Cloning And Expression Analysis Of The Chloroplast Glutathione Reductase Gene In Tomato

Enviromental stress is one of the most significant problem in agriculture production practices. Exposed to stressful conditions, plants tend to produce and accumulate reactive oxygen species (ROS) which can attack protein, lipid and other components of cell. Glutathione reductase (GR), as key enzyme in ROS cleaning system, plays a significant part in protecting plants from damaged by ROS. In this study, we isolated and characterized chloroplast glutathione reductase gene from tomato. The main results are as follows:1. Two degenerate primers were designed to amplify specific DNA fragment using cDNA prepared from tomato leaves according to the homologous sequences from other plants. The middle fragment of interested cDNA was obtained by RT-PCR. The 5'and 3'fragment of the cDNA was isolated by 5'and 3'RACE. The clone, which was named LeGR (Acession numeber: EU285581), contains 1772 bp nucleotides with an open reading frame (ORF) of 765 bp, comprising 255 amino acid residues with the predicted molecular mass of 33 kDa. The deduced amino acid sequence showed high identities with GR from Nicotiana tabacum, Pisum sativum, Arabidopsis thaliana, Oryza sativa, Brassica juncea.2. Northern hybridization showed that LeGR constitutively expressed in stems, sepal, petals, fruits and leaves of wild type plants. The transcripts were plentiful in the tissues abundant of chlorophyll.3. The expression of LeGR was obviously induced by low temperature and high temperature stresses and the expression levels varied with the time of treatment.4. The full-length LeGR cDNA was subcloned into the expression vector pBI121 downstream of the 35S-CaMV promoter to form sense and antisense constructs. The constructs were first introduced into Agrobacterium tumefaciens LBA4404 by the freezing transformation method. Then the gene was introduced into the tomato of Zhongshu - 4 by A. tumefaciens-mediated leaf disk transformation.5. A recombinant of prokaryotic expression vector pET-LeGR was constructed and transformed to E.Coli. to express LeGR protein. SDS-PAGE indicated that the molecular weight of the target protein accorded with theoretic molecular weight.