Cloning And Functional Analysis Of The Chloroplast LesAPX And Cu/Zn-SOD In Tomato

Photosynthetic apparatus of chilling-sensitive species produces more reactive oxygen species (ROS) under chilling stress in low irradiance, damaging plants. Two key enzymes for ROS detoxification in the chloroplast are SOD and APX. SOD catalyzes the dismutation of two molecules of superoxide anion radical into oxygen and hydrogen peroxide. Utilizing ascorbate as an electron donor, APX reduces hydrogen peroxide to water.In this reseach, a full length cDNA of chloroplast stroma ascorbate peroxidase gene(LesAPX) was cloned from tomato using RT-PCR approach with degenerate primers based on the conserved motifs found in a number of sAPX genes. The homology comparison indicated that the cloned gene is the expected one. And Cu/Zn-SOD gene from tomato was also isolated. These two genes were transformed into tobacco and characterized. 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 of sAPX 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 named LesAPT (Acession Numeber: EU251405), contains 1435 bp nucleotides with an open reading frame (ORF) of 960 bp comprising 320 amino acid residues with the predicted molecular mass of 28 kDa. The deduced amino acid sequence showed high identities with sAPX from Nicotiana tabacum, Arabidopsis thaliana, Vigna unguiculata, Spinacia oleracea.2. The full-length LesAPX cDNA was subcloned into the expression vector pBI121 downstream of the 35S-CaMV promoter to form antisense construct. The construct was first introduced into Agrobacterium tumefaciens LBA4404 by the freezing transformation method and verified by PCR. It was indicated that the LesAPX had been recombined into tobacco genome and antisense transgenic tobacco plants were obtained. Antisense- transgenic lines could maintain higher activities of chloroplastic SOD and APX compared with WT. The maximal photochemical efficiency of PSâ…¡(Fv/Fm)and photosynthetic rates(Pn)in A(-)60 and A(-)40 lines decreased more under chilling stress than WT.3. The full-length Cu/Zn-SOD 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. It was indicated that the Cu/Zn-SOD gene had been recombined into tobacco genome, and both sense and antisense transgenic tobacco plants were obtained.4. A recombinant of prokaryotic expression vector pET-Cu/Zn-SOD was constructed and transformed to E.Coli. BL21. 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 Cu/Zn-SOD in sense transgenic tobacco plants.5. WT, sense- transgenic line S(+)21 and antisense- transgenic line S(-)8 of tobacco were used to measure the photosynthetic and chlorophyll fluorescence parameters, fast OJIP chlorophyll a (chl a ) fluorescence parameters, chloroplastic reactive oxygen species and activities of antioxidant enzymes under chilling stress. The maximal photochemical efficiency of PSâ…¡(Fv/Fm)and photosynthetic rates(Pn)in S(+)21 lines decreased less under chilling stress, while Fv/Fm and Pn in S(-)8 lines decreased obviously. Compared with WT, the sense-transgenic lines could maintain higher activities of chloroplastic SOD and APX, lower content of chloroplastic O₂^? and H₂O₂ under chilling stress. By using fast OJIP chorophylla a (Chl a) fluorescence transients, the effect of chilling stress on the tobacco leaf photosystemâ…¡(PSâ…¡) functions was investigated. Compared with WT, sense-transgenic lines could endure chilling temperature, and maintain higher electron transport capacity. Those results indicated that Cu/ZnSOD gene enhanced the tolerance of tobacco photosynthetic apparatus to chilling stress.