Tuo Zeng^1,2,3 , Shenkui Liu² , Ruye Luo^1,3 , Pengtao Gong^2,3 , Degang Zhao¹ , 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
Author    Correspondence author
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.

Zeng et al., 2011, Cloning and Expression of An Alcohol Dehydrogenase from Lotus japonicus and Characterization of LjADH1, Legume Genomics and Genetics, Vol.2, No.2 (doi: 10.5376/lgg.2011.02.0002)

Alcohol dehydrogenase (ADH), usually using NAD⁺ and NADP⁺ as coenzymes, widely exists in living 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  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.

Lotus japonicus; Alcohol dehydrogenase; LjADH1 (GenBank Accession No.: JN165714); Prokaryotic expression; Yeast expression; Abiotic stresse