The Research Of Salt Tolerance Of HKT1 In Thellungiella Halophila

Ion transportation and homeostasis of sodium and potassium is the key on salt-tolerance of plants under salt stress. One of the important features of salt stress is Na~+ poisoning on the plant tissue. Because Na~+ influencing on the plant metabolism including osmotic and ionic stress, and also cause a series of secondary stress such as nutritional imbalance and oxidative stress which are so serious that can disturbed water and ion homeostasis on the level of cells and whole plants. Therefore,the plants cells and molecular were damaged, and plants growth slowly or even death. But the K~+ is the most abundant free ion in plant cells and plays an important role including the plant growth and metabolism under salt stress.There are many K~+ transporters and K~+ channels in the roots and shoots of plant. Most of these transporters and channel which located in the plasma membrane and vacuole membrane can absorb and distribute K~+ from the soil in the plants. Therefore, the plant can maintain a high K~+ / Na~+ ratio in the level of cells. HKT family has played a very important role as one of the cation transporters in plants.HKT family involved in absorbing the external potassium ion in the plants under conditions of low potassium, and its function is K~+-Na~+ co-transporters or Na~+ transporters. Recently, Study of HKT protein showed that it plays an important role on improving the plant salt tolerance. AtHKT in Arabidopsis thaliana is a key factor that can determine salt-tolerant of plants, and it involved in Na~+ absorption of root. Thellungiella halophila is one of the halophytes, that is close relative of Arabidopsis thaliana, it is a model plant of the moleculer and genetic research. Studying the function and mechanism of the ThHKT1 can help to further reveal the mechanism of salt-tolerant in Thellungiella halophila.The research includes the following contents:1. ThHKT1 full cDNA sequence in Thellungiella halophila was cloned.2. Study the functions of ThHKT1 by using the over-expression of ThHKT1 in Arabidopsis sas2-1 mutant.1) The plant expression vector of 35S promoter﹕ThHKT1 and AtHKT1 promoter﹕ThHKT1 in pCAMBIA3301 were constructed。2) The plant gene over-expression vector of 35S promoter﹕ThHKT1 and AtHKT1 promoter﹕ ThHKT1 in pCAMBIA3301 were transformanted into Agrobacterium tumefaciens strain GV3101, and furtherly them were introduced into AtHKT1 mutant sas2-1 (ecotype Landsberg) by dipping method; and screening the homozygotes to study the function of ThHKT1 by using 0.2% basta.3) The molecular identification of 35S promoter﹕ThHKT1 of pCAMBIA3301 in Arabidopsis AtHKT1 mutant( sas2-1) are as below: (1) The strong positive signals that about 500bp of basta were obtained in 35S promoter﹕ ThHKT1 transformants in Arabidopsis, and no signal was shown in sas2-1 mutant and wide type plants by PCR. It was showed that ThHKT1 all full genes into Arabidopsis genome. (2) Measurements on normal MS medium and different K~+ concentrations (50, 100 mmol / L). The K~+ content in shoot and root of ThHKT1 transgenic lines are much higher than in sas2-1 under the different concentration K~+. (3) The Na~+ content of shoot and root in ThHKT1 transgenic lines are similar to that in sas2-1, but higher in shoot and lower in root than that in the wild type under the different concentration NaCl (0, 50, 100, 150mmol/L) .3. Study the functions of ThHKT1 by using the gene silencing of ThHKT1 in the Thellungiella halophila.1) The plant gene silencing vector ThHKT1:pCAMBIA3301 was constructed。2) Gene silencing vector of ThHKT1:pCAMBIA3301 was transformated into wide type of Thellungiella halophila by dipping method. The molecular identification of these Thellungiella halophila basta-resistant plants are as below: (1) PCR result showed the strong positive signals that about 500bp of basta were obtained in transformants plants, but no signal was shown in wild type plants. (2) Comparing the expression level between transgenic lines and wide type by RT-PCR, we found expression level of ThHKT1 has decreased transgenic plants. (3) Under the treatment of different concentrations NaCl (0,200,400 mmol/L), the growth of the transgenic lines was worse than that in wild type plants, and the transgenic plants showed that accumulate less K~+ than that in wild type plants under 0 and 200 mmol/L NaCl. But under the 400 mmol/L NaCl ,the difference between transgenic plants and wide type is not very obvious.These results show that ThHKT1 under the condition of the normal and high-K~+ improve content of the K~+ in all transgenic plants, and also reduce the Na~+ transport into the plant cell. Therefore it is suggested that HKT1 in Thellungiella halophila as function of K~+ transporter,and it can control Na~+ and K~+ ion homeostasis.