The Cloning And Function Analysis Of Suaeda Salsa SsHKT1 Promotor

Soil salinization is one of the main abiotic stresses that affect the crop growth and yield, and NaCl is considered as the key toxic factor. Under salt stress, maintaining high K⁺/Na⁺ in the cytosol is highly related to salt-tolerance in plants. More and more attention has been paid on HKT family in plant salt-tolerance. Nowadays nonhalophytes such as Arabidopsis thaliana, wheat and rice have been used to study the role of HKT1, and it is considered that HKT1 plays an important role in Na⁺ transportation and Na⁺ homeostasis in plants under salinity. In recent years, a great attention has been paid to salt-tolerance of halophytes, and much progress has been made in the HTK1 gene of halophytes such as Thellungiella salsuginea and Suaeda salsa. It has been showed that the function of halophyte HKT1 is K⁺ carrier, which is obviously different from non-halophytes. Therefore, further study on halophyte HKT1 will play a critical role in revealing the mechanism of plant salt-tolerance.In our laboratory, SsHKT1 has been isolated from euhalophyte Suaeda salsa L. The results proved that SsHKT1 mediated K⁺ uptake instead of Na⁺. Moreover, SsHKT1 expression is induced by a low K⁺ level and NaCl treatment, the transcript of SsHKT1 maintained at a high level under NaCl stress. In order to examine the molecular mechanism of SsHKT1 expression and and its function in salt-resistance, we cloned SsHKT1 promoter on the basis of SsHKT1 sequence. SsHKT1 promoter characteristic such as cis elements has been analyzed. Promoter activity was examined using PCAMBIA 1391 vector containing SsHKT1 promoter and GUS gene. In addition, SsHKT1-overexpressed rice plants have been gengerated by Agrobacterium tumefacians to further clarify the function of SsHKT1 in plant plant slat tolernace. The main results are as follows: 1. The amplification of SsHKT1 promoterA series of specific primers were designed according to published SsHKT1 cDNA sequence. Using total DNA isolated from Suaeda salsa as the template, a 1044 bp DNA fragment upstream of the coding sequence of SsHKT1 gene was amplified by three times of TAIL-PCR. BLAST comparison suggests that the 1044 bp fragment is SsHKT1 promoter.2. The element analysis of SsHKT1 promoterThe software PLACE and PlantCARE were used to analyze the elements of SsHKT1 promoter. The result suggests that many kinds of important cis-acting elements are found in the promoter, such as NaCl-inducible element GT-1, defense- and stress-responsible element TC-rich repeats, light inducible element GATA-box and G-box, meristem expressing elements CAT-box, and special endosperm element Skn-1-motif in addition to typical cis-acting elements of a promoter such as TATA-box,CAAT-box.3. Activity and expression analysis of SsHKT1 promoterSsHKT1 promoter digested by BamHâ… and Pstâ… was introduced into plant transformation vector pCAMBIA 1391 which contains GUS gene to replace original 35S promoter. Therefore, SsHKT1 promoter-GUS recombinant vector was constructed.The SsHKT1promoter-GUS construct was transferred into Agrobacterium tumefaciens EHA105, the positive clones were selected to infect tobacco leaves, the leaves were tested by GUS histochemical staining. The result shows that GUS gene is expressed in tobacco leaves, suggesting that SsHKT1 promoter is correctly inserted into the tobacco DNA and has promoter activity.Arabidopsis inflorescence was infected by Agrobacterium tumefaciens containing SsHKT1 promoter-GUS construct, the seeds were screened by hygromycin. Resistant plants were analyzed by GUS staining. The result showed that GUS gene was mainly expressed in vascular tissue of the tansformed Arabidopsis.4. The analysis of SsHKT1 promoter transforming riceRice callus were induced on NBD2 medium with mature seeds, and subcultured on NBD0.5 medium. PROKâ…¡-SsHKT1 plasmid was transferred into Agrobacterium EHA105, positive clones were selected to infect the callus. Callus was selected with 50 mg/L G418. G418-resistant callus was induced on RE medium. The seedlings were cultured on MS medium and nutrient soil to obtain T₀ transgenic rice plant. It laid the foundation of the role in plant salt tolerance of SsHKT1.