Molecular Cloning And Function Characterization Of SsVP And SsMAPKK Genes In Suaeda Salsa

Salinity stress disrupts the homeostasis in water potential and ion distribution, results in osmotic and ionic stress in plants. It can arose growth arrest, and in some extreme conditions, even death of plants. To cope with salt stress, plants have developed a variety of adaptation mechanisms. One is the accumulation of the solute osmo-protectants, the other is the mechanisms of ion homeostasis including Na+ extrusion system and Na+ compartmentation into the vacuolar to reduce the toxic effects of this cation. The active transport of Na+ and solute molecules depends on the proton electrochemical gradients established by proton pumps. In plants, three distinct proton pumps generate proton electrochemical gradients across cell membranes: the P-type ATPase pumps, the vacuolar H+-ATPase and the vacuolar H+-pyrophosphatase. The vacuolar H+-PPase plays a role in establishing the H+ electrochemical gradient across the vacuolar membrane.Environmental stresses induce various biochemical, physiological and molecular responses, including gene expression. It is thought that these responses are promoted as a result of the perception of environmental stimuli and signal transduction in plant cells. In eukaryotes, it is very clearly that MAP kinase (mitogen-activated protein kinase) signal transduction pathways are important routes for the channelling of extracellular stimuli to cytoplasm or nucleus. In plants, MAPK module can be used for the transmission of multiple signals, including osmotic and oxidative stress.In this research, we isolated two cDNAs that may encode a MAP kinase kinase (SsMAPKK, AY093683) and a vacuolar H+-PPase (SsVP) from a X Zap-cDNA library constructed from a 400 mmol/L NaCl-treated Suaeda salsa aerial tissue. We analyzed their sequence characterizations, genomic structures and transcription levels under salinity stress. The results indicated that SsVP showed highest homology to the vacuolar H+-PPase (CVP) gene from Chenopodium rubrum and phylogenetic analysis indicated that Ss VP and CVP share a cluster, which showed that they might be more similar in evolution. Southern blot analysis showed that there was more than one copy of SsVP in the Suaeda salsa genome. Northern blot analysis indicated that the expression level of SsVP in S. salsa aerial tissue was significantly increased after 48h being treated with 400mmol/L NaCl. The activity of vacuolar H+-PPase of Suaeda salsa increased significantly in response to salt stress. The expression level of SsVP in S. salsa aerial tissue was also significantly increased after 4 days droughtstress. SsMAPKK showed highest homology to the NPK2 gene from Nicotiana tabacum . Genomic southern blot analysis suggested there was only one copy SsMAPKK gene in Suaeda salsa genome. Previously, we haved identified that SsMAPKK was induced in stems by salt stress.Then the SsVP ORF was integrated into the plant expression vector pCAMBIA1300 and SsMAPKK ORF was integrated into the plant expression vector pROKII. The integrated vector was introduced into Arabidopsis thaliana by in planta transformation method mediated by Agrobacterium tumefaciens GV3101. SsVP transformations were screened on media with hygromycin(25mg/L). we obtained TI plants. SsMAPKK transformations were screened on media with kanamycin(30mg/L). Nineteen individual kanamycin resistant plants were obtained. T2 plants were checked for integration of foreign gene by counting ratio of the number of tolerant plants to the number of non-tolerant plants on selection medium with kanamycin(30mg/L). PCR analysis indicated that all lines had been integrated of SsMAPKK. Northern analysis revealed the presence of expression of SsMAPKK mRNA in transgenic lines. In principle, SsVP overexpression can increase proton electrochemical gradients across the vacuolar membranes, which permit the secondary active transport of Na+ and solute molecules. SsMAPKK overexpression can active downstream stress-related gene to enhance transgenic plants tolerance to multiple environmental stress conditions.