| Drought, salinity, high and low temperature and oxidative stresses which influence plant growth and development seriously, and then lead to the productivity declining and poor quality of crops. Sucrose non-fermenting 1-related protein kinase 2(Sn RK2) is a kind of protein kinase play crucial roles in abiotic stress response in plants. In previous work, the Sn RK2 gene family was cloned from potato(Solanum tuberosum L.), a total of eight family members were St Sn RK2.1~St Sn RK2.8. Promoter is an important regulatory element on transcription level, combine with many kinds of transcription factors, and determine the expression pattern and expression intensity. At present, a large number of Sn RK2 genes have been isolated and studied, but there is rarely to study Sn RK2 gene promoter. We analyzed St Sn RK2 transgenic tobacco plants resistance to adversity and explored the Sn RK2 gene promoter’s expression characteristics in plants. We cloned the Sn RK2 gene promoters from potato and analyzed preliminary their function. The main results are as follows:1. Under PEG and Na Cl treatment, biological characteristic and physiological and biochemical characteristics of the St Sn RK2.3, St Sn RK2.5, St Sn RK2.6 and St Sn RK2.8 transgenic tobaccos were studied. The results showed that these four transgenic tobacco plants had significant resistance compared to non-transgenic tobacco plant, clarifying these four genes play an important role in improving drought- and salt-tolerance.2. The gene upstream sequence from the translation start site was cloned from potato genome by conventional PCR, the size are 977 bp, 961 bp, 855 bp, 932 bp, 766 bp, 763 bp, 686 bp, and named p St Sn RK2.1, p St Sn RK2.2, p St Sn RK2.3, p St Sn RK2.4, p St Sn RK2.5, p St Sn RK2.6, p St Sn RK2.8, respectively. Using the software for sequences analysis, the results showed that the promoters’ sequences contain basic component TATA-box, CAAT-box and a plurality of cis-acting elements in response to abiotic stresses in plants.3. In order to deeply study the function of Sn RK2 gene promoters, the plant expression vector with the fusion of gene promoter and GUS gene were constructed. In this study, four vectors were successfully constructed; there were p BI121-p St Sn RK2.3, p BI121-p St Sn RK2.5, p BI121-p St Sn RK2.6 and p BI121-p St Sn RK2.8. Then they were transferred into Agrobacterium tumefaciens and introduced into tobacco by leaf disc transformation method. Transgenic tobacco plants were obtained through a series of antibiotic kanamycin screening and PCR amplification.4. Different tissues of GUS staining were performed on transgenic tobacco. The results showed that p St Sn RK2.3, p St Sn RK2.5, p St Sn RK2.6 and p St Sn RK2.8 all detected GUS staining in different tissues(root, stem and leaf). Then measured different levels of GUS activity, the GUS activity was the highest in stem, followed by leaf, root was the lowest. It indicted that these promoters driven gene expression did not have tissue specificity in transgenic tobacco plants.5. GUS activity of transgenic tobacco plants were measured for abiotic stresses treatments, the results showed that GUS activities of p St Sn RK2.3, p St Sn RK2.5 and p St Sn RK2.6 exhibit different trends. The p St Sn RK2.3 is relatively sensitive to ABA and Na Cl treatments, and not activated by PEG treatment. The p St Sn RK2.5 and p St Sn RK2.8 are not activated by ABA treatment and Na Cl treatment, but they are strongly activated by ABA treatment. The p St Sn RK2.6 is not activated by ABA treatment, but it is activated by Na Cl treatment and PEG treatment, and the reaction of Na Cl treatment is stronger than PEG treatment. |
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