CDNA Isolation And Function Analysis Of GDP-mannose Pyrophosphorylase Gene(GMPase) Of Tomato Leaves

Temperature is the major factor limiting the productivity and geographical distribution of plant. L-ascorbate (Vitamin C; AsA), one of the best known of the plant metabolites, is a major antioxidant that serves many functions in plants and has many proposed roles in plant growth and metabolism.. AsA is involved in the detoxification of reactive oxygen species (ROS), e.g., superoxide, singlet oxygen, ozone, and H₂O₂, that are produced during aerobic metabolic processes such as photosynthesis or respiration (Asada and Takahashi, 1987). AsA also participates in the regeneration ofα-tocopherol (vitamin E) from the tocopheroxyl radical (Asada, 1994) and act as a substrate for AsA peroxidase (Conklin et al., 1996, 1997; Noctor and Foyer, 1998; Smirnoff, 2000; Sanmartin et al., 2003).. In addition, AsA functions as a co-factor for enzymes such as prolyl and lysyl hydrolases, violaxanthin de-epoxidase (VDE), and ethylene-forming enzyme (Davies et al., 1991;Smith et al., 1992; McGarvey and Christoffersen, 1992; Eskling et al., 1997) as well as for 2-oxoaciddependent dioxygenases required for the synthesis of abscisic acid and gibberellic acid (Arrigoni and De Tullio, 2000; Arrigoni and De Tullio, 2002; Smirnoff, 2000). It also preserves the activity of a number of different enzymes by maintaining prosthetic group metal ions in the reduced state (Padh, 1990; Davey et al., 2000; Smirnoff, 1996).GDP-D-mannose pyrophosphorylase is an enzyme in initial committed step in the biosynthesis of AsA. Furthermore, GMPase which results in the production of GDP- -mannose, is also used for cell wall carbohydrate biosynthesis and protein glycosylation. Many experiments have suggested that GMPase is related to AsA biosynthesis. In this study, we isolated and characterized GMPase gene from tomato using homological clone. The functional analysis showed that expression of the gene was induced by temperature stress, The depletion of GMPase decreased the resistance to temperature stress. The main results are as follows:1. Two mixed primers were designed to amplify specific DNA fragment using cDNA prepared from the leaves of tomato on the homologous sequences from other plants. The middle fragment of interested cDNA was obtained by RT-PCR. The full length of the cDNA was isolated by 5'–RACE and 3'-RACE. The clone contains 1402nucleotides with an open reading frame (ORF) of 1083 bp comprising 361 amino acid residues with the predicted molecular mass of 43 kDa. The deduced amino acid sequence showed high identities with GMPase from Solanum tuberosum, Nicotiana tabacum, Medicago sativa, Arabidopsis thaliana and Zea mays. This indicated that the full length of the cDNA encoded a GMPase, which was designed as GMPase.2. Northern hybridization shows that GMPase constitutively expressed in stems, petals, fruits and leaves of wild tomato plants. The transcripts were high in the leaves. GMPase expressed extensively under 4℃and 40℃in leaves and the expression of GMPase was obviously induced by temperature stress3. The full-length GMPase 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 and verified by PCR and Northern hybridization. It was indicated that the GMPase gene had been recombined into tobacco genome and both sense and antisense transgenic tobacco and sense transgenic potato plants were obtained.4. RNA gel blot showed that WT plants had positive signals, while no signal was found in all antisense transgenic tobacco plants. GMPase plays an important role in protecting plants from damage of temperature stress.5. A recombinant of prokaryotic expression vector pET- GMPase was constructed and transformed to E.Coli. BL21 to express. The strong induced fusion protein bands were collected into PBS solution and used to immunize white mice to obtain antiserum. The value of antibody reaches 1: 500. Western hybridization revealed the presence of the strong positive protein signals corresponding to GMPase in sense transgenic plants ,Whereas no such protein was detected in antisense transgenic plants..6. Leaves of the antisense transgenic tobacco plants had AsA contents that were reduced to 30–40%. Antisense Transgenic tobacco developed a phenotype different to that of the WT tobacco. Transgenic plants began to flowering when kept in tissue culture, and all transgenic tobaccos had flowered when they have 7-8 leaves, whereas no such phenomenon happened in WT tobaccos. WT tobaccos flowered when they had 25-28 leaves. Furthermore, the internode length of antisense transgenic tobacco was significantly longer than that of wild-type when all have 7-8 leaves.7.Under temperature stress(4℃and 40℃), the leaves of the antisense transgenic tobacco plants had lower SOD (superoxide dismutase; EC 1.15.1.1), CAT (catarase, EC 1.11.1.6), APX (ascorbate peroxidase; EC 1.11.1.7) activity and AsA content and higher H₂O₂ content and MDA content. Results from different tests indicated that depletion of GMPase promoted a lower level of temperature tolerance in transgenic tobaccos.8. These results indicate that GDP-mannose pyrophosphorylase (GMPase) is the key enzyme of L-ascorbate synthesis. This further proved that GMPase gene had tightly touch with the tolerance to temperature stress.