The Characterization Of ZmPIS Promoter From Maize Response To Stress And Identification Of The Core Functional Region

Drought and soil salinization severely limits plant growth and development,, which have become important factors affecting the quality and yield of crop. The biological process of crop adaptation to drought and soil salinization and promoting yield are regulated by multiple genes. Discovering and cloning genes and promoters responded to adversity and developing drought and salt tolerant cultivars by transgenic technology is an effective way to solve the above problems. Using plant genetic engineering means to improve crop traits, target gene highly expressed in the host cells and tissues or temporal and spatial specificity has attracted a lot of attention. The regulation of gene expression could occur in the DNA, transcription, translational and post-translational levels. The regulation of transcription level is the interactions between cis-acting elements of promoter and trans-acting factor to achieve gene expression in specific time or tissue. Cloning promoter with independent intellectual property rights, which could be used for genetic improvement of crop resistance, and discovering its core functional sections and regulatory elements have important theoretical significance and applicational value.In previous work, Zhai et al. found that maize ZmPIS gene could be induced by salt and drought stress. Guan et al. cloned the promoter sequence of ZmPIS gene and preliminary analyze its activity. On the basis of the above work, the function analysis and characterization of ZmPIS promoter are performed systematically.Bioinformatic analysis showed that ZmPIS gene promoter contains multiple stress response elements. Series of 5’deleted promoter-Gus mutants (PZ1-PZ8) were constructed and transformed into tobacco (Nicotiana benthamiana). GUS fluorescence activities of PZ1-PZ8 transgenic tobacco leaves were determined. The results showed that PZ7 had a significantly higher activity, compared to the PZ1-6 and PZ8. GUS staining with flowers, fruits, seeds and 2-week-old plants of PZ1, PZ7 and 35S transgenic tobacco indicated that PZ7 had a higher GUS expression intensity in all above tissues, compared to PZ1 full-length promoter.To further study the stress-response characteristics of ZmPIS promoter, the expression levels of Gus gene in PZ1-PZ8 transgenic tobacco detached leaves were determined under 200 mM NaCl or 18% PEG treatment. The results showed that PZ1-PZ7 had similar induced-expression pattern under salt and osmotic stress. Compared to untreated controls, GUS staining was significantly deeper after 24h、 48h、72 h stress treatment. However, GUS staining intensity of PZ8 and 35S promoter transgenic tobacco leaves had no obvious differences during the stress treatment. Moreover, PZ7 showed the highest GUS staining intensity contrast with PZ1-PZ6 and PZ8 under different conditions. To further verify the above results of experiments in vitro, GUS staining and fluorescence activities of PZ1, PZ2, PZ6, PZ7 and PZ8 transgenic tobacco plants were determined with 200 mM NaCl or 18% PEG treatment. The results showed that GUS enzyme activities of PZ1、PZ2、PZ6 and PZ7 were obviously induced up to more than 2.0 fold in the leaves under salt or osmotic stress condition and maintained at a relatively high expression level with 48 h and 72 h treatment. That is to say, PZ1-PZ7 had obviously salt and osmotic stress-inducing activity while the stress- inducing activities of PZ8 were loss significantly. Notably, compared with other mutants, PZ7 had highest promoter activity and reached 20 times more than PZ1 and 3 times more than PZ6 under both normal and stress conditions. Under normal conditions, the activity of PZ7 was about 1.1 fold of PZ8 and 0.2 fold of 35S promoter, while reached 2-fold more than PZ8 and 0.5 fold of 35S promoter under salt or osmotic stress. These results suggested that the 110bp fragment of ZmPIS promoter between PZ7 and PZ8 (-466 to-357 bp) may contain functional elements involved in salt and osmotic stress response. PZ7 (-466 bp) is the key sequence fragments of ZmPIS promoter with high promoter activity and the characterization of salt and osmotic stress response, which showed the applicational prospect.In the study, we found that -466 bp (PZ7) fragment had high promoter activity and salt- and osmotic-stress responsive activity. Thus,-466 bp was a core functional region of ZmPIS promoter. The 110 bp sequence between -466 to-357 bp could contain salt and osmotic stress responsive cis-acting element. These results will deepen our understanding on core functional region and stress-responsive cis-acting element of ZmPIS promoter and provide new promoter resources and valuable information for crop genetic improvement, thereby contributing to resolve the status of salt or drought stress in the agricultural production of China.