Studies On A Jasmonates-responsive Cis-acting Element Of PDF1.2 Promoter And Its Interactions With Relevant Transcription Factors

The plant hormone jasmonatic acid affects development and mediates plant responses to environmental stresses. Transcriptional regulation is controlled by the interactions of cis-acting elements with the relevant factors. In this paper, an important cis-acting element within the promoter of PDF7.2, which is activated by JAs, was analyzed ,and the interaction of this element with relevant factors (JERFs) was also studied. The results were as fellows:LA JA responsive cis-acting element within the promoter of PDF 1.2 was investigated via comprehensive mutant analysis.(1)A strategy based on cDNA sequence was used to amplify the promoter ofPDF1.2 gene with Arabidopsis gDNA as template. In transgenic tobacco plants ,the analysis of transient expression by monitoring 3 -glucuronidase activity revealed that a chimeric gene construct containing a 1.2 kb PDF1.2 promoter fused to a GUS reporter gene was induced by MeJA.(2)Deletions of four different lengths were conducted using PCR amplification with the information of sequence of PDF 1.2 gene promoter. These mutated promoters were fused to the GUS reporter gene and introduced into tobacco plants. The results of GUS activity driven by these different constructs showed that the promoter sequence between positions-300 and -243 (containing a GCC box and a G box-Like sequence)was an essential JA-responsive element region to activate the expression of GUS reporter gene, and the -300 bp position was defined as the boundary of the minimal functional promoter in response to JA signaling.(3)On the basis of the deletion analysis, three substitution mutants (Ml: 6bp sequence upstream of GCC box M2: GCC box and M3: G box-like sequence)by PCR were designed to isolate the essential JA-responsive element. Transgenic tobacco plants containing promoter substitution constructs were generated by Agrobacterium-rnQdiaied leaf transformation. Loss-of-function experiment, using transient expression analysis of GUS reporter genes, confirmed that GCC box act as an essential element to respond JAsignaling in PDF1.2 promoter. Compared \\ith the GCC box. the G box-like sequence and the imperfect palindrome (GATTAACC) had less effect on the promoter to respond JA signaling.(4)A chimeric promoter containing 4 GCC box repeat fragment and a 35S minimal promoter was constructed. Furthermore, the chimeric promoter replaced the 35S promoter in pBI121 vector. In transgenic tobacco plants, the transient-expression assay of the chimeric gene (4 X GCC-35S min::GUS) demonstrated that the 4 X GCC-35Smin promoter could respond to MeJA treatment and the GCC box is an important element in response to JA signaling. Moreover, this experiment results would be meaningful to improve the crops characterization of resistance against various environment stresses or to study the regulation of gene expression in transgenic plants.2.Using yeast one-hybrid system ,the interactions between the GCC box and its relevant factors(JERF 1/2/3/4) were assayed, The main results were as fellows:(l)In the experiment xGCC-box sequence (target biding sequence) was synthesized and separately inserted into (Sma I ) and pHISi-1 (EcoR I and Xba I) plasmids. These linearized reporter plasmids were separately integrated into yeast YM4271 genomes , and different yeast colonies with integrated pLacZi-4GC pLacZi-4>