR products were separated by agarose
gel electrophoresis and ligated into pMD 18-T vector
(Takara), then transformed into E. coli JM109 strain.
The positive clones were selected to be sequenced at
Beijing Genomic Institute, China). Sequence analysis
was conducted online in NCBI for Blast and phy-
logenetic analysis.
3.3 Construction of prokaryotic expression vector
We designed a pair of primers with the restriction sites
of BamH andⅠ Sal by Primer Premier (Version 5.0),
Ⅰ
forward primer with BamH Ⅰ as 5'-GGGATCCA
TGTCGACCACAGCT-3', and reverse primer with
SalⅠ as 5'-CGGAGCTCACACATCATTGTTTTTG-3',
respectively. The plasmid for sequencing was used as
templates to amplify the gene following the pro-
cedures as pre-denaturation at 95℃ for 3 min, fo-
llowed by 30 cycles (95℃ 30 sec, 54℃ 30 sec and
72℃ 2 min), and ended at 72℃ for 10 min. The PCR
products were recovered to ligated into pMD18-T
vector named as pMD18T-LjADH1, and then trans-
formed into E. coli JM 109 strains by the approach of
thermal stimulation. Meanwhile the recombinant plas-
mids and pQE30 vectors were extracted to be digested
by Sac and
Ⅰ BamHⅠ, respectively, and then have
the LjADH1 ligated to the pQE30 vector to make
recombinant vector, pQE30-LjADH1. We transformed
pQE30-LjADH1 into E. coli JM109 competent cells.
Monoclonal was randomly picked up for PCR ampli-
fication to identify whether the gene transformed into
E. coli M15 strains by BamHⅠ and Sac Ⅰ digestion.
3.4 Expression of fusion protein and concentration
determination
The recombinant E. coli M15 strains harboring pQE30-
LjADH1 were inoculated into resistant medium and
incubated overnight at 37℃ and 200 r/min shaking
culture. Then 200 μL overnight incubated strains was
added into 5 mL LB liquid medium containing 100
μg/mL ampicillin and 25 μg/mL kanamycin to con-
tinue shaking culture at 37℃. While the OD 600 value