Activity analysis of different length fragments of walnut JrTT1-1 promoter in response to drought stress

  Objective  TT1 is a C2H2-ZFP (WIP-type zinc finger structure) transcription factor regulatory protein. Walnut JrTT1-1 gene promoter contains drought response elements and has the function of regulating drought stress. In this study, the different length promoter fragments of JrTT1-1 gene were isolated and their expression activity after drought stress was analyzed to explore the mechanism of JrTT1-1 gene response to drought stress.

  Method  According to the distribution of WRKY cis-acting elements, the promoter of JrTT1-1 gene was divided into 5 fragments of different lengths: 1 002 bp (−1 − −1 002), 720 bp (−1 − −720) , 448 bp (−1 − −448), 174 bp (−1 − −174) and 149 bp (−1 − −149), and they were denoted as S1, S2, S3, S4, S5, respectively. The recombinant vector was constructed by replacing the CaMV35S promoter of pCAMBIA1301 vector with S1, S2, S3, S4 and S5, and transformed into Arabidopsis thaliana by Agrobacterium-mediated dipping method. The transgenic lines were confirmed by hygromycin screening, PCR verification and GUS gene expression evaluation. Then the transgenic lines were cultivated to T3 generation for further analysis. GUS activity was measured in different tissues at different growth stages to evaluate the temporal and spatial expression activity of different fragments. The seeds of S1, S2, S3, S4, and S5 transgenic plants were germinated and grown to 30-d-old and then subjected to drought treatment (50 mM mannitol), no drought treatment was set as the control (CK), the GUS enzyme activities of the whole plant, roots and aerial parts were analyzed to evaluate the difference of varied fragments in response to drought.

  Result  Under normal growth conditions, GUS activity can be detected in S1, S2, S3, S4, S5 transgenic Arabidopsis in different growth stages and in varied tissues and organs, but the GUS activity of different fragments was different, and the activity decreased as the fragment became shorter; but the difference between S1 and S2 was not significant. Comparing the GUS activity in mature seeds, fresh seeds, 35-d-old roots, stems, leaves, and flowers, it was found that there were differences between different tissues, which reflected the specificity of the tissue expression of five fragments. Under drought stress, the GUS activities of the whole plant, roots and aerial parts of S1, S2, S3, S4, and S5 transgenic plants were significantly increased: the GUS activities of the whole plant were increased by 1.50-, 1.46-, 1.47-, 1.46-, 2.23-fold, the roots were enhanced by 1.29-, 1.29-, 1.28-, 1.53-, 1.36-fold, and the aerial parts were strengthen by 1.62-, 1.59-, 1.57-, 1.59-, 2.30-fold, respectively, of those under normal conditions.

  Conclusion  The expression activity of the JrTT1-1 gene promoter fragment is positively correlated with its length, and the activity of each length promoter fragment is specific to tissues of root, stem, leaf, flower, and seed. WRKY elements and their numbers may be related to the regulation of drought stress, and the expression of JrTT1-1 promoter segments under drought stress also has tissue differences.