Functional Analysis Of Na~+/Pi Transporter Gene From T.halophila In Transgenic Tobacco

Phosphorous (Pi) is an essential macronutrient for plants growth and development. It serves various basic biological functions as a structural element in nucleic acids and phospholipids, in energy metabolism, and in the activation of metabolic intermediates, as a component in signal transduction cascades, and the regulation of enzymes. So it has become the focus on the absorption and metabolism of Pi in the plant science.Na~+/Pi transporter gene was cloned from T.halophila in our lab. Its function and activity of Pi transport has been studied in yeast mutant PAM2. The results showed that the gene encodes a high affinity Pi transporter. The Km of Na~+/Pi transporter under pH6.0 and pH7.5 is 7.4μM and 8.7μM, respectively. This gene was mainly expressed in leaves. And the protein was located in chloroplast by expressing N terminus GFP fusion protein in tobacco cells. But in higher plants, the reports on studying about Na~+/Pi transporter gene is few and its concrete function still needs further study.The whole gene DNA sequence of Na~+/Pi transporter gene was cloned by PCR in this work. Sequence analysis showed that this gene had 8 introns and 9 exons. And it encoded a peptide. We found that there was only one copy of Na~+/Pi transporter gene in T.halophila by Southern blotting analysis. Excising its signal peptide and heterogeneously expressed in yeast, Na~+/Pi transporter was located in the membrane of yeast cell. The result provided cytologic evidence for Pi transport in functional complementarity to mutant yeast.Na~+/Pi transporter gene was transformed into tobacco by Agrobacterium mediated method. By PCR and Southern blotting analysis, a number of transgenic lines were identified. And the expression of Na~+/Pi transporter gene was analyzed by quantitative fluorescent RT-PCR. The results showed that Na~+/Pi transporter gene presented high expression in transgenic tobacco lines. Intact chloroplasts were isolated to determinate the Pi transport activity of Na~+/Pi transporter. The results showed that the Pi transport activity of chloroplasts in transgenic tobacco lines was 10-20% higher than that in wild tobacco line.In low-Pi medium, the transgenic tobacco lines showed higher growth rates compared with wild tobacco lines and the number of leaf was more than that of wild type tobacco lines at the same stages. This presented that the transgenic tobacco lines increased the absorption of Pi and kept high growth rate. By nutrient solution culturing method, the physiological characteristics of transgenic and wild tobacco lines under lower Pi conditions were studied. The results showed that after 14 days treatment, the rate of root to shoot of transgenic tobacco lines was higher than that of wild lines. The results showed that the gene could increase the response of transgenic tobacco to lower Pi conditions. Further more the photosynthetic characteristics was analyzed, showing that the rate of photosynthesis of transgenic tobacco lines was higher than that of wild lines. The Pi transport activity of chloroplasts of transgenic tobacco lines was higher than that of wild tobacco lines. The gene could increase the usage of Pi of transgenic tobacco. So it may increase the rate of photosynthesis of transgenic tobacco directly or indirectly.In addition, the RNAi vector was constructed according to the sequence of the gene and planned to transform T.halophila or Arabidopsis thaliana for further analysis. It could be said that the partly job in the process of studying the function of Na/Pi transporter has been done.