Cloning, Genetic Transformation And Function Preliminary Analysis Of Lgnhx1 Gene From Limonium Gmelinii (willd.) Kuntze

Soil salinization is one of the most serious problems of the world's agriculture. One-third area of China is arid. The soil salinization and secondary salinization caused by irrigation are the main obstacles which constraint agricultural development and the important factor which impact the stability of ecological environment. So, improving the plants especially the crops'salt-tolerance is the most effective method of the development and utilization of saline land, and also the major issue of current and future agricultural development. Limonium gmelinii (Willd.) Kuntze. is the unique plant of Xinjiang. Most soil in its habitats is saline soil meadow. L. gmelinii had formed stably special structure, function and genetics. In the long-term natural selection conditions L. gmelinii had formed the physiological and biochemical mechanisms to adapt to specific salinity. Therefore, study on the salt resistance gene of L. gmelinii is of great theoretical and practical significance.The Na~+/H~+ antiporter gene (NHX1) energized by the pH across the tonoplast, facilitates vacuolar compartmentalization of the cation. As a fundamental mechanism in salt tolerance, an active antiporter would function to sequester Na~+ into the vacuole, which results in avoidance of cytoplasmic Na~+ toxicity and maintenance of a high cytoplasmic K~+/Na~+ ratio. In parallel, vacuolar Na~+ would serve as an osmoticum necessary for cellular H2O homeostasis. In this study, specific primers were designed according to gene LgNHX1 of L. gmelinii cloned by the key laboratory of agricultural biotechnology. Then, the gene was cloned by means of RT-PCR. The length of its coding region is 1656bp, and the gene encodes 551 amino acids. The protein hydrophobicity analysis showed that LgNHX1 highly hydrophobic. Transmembrane domain analysis showed that LgNHX1 contained 12 transmembrane domains, and it's a kind of transmembrane protein.Using conventional molecular cloning techniques, the prokaryotic expression vectors pET28a-N1, pET32a-N1, pET28a-N2, pET32a-N2 were constructed. Recombinant plasmid was transformed into BL21, using IPTG to induce protein expression.Eukaryotic expression vector pCAMBIA1301-LgNHX1 had been constructed. The recombinant plasmid pCAMBIA1301-LgNHX1 was transformed into Agrobacterium tumefaciens GV3101. The gene was transformed into tobacco and tomato genome. Transformed materials were selected on solid medium containing Hygromycin B. Transgenic tobacco and tomato plants were identified containing purpose gene by PCR. We used RT-PCR methold to test the purpose gene transcription level.By means of tissue culture, we cloned 4 lines of transgenic tobacco, and treated them with NaCl. The result showed that compared with non-transgenic tobaccos, the transgenic ones had higher chlorophyll content and lower MDA and membrane permeability. It shows that the transformation of LgNHX1 had improved the salt tolerance of tobacco in a certain extent.This study not only laid a theoretical foundation for further analysis and application of L. gmelinii NHX1 gene, but also provide a certain experimental basis to L. gmelinii used to genetic engineering as a germplasm resource of salt resistance.