Characterization And Functional Analysis Of Cucumber CsNMAPK Gene Under NO₃^- Stress

Excess application of chemical fertilizer has caused secondary salinization in Chinese greenhouse. Soil secondary salinization is one of the major limited factors affecting vegetable production in the greenhouse. Unlike the seashore, NO₃^- is the main accumualated anion in greenhouse soil. The mechanism of plant resistant to salt stress has been widely investigated, which mainly focused on the NaCl treatment and little was known about the molecular mechanism resisting to NO₃^- stress in plants. Cucumber is one of the most important vegetables cultivated in the Chinese greenhouse and was easily damaged by salt stress. Therefore, it is important to investigate the mechanism of cucumber tolerant to NO₃^- stress.Mitogen-activated protein kinase (MAPK) cascades are one of the major pathways in signal transduction in all eukaryotes. The MAPK cascades are composed of MAPK, MAPKK (MAPK kinase) and MAPKKK (MAPKK kinase). They transfer signals through phosphorylations of MAPKKK-MAPKK-MAPK in turn. In plants, MAPK cascades interlace with other signal transduction pathways and form a molecular interaction network, and regulate various cellular functions, including growth, development, biotic and abiotic stress responses. To date, numerous MAPKs have been isolated from different plants, and considerable progress has been made in understanding plant MAPKs. The regulation of MAPK signaling has been discussed most in dicot plant species (Arabidopsis, tobacco and alfafa etc.) and the study on cucumber MAPKs was virtually lacking. In this research, cucumbers were used as experimental materials. Research about the gene clone, expression characteristics and functional analysis of CsNMAPK was done. The main results were summarized as follows:1. Two degenerate primers were designed to amplify specific DNA fragment using cDNA prepared from cucumber root according to the homologous sequences from other plants. The middle fragment of the cucumber 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 CsNMAPK (Acession Numeber: DQ812086), contains 1636 bp nucleotides with an open reading frame (ORF) of 1113 bp comprising 370 amino acid residues.2. Comparing of the predicted protein sequence of the CsNMAPK with MAPKs from other plants showed that CsNMAPK was most homologous to the Glycine max (GmMAPK1) (85.18 %), Pisum sativum (PsMAPK3) (84.91 %), Medicago sativum (MsMAPK ) (84.64 %), Solanum tuberosum (StWIPK) (83.65 %), Lycopersicon esculentum (LeMAPK) (83.11 %), Capsicum annuum (CaMAPK1) (81.60 %). Construction of a dendrogram based on the homologous full-length amino acid sequences revealed that CsNMAPK belong to subgroupâ… MAPK in plants, which may play important roles in environmental stresses and hormone signal transduction.3. Northern blot analysis revealed that excess NO₃^- induced the expression of CsNMAPK. When treated with different concentrations of NO₃^-, the mRNA level of CsNMAPK was up-regulated by 56, 98, 140 mM NO₃^-, but down-regulated by 182 mM NO₃^-, compared to the normal NO₃^- concentration of 14 mM in the hydroponic systems. To determine the main location of cucumber CsNMAPK expression under excess NO₃^-, the expression pattern was examined in various plant organs, including roots, leaves and stems. Tissue specific expression showed that CsNMAPK expressed in all organs and little difference of the expression could be seen. When treated the salt tolerant cucumber cultivar (Xintaimici) and salt sensitive cucumber cultivar (Shennongchunwu) seedlings with 182 mM NO₃^- for different times, the two cultivars showed slight differences in expression kinetics. The CsNMAPK expression was a little up-regulated in Xintaimici within 30 min and then the expression decreased gradually, while Shennongchunwu maintained its expression until 1 h after down-regulation. The expression of CsNMAPK could also be induced by NaCl, ABA, H2O2, PEG and SA.4. The full-length CsNMAPK cDNA was subcloned into the expression vector pBI121 downstream of the 35S-CaMV promoter to form sense constructs. The constructs were first introduced into Agrobacterium tumefaciens LBA4404 by the freezing transformation method. The transgenic tobacco plants were verified by PCR, Northern blot and Western blot. It indicated that the CsNMAPK gene had been recombined into tobacco genome and transgenic tobacco plants were obtained. Transgenic seeds were germinated on MS media with 150 mM NO₃^- to score their tolerance to NO₃^- stress. The results showed that the transgenic seeds exhibited enhanced resistance to NO₃^- stress during seed germination. Experiments were also done during the seedling stage with 98 mM and 182 mM NO₃^-. The results indicated that there was little difference of the transgenic and wild type tobacco plants under normal enviroment. When treated with 98 mM and 182 mM NO₃^-, transgenic plants showed more resistance. To investigate the effect of CsNMAPK over-expression in tobacco on the physiological response to NO₃^- stress, a panel of physiological parameters, including malondialdehyde (MDA) contents, relative electrolyte leakage, H2O2 content, antioxidant enzyemes, net photosynthesis rate and free proline content were investigated. The resultes indicated that relative electrolyte leakage, MDA content and H2O2 content in the transgenic plants were lower than that in the wild type tobacco plants. Under NO₃^- stress treatments, the enzyme activities of SOD, CAT, POD and APX of transgenic tobacco were higer than the wild type tobacco. Free proline accumulation increased in both transgenic and wild type plants, but the increase was higher in the transgenic lines, demonstrating that they have higher osmotic adjustment ability.5. A recombinant of prokaryotic expression vector pET-CsNMAPK was constructed and transformed into E.coli BL21. The strong induced fusion protein bands were collected into PBS solution and used to immunize white mice to obtain antiserum. Western hybridization revealed the presence of the strong positive protein signals corresponding to CsNMAPK in sense transgenic plants of tobacco.6. CsNMAPK was also introduced into 6 different cucumber cultivars by overy injection pathway with the sense-expression vector and antisense-expression vector. The T1 plants were selected by resistance to 2500 mg/L kanamycin. 8 of kanamycin-resistant transgenic cucumbers were identified by PCR and Real-time PCR including 3 of antisense transgenic plants and 5 of sense transgenic plants.