Cloning and Expression of An Alcohol Dehydrogenase from Lotus japonicus and Characterization of LjADH1

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 H2O2 stress, while LjADH1 expressed in recombinant yeast can promote growth of the recombinant yeast under the stress of H2O2 and some heavy metal salts such as CuCl2 and CdCl2 except for NiCl2. 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.


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 nicotinamide 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 dehydrogen and become NADH.
ADH widely occurs in all kinds of organs in plant, which involves in development, maturation and senescence of fruits (Chervin et al., 1999), as well as in the resistant responses to stresses. ADH can be induced 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 sequence 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 conservative function domains. Even though there is a few differences in amino acid sequences among individuals, 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. 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 chromosome 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 chromosome 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 tetraploid 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 japonicus has become a convenient model plant for the studies of genome and genetics in legumes, particularly in reference to rhizobial and arbuscular mycorrhizal symbiosis since in the early 1990s (Handberg and Stougaard, 1992). Based on the conservative characteristics of ADH genes, we used MG20, a Lotus japonicus germplasm originated from Miyako Island of Japan (Kawaguchi, 2000), as experimental materials to clone homologous ADH gene from Lotus japonicus cDNA, to express the gene in E. coli and yeast and to characterize the enzyme in this study.

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 length encoding 380 amino acids. The further sequence alignment indicated that the cloned sequence had 99% homology to an unannotated Lotus japonicus sequence deposited in the GenBank (GenBank ID: CAG 30579.1). We conferred the sequence as ADH1 of Lotus 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 indicated 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).

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 2011), to make the heterologous expression construct pQE30-LjADH1. The construct was validated by digestion of BamH 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.

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 expression. The result showed that fusion proteins were expressed 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 Figure 1 Homologous analysis of amino acid sequences among plant ADH family was measured at 1.12 mg/mL by the BCA method, the concentration of 6×His-LjADH1 under the optimum expression conditions.

Enzymatic activity of fusion LjADH1 protein
We proposed the expressed fusion protein of LjADH to be a kind of dehydrogenase, therefore we employed the Vallee and Hoch method to measure enzymatic ac-tivity (Vallee and Hoch, 1955). The principle of this method is that NADH and NAD + have their own maximum absorption peaks at 340 nm and 260 nm, respecttively, so as NAD + was converted to NADH, the maximum absorption peaks at 260 nm will be shifted to 340 nm. The enzymatic activity of LjADH1 was calculated following the formula, U/mg=E 340 ×3.1/(6.2×E W ), here E 340 is the absorbance increase within 5 minutes at 340 nm (absorbency units 0.001), E W is the weight (mg) of enzyme per mL enzyme solution. In this re-search we measured that absorbance change of Lj-ADH1 in a minute was 0.108, so LjADH1 enzymatic activity come out 48.2 U/mg (figure 5). Figure 5 The absorbance of recombinant fusion protein at 340 nm

Resistant analysis of the prokaryotic fusion protein
We added H 2 O 2 into E. coli M15 recombinant strain which harboring pQE30-LjADH1 and reference strain with pQE30. The reference strain grew better than that of recombinant strain in absence of H 2 O 2 . This phenolmena might be due to the effect of overexpressed heterologous proteins during bacteria growth stages. Whereas when we added H 2 O 2 into recombinant strain solution at 1 mmol/L H 2 O 2 final concentration, the recombinant strain obviously grew better than that of the reference strain ( Figure 6). The results suggested that alcohol dehydrogenase might have some functions in the survival capability of prokaryotes under oxidative stress.

Construction of eukaryotic expression vector pYES2-LjADH1
In this research, we constructed a yeast expression construct named pYES2-LjADH1, The construct was validated by digestion of restriction enzymes BamH Ⅰ and Xba Ⅰ to produce 5.9 Kb and a 1.1 Kb fragment by agarose gel electrophoresis ) (figure 7A), those fragments in size were in accord with the size of pYES2 and LjADH1 sequence. that was confirmed by digestion of restriction enzymes BamH and Ⅰ Xba . The recombinant str Ⅰ ains were also identified by PCR to confirm that the pYES2-LjADH1 were transferred into yeast (figure 7B).

Resistant analysis of LjADH1 protein expression in yeast
In order to understand how do the LjADH1 protein response to different ion stresses, we inoculated the Ⅰ -LjADH1 by PCR recombinant yeast strain with pYES2-LjADH1 and wild type strain pYES2 in the YPG media with differrent ion concentrations, respectively (Figure 8). The responses showed that the growth status between the recombinant and its wild type yeast had very similar in the YPG medium without any treatment, but the recombinant yeast grew better than the wild type in the YPG media with final concentrations of 10 mmol/L CuCl 2 , 100 μmol/L CdCl 2 , 150 μmol/L CdCl 2 and 3.5 mmol/L H 2 O 2 , respectively. There is no any response between the recombinant and wild type in con- Figure 8 Over expression of pYES2-LjADH1 under different abiotic stresses Note: A: The controls, Yeast cells harboring pYES2-LjADH1 and pYES2 vector, respectively, were inoculated in YPD medium, growth status was observed after incubated 2~3 days at 30 ; B~F: Yeast cells harboring pYES2 ℃ -LjADH1 and pYES2 vector were respectively inoculated in YPD media with different concentrations of 10 mmol/L CuCl 2 , 1.8 mmol/L NiCl 2 , 100 μmol/L CdCl 2 , 150 μmol/L CdCl 2 , and 3.5 mmol/L H 2 O 2 , growth status observation after 2~3 days incubation at 30℃ centration of 1.8 mmol/L NiCl 2 . The results indicated that the LjADH1 protein has some resistant functions to oxidative stress in yeast. With reference to evidence of the recombinant yeast growth in the concentration of 3.5 mmol/L H 2 O 2 we considered that LjADH1 might have certain resistant functions to abiotic stresses. There was a report that the plants will lack of resistance to waterlogging while the ADH gene in plant is going to silence, whereas overexpressing ADH alone might not yet improve the plant resistance to waterlogging (Ismond et al., 2003). Therefore the functions of LjADH1 in plants need to be further studied in future.

Conlusions
In this research, we cloned alcohol dehydrogenase gene from Lotus japonicus MG20, and considered that the gene encodes a typical zinc-binding alcohol dehydrogenase, named as LjADH1.
In this research, the gene was ligated to pQE30 expression vector with a 6×His tag, and a batch of nondenaturalized proteins was expressed in E. coli and purified by Ni + -NTA gel column under the optimum induced expression conditions. The enzymatic activity of the LjADH1 was determined by method proposed by Vallee and Hoch, which demonstrated strong alco-hol dehydrogenase activity. In respect to stress tolerance 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 CuCl 2 , 100 μmol/L CdCl 2 , 150 μmol/L CdCl 2 and 3.5 mmol/L H 2 O 2 , respectively, but except for 1.8 mmol/L NiCl 2 . It was obvious that the LjADH1 protein over-expressed in yeast might increase the yeast cells' tolerance to abiotic stresses. 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.

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.

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 phylogenetic analysis.

Construction of prokaryotic expression vector
We designed a pair of primers with the restriction sites of BamH and Ⅰ 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 procedures as pre-denaturation at 95 ℃ for 3 min, followed 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 transformed into E. coli JM 109 strains by the approach of thermal stimulation. Meanwhile the recombinant plasmids 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 amplification to identify whether the gene transformed into E. coli M15 strains by BamHⅠ and Sac Ⅰ digestion.

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 continue shaking culture at 37℃. While the OD 600 value reached 0.6, IPTG was added with a final concentration of 0.1mmol/L to induce target fusion protein.
Under the shaking culture at the speed of 200 r/min at 30℃, the recombinant strains were collected after induced for 0 min, 30 min, 60 min, 120 min, 180 min, 240 min, 300 min and 360 min, respectively. The supernatant was discarded by centrifugation at 13 000 r/min at 4 for 1 min ℃ , and precipitate was re-suspended with PBS, then added equivalent 2×SDS sample buffer, denatured for 5 min at 100℃ and cooled on ice bath for 5 min, finally centrifugated at 13 000 r/min at 4 for 5 min. Took about 20 μL of t ℃ he samples used for SDS-PAGE electrophoresis.
Protein concentration was measured by the BCA method (Walker, 1994). 37.5 μL (8 mg/mL) BSA was added into 262.5 μL ddH 2 O to make protein standard solution with final BSA concentration 1 mg/mL, took 150 L solution diluted 7 times consecutively to prepare BSA diluting standard solution with the concentrations at 500 g/mL, 250 g/mL, 125 g/mL, 62.5 g/mL, 31.25 g/mL, 15.625 g/mL, and 7.813 g/mL, respectively. The purified fusion proteins was also diluted and then took 100 μL diluted standard solutions respectively to mix with 2 400 μL CBB (Coomassie Brilliant Blue), the OD 595 value was read three times by spectrophotometer using ddH 2 O as reference.

Enzymatic activity of Fusion protein
Enzymatic activity of fusion protein was measured by the method of Vallee and Hoch (Vallee and Hoch, 1955), 150 μL pyrophosphate buffer, 50 μL substrate solution and 100 μL solution of NAD + were mixed to a cuvette. The cuvette was placed in water bath at 37 for 5 ℃ min and then added 10 μL purified fusion protein solution preheated under the same conditions, immediately count the time and read the increase of absorbance at 340 nm every 1min interval in the successive 5min until the values of absorbance is stable.

Resistant analysis of the fusion protein expressed in E. coli
Employ Echave's method to be slightly modified to analyze the prokaryotic resistance (Echave et al., 2003) in this reserch. 2.5 mL recombinant strain solution and its reference respectively cultured overnight until the OD 600 reached 0.3, then added 22.5 mL LB media containing 100 μg/mL ampicillin and 25 μg/mL ka-namycin cultured at 37 . then 0.1 ℃ mmol/L IPTG was added to dilute ten times, The cultured media were divided into two groups, one group of the recombinant added H 2 O 2 with a final concentration of 1 mmol/L and the other group added water as control, the OD 600 values were measured in every 30 min to calculate the resistant activity.

Authors' contributions
TZ and SKL designed and conducted this experiments. RYL, PTG and DGZ participated the experiment design and data analysis; XJF is the person who takes charge of this project, including experiment design, data analysis, writing and modifying of the manuscript. All authors have read and approved the final manuscript.