Thellungiella Halophila ThPIP1 Gene Transferring Rice And Mechanism Of Salt Stress Tolerance

High salinity is one of the major abiotic stress factors existing in the areas of costal, arid and semi-arid regions,and affects the growth and development of plants, such as rice, which is sensitive to salt stress. Usually, rice growth and yield are always constrained by salt stress. AQPs family are mainly composed of membrane proteins which specifically transport water molecular and have been widely focused attention due to its participation in various physiological processes. AQPs not only transfer water but also some small molecular substances, such as urea, CO2 and NH3, and also play an important role in response to abiotic stresses. Thellungiella halophila is one of typical extremophile plants which could adapt to the high-salt environment and complete their life cycle. Therefore, it is meaningful to investigate the function of AQPs from T. halophila which is considered to have the adaptability of plants to the unfavorable environment.In the current study, an aquaporin gene ThPIP1 from T. halophila was cloned. ThPIP1 gene encodes 284 amino acids with 855 bp nucleotides and the deduced molecular weight of Th PIP1 protein is 30.345 k D(Gen Bank accession number:JX133234.1). Bioinformatics analysis indicates that Th PIP1 belongs to a typical family member of plasma membrane intrinsic protein(MIP) which is also known as AQPs. Th PIP1 has two conserved “NPA” motifs and six putative transmembrane structures and shares high homology with PIP1 subfamily members from Brassica rapa and Arabidopsis. In order to clarify whether Th PIP1 clusters to PIP subclass, subcellular localization experiment was conducted, the result showed that Th PIP1 is localized at plasma membrane, indicating that Th PIP1 belongs to a member of PIP subfamily.The expression patterns of T. halophila Th PIP1 under drought, low temperature and Na Cl stress were investigated by the method of q RT-PCR. Results showed that the expression of Th PIP1 responses to drought, low temperature and Na Cl stress. However, Th PIP1 has difference in responding to different stress factors and stressing time. The expression of Th PIP1 gene in the roots exhibited a peak value after 2 hours with a quick response to the salt stress.To further study the physiological functions of ThPIP1 under salt stress, ThPIP1 was transferred into Nipon Kitake Cultivar Japonica and transgenic rice lines were obtained. The salt tolerances of transgenic rice lines were evaluated based on the analyisis of osmotic regulation and ion balance. When high concentration of salt solution was used to culture the rice, the transgenic lines exhibited better growth than the wild type. Although the transgenic lines and wild type under salt stress were inhibited in different degrees, the inhibition extent in transgenic lines was lower than that in wild type, and the transgenic lines have longer roots relative to wild type. Additionally, the biomass weights and SPAD values from the transgenic lines are significantly higher than those from wild type. Generally, SPAD value is an index reflecting chlorophyll content in plant leaves, and higher SPAD values in the transgenic lines were observed, indicating that the transgenic lines have sronger photosynthesis rates under salt stress. Interestingly, when the rice was culture with high salt concentrations, the osmotic potential of the transgenic lines significantly decreased, implying that the transgenic lines to absorbed more water to meet the water requirement for normal metabolism. In addition, the transgenic rice lines under salt stress accumulated organic substances, such as proline and soluble sugar which play important physiological regulatory roles in decreasing the osmotic potential and ensuring water adsorption.Malondialdehyde(MDA) is an important index indicating damage degree of cell membrane and the higher MDA values represent that the damage on cell membrane is serious. The results showed that the MDA contents in the transgenic lines were significantly lower than those in the wild type, suggesting that the damage on cell membrane in the transgenic lines were relatively slight, which was considered to be beneficial to maintaining ionic balance. Furthermore, the ratios of K+/Na+ in the transgenic lines were much higher than those in the wild type, and former was benefit to alleviate the toxicity of Na+ in the plant cells. For further explaining the mechanism of ions regulation in the plants, the Na+ and K+ fluxes in the root meristematic zones of 5-d-old rice seedlings were determined by non-invasive test. Data showed that the Na+ efflux in the roots of some transgenic lines was significantly increased under salt stress, but the K+ efflux rates were decreased suggesting that the transgenic lines could regulate the balance of Na+ and K+ through an increase in the efflux of Na+, and positively absorbed more K+ to maintian higher ratios of K+/Na+. Therefore, the expression of Th PIP1 gene in the transgenic rice could improve the ability of salt resistance by reducing the damage of cell membrane as well as improving the osmotic regulation substances and ratio of K+/Na+.To profile the regulatory role of ThPIP1 protein in participating in the salt tolerance physiological process, the split-ubiquitin yeast two-hybrid was performed. Data showed that two target proteins of Th PIP1, Th PIP2 and nonspecific lipid protein ns LTP2, were obtained and identified. The interaction between Th PIP1 and Th PIP2 might play an important role in regulating the activity of water transport by forming heterologous tetramer. But, ns LTP2 might participate in various physiological processes, such as the transportation of lipid, the synthesis of wax and stress resistance. Therefore, Th PIP1 could play positive roles in the stress resistance through interaction with ns LTP2.