The Poplar Xylem-specific Promoter Function And The Key Regulatory Domain Analysis Of JCesAP

As one of the main tree species of China, Poplar has the fine features of rapid growth, adaptability, easy hybridization and improvement.With the ever-increasing development of landscaping, poplar play an important role. But due to the beetles stem borers severely damaging poplar, and the use of traditional control technology is difficult to prevent and inevitable to environmental pollution, plant genetic engineering technology controlling the boring pests become the effective measures to solve this problem effectively.In this study, the plant expression vectors fused with report gene and promoters isolated from poplar were transformed into poplar and tobacco. To screen the xylem-specific promoter, the GUS activity of different tissues and organs were detected quantitatively. The GUS activity of different organs of transgenic poplar under the stimulation of trauma and ABA were quantitatively analyzed to learn the response of the promoter to trauma and ABA hormone. At the same time, using deletion analysis, the different subsegment of the xylem-specific promoter JCes AP isolated early from Populus Canadensis Aanalysis were acquired. Using model plants as transgenic system,expression ability and level of the subsegments driving GUS gene in transgenic plants were analyzed. The structure and function of the regulatory elements and the relationship between the structure and the interaction would be clarified. The regulatory element for determining the specificity and the inducing activity of the xylem tissue were determined.The main results are as follows:(1) With Agrobacterium mediated method, transgenic plants, including PMDCes AP-GUS 、 PJCes AP-GUS and PDMDCes AP-GUS, were obtained. The GUS histochemical staining result showed that plant material leaf are displayed blue, which indicated that MDCes AP, JCes AP and DMDCes AP have promoter activity. But MDCes AP shows obvious mesophyll vein and deep blue, indicating the xylem tissue specificity obviously. Through quantitative GUS activity detection, the results show that MDCes AP is the most powerful promoter and with xylem tissue specificity; Compared with leaf and stem, DMDCes AP shows the highest specificity in the root; compared with root and stem, JCes AP shows highest specificity in leaf tissue.(2) The quantitative detection of GUS activity in the roots, stems, and leaves of transgenic poplar harboring PMDCes AP-GUS, PJCes AP-GUS and PDMDCes AP-GUS constructs after wounding and ABA treatment were performed. The results show that 12 h after wounding treatment, GUS activity was highest in the roots of PMDCes AP plants.After ABA treatment, GUS activity of roots in PDMDCes AP and PJCes AP plants increased first, and then decreased, increased, idecreased. GUS activity in PDMDCes AP was highest at 0h. In roots and stems, GUS activity in PMDCes AP plants was highest before ABA treatment.(3) The quantitative detection of GUS activity in the roots, stems, and leaves of transgenic tobacco harboring PMDCes AP-GUS, PJCes AP-GUS and PDMDCes AP-GUS constructs after wounding and ABA treatment were performed. The results show that 24 h after wounding treatment, GUS activity was highest in the roots of p JCes AP plants.After ABA treatment, GUS activity of roots in PMDCes AP plants decreased first, and then increased, decreased, increased. In roots and leaves, GUS activity in PJCes AP increased first, and then decreased. In roots, GUS activity in PDMDCes AP plants was highest before ABA treatment.(4) GUS was used as the reporter gene, and a series of plant deletion expression vector of JCes AP promoter were constructed. The activity of different subsegment of JCes AP was analyzed by Agrobacterium mediated transformation. The function of the deletion fragments was analyzed by instantaneous detection. The results showed that the deletion fragments of L2, L3 and L4 had the promoter function. Using GUS histochemical staining method, stable expression transgenic tobacco was detected. The results showed L2 blade surface has obvious blue region. Under the microscope, L3 and L4 could be seen the blue, not too obvious, indicating that the L2, L3 and L4 subsegments have promoter activity, L2 activity is stronger than i L3 and L4.