| Tomato(Solanum lycopersicum) is one of the most widely cultivated vegetable crops in the world. In spring and winter, low temperature is a major threat to growth, development and quality production of tomato. Recent studies have shown that Mitogen Activated Protein Kinase(MAPK) cascade plays an important role in plant defense signaling.It has been reported that MPK3 involved in resistance to low temperature stress. In this study, Sl MPK3 gene, cloned from Solanum pimpinellifolium, was transgenetically overexpressed in cultivated tomato(Solanum lycopersicum) M82 using the agrobacterium mediated transformation leaf disc method. T1 generation was used to investigate the role of Sl MPK3 under chilling stress. The subcellular localization was characterized using agrobacterium mediated transient expression analysis in onion epidermal cell by constructing Sl MPK3 and green fluorescent protein(GFP).The results showed as follows:1. The tomato Sl MPK3 bioinformatics was analysed by Mega 6.0 software. As can be seen from thephylogenetic tree, different MPK3 evolution process exist between monocotyledon and dicotyledon plants. Tomato Sl MPK3 gene and pepper Ca MPK3 have the highest homology. The secondary structure of tomato Sl MPK3 included alpha helix, random coil and extended strand. The p VBG2307-MPK3-GFP2 vector was constructed. The result of subcellular localization showed that Sl MPK3-GFP fusion protein was localized in the nucleus and the membrane.2. Sl MPK3 gene was introduced into cultivated tomato M82 by high efficient Agrobacterium tumefaciens transgenic system. T0 generation regeneration plants were identified by kanamycin and genomic PCR, and five positive transgenic plants were obtained.The fluorescent quantitative PCR detection showed that Sl MPK3 expression was significantly higher in three transgenic lines(OE-4, OE-6 and OE-7) than control.3. The expression pattern of Sl MPK3 gene induced by low temperature stress was analyzed in T1 transgenic plants. The Sl MPK3 transcript was up-regulated rapidly under low temperature stress in leaves of transgenic plants and control. The Sl MPK3 expression quantity increases with the increase of low temperature stress time from 0 to 8 h, and its peak at 8 h. Moreover, the relative expression fold was higher in transgenic plants as compared to the control plants.4. Seedling cold resistance was identified using T1 generation transgenic plants.Chilling injury level and cold tolerance index was calculated after 48 h low temperature treatment. Cold tolerance index of transgenic plants was significantly higher than control(0.8 and 0.37, respectively) after identification of cold resistance in seedling.5. The growth of transgenic plants was observed under 4 ℃ stress. After 8 h low temperature treatment, wild type plants blade began to appear scattered necrosis, while in transgenic appeared normal. After 24 h, leaves from the control plants started wilting and produce symptoms of leaf necrosis, chlorosis and yellowing. However, the transgenic plants were still keet normal symptom without obvious yellowing.6. The plasma membrane stability and oxidation resistance were determinated T1 generation transgenic plants after 4℃ low temperature stress. After 120 h treatment at 4℃low temperature, the relative electrolyte leakage was 59.79% in transgenic plants, which was14.87% lower than control. The malondialdehyde(MDA) content was 6.76 μmol/g FW on average in the leaves of transgenic plants, which was 30.81% lower than control at 120 h low temperature treatment. Hydrogen peroxide(H2O2) content is significantly higher in wild type plants leaves than that of the transgenic plants( 2.02 and 1.57, respectively) at 120 h.7. At the same time, the antioxidant enzymes activity, soluble sugar and soluble protein content increased significantly in the transgenic plants and wild type plants. At 120 h after low temperature treatment, the activities of SOD, POD and CAT in the leaves of transgenic plants were 29.40%, 24.24% and 29.40% respectively higher than control.8. The content of soluble protein and soluble sugar gradually increased with the increasing of the treatment time in the transgenic plants and wild type plants after low temperature. After 120 h treatment at 4 ℃, the soluble protein content in the transgenic plants was 40.02 mg/g FW(OE-4), 42.21 mg/g FW(OE-6), 40.33 mg/g FW(OE-7),respectively, which were significantly higher than that of control(36.86 mg/g FW). The soluble sugar content was 51.87 mmol/g FW averaged in transgenic plants, which was21.82% higher than that in control at same treatment-point. |
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