Co-expression Of The Sesuvium SpSOS1 And SpAHA1 Gene Enhances Salt Tolerance In Arabidopsis

Saline plants are an important material for studying the salt tolerance of plants.The study of salt tolerance genes in salt plants can give us a better understanding of the salt tolerance mechanism of plants. For example, the plant SOS1 ( Salt Overly Sensitive 1 ) protein is the only known Na+ / H+ transporter protein on the plasma membrane and plays a very important role in the process of efflux of sodium ions.Previous studies have shown that the SpAHA1 protein can produce H+ transmembrane gradient by decomposing ATP to form the transmembrane electrochemical potential gradient. While the SpSOS1 protein enhances the salt tolerance of the transgenic yeast using the energy which is generated by this gradient. Whereas, this theory has not been verified in plants, we transferred the SpSOS1, SpAHA1 and SpSOSl-AHA1 gene into the Arabidopsis to obtain the resistant seedlings for salt tolerance analysis.Herein, we introduce a kind of significant saline plant, Sesuvium portulacastrum,which has very high salt tolerance as the model creature. In this study, Sesuvium portulacastrum Na+/H+ antiporter gene SpSOS1?plasma membrane ATPase gene SpAHAl?SpSOS1-AHA1 were PCR-amplified and constructed into the binary vector,pCAMB1A1304. Then these genes were transformed into Arabidopsis by Agrobacterium tumefaciens strain GV3101 mediating. Three overexpressing transgenic plants were obtained using SpSOS1?SpAHA1 gene-specific primers and Hygromycin B screening marker primers to identify resistant seedlings. Subsequently,a series of salt tolerance analysis was carried out using T3 transgenic seedlings. The SpSOS1 protein was expression on the plasma membrane was verified by fusion SpSOS1 protein with GFP and observed the fluorescence signal was only on the cell membrane.Previous research showed that:1.Overexpressing SpSOS1 Arabidopsis had a above 87.5% germination rate and was higher than 60% germination rate of wild type plants in 75 mM NaCl culture medium . Meanwhile, the SpSOSl transgenic plants had a greater salt tolerance than wild type plants, specific manifestations in the mutants had higher fresh weight,longer root length and more lateral root number which could grow well in 100 mM NaCl culture medium. In contrast, wild-type Arabidopsis was wilted, lost green, and even died under salt stress of 50 mM NaCl. In addition, the transgenic Arabidopsis thaliana was cultured for 4 weeks and then treated with 200mM NaCl for 10 days.The phenotypic analysis showed that the plant growth was slowed down under the condition of salt treatment, but the transgenic Arabidopsis SpSOSl was less affected and the leaves were well growing and could complete the growth period, and the wild-type Arabidopsis has been dead. The Na+ and K+ contents of the transgenic Arabidopsis thaliana SpSOS1-1304 were significantly higher than those of the wild type Arabidopsis thaliana, and the total Na+/K+ was higher. The activity of antioxidant enzymes in transgenic Arabidopsis thaliana seedlings was also determined.The results showed that the activity of related enzymes in Arabidopsis thaliana was significantly increased.2. The expression level of related salt tolerance genes in transgenic Arabidopsis thaliana was significantly higher than that in wild type Arabidopsis thaliana by real-time quantitative PCR, such as AtSOS1, AtSOS2, AtSOS3 and so on.3 .The expression of SpSOS1 protein in the Sesuvium portulacastrum was similar to that of the fluorescent protein GFP, but no fluorescence signal was found in the other parts of the cell. The fluorescence of the positive control was expressed in all the parts of the cell.4.We found that wild-type Arabidopsis got yellow and was died with the treatment of 50 mM NaCl,and the leaves of SpAHA1-1304 transgenic seedlings were turn yellow, and the other two kinds of transgenic seedlings had a normal growth.In addition,the SpSOS1-AHA1-1304 transgenic seedlings had better growth than single gene transgenic seedlings SpSOS1-1304 with the treatment of 100 mM NaCl. At the same time, we measured Na+ and K+ contents in Arabidopsis thaliana. We also found that SpSOS1-AHA1-1304 transgenic seedlings had relatively high K+ and lower Na+.These results demonstrate that the Sesuvium portulacastrum SpSOS1 protein is expressed on the cell membrane and is a transport protein on the cell membrane.Transgenic Arabidopsis thaliana SpSOS1 can prevent the excessive accumulation of Na+ in the plant to prevent salt damage, and through the activation of antioxidant defense system, regulate intracellular Na+ / K+ balance, thereby enhancing plant salt tolerance. And we also found that SpAHA1 protein can regulate the SpSOS1 protein to provide energy for the latter, into the plant can further improve the transgenic Arabidopsis salt tolerance,this result was first validated in plants and provided experimental evidence for the "ATPase enzyme provides energy for transporter".