Advanced search
    Xie Muhong, Li Wenkai, Zhang Zhaolong, Cui Maokai, Wang Yi, Sun Yawei, Yang Guiyan. Activity analysis of different length fragments of walnut JrTT1-1 promoter in response to drought stress[J]. Journal of Beijing Forestry University, 2022, 44(8): 31-38. DOI: 10.12171/j.1000-1522.20210126
    Citation: Xie Muhong, Li Wenkai, Zhang Zhaolong, Cui Maokai, Wang Yi, Sun Yawei, Yang Guiyan. Activity analysis of different length fragments of walnut JrTT1-1 promoter in response to drought stress[J]. Journal of Beijing Forestry University, 2022, 44(8): 31-38. DOI: 10.12171/j.1000-1522.20210126

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

    More Information
    • Received Date: April 01, 2021
    • Revised Date: April 25, 2021
    • Accepted Date: July 07, 2022
    • Available Online: July 11, 2022
    • Published Date: August 24, 2022
    •   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.
    • [1]
      Pelin A, Panos D. DNA sequence and structural properties as predictors of human and mouse promoters[J]. Gene, 2008, 410(1): 165−176. doi: 10.1016/j.gene.2007.12.011
      [2]
      Coca M A, Almoguera C, Thomas T L, et al. Differential regulation of small heat-shock genes in plants: analysis of a water-stress-inducible and developmentally activated sunflower promoter[J]. Plant Molecular Biology, 1996, 31(4): 863−876. doi: 10.1007/BF00019473
      [3]
      吉仁花, 张文波, 林晓飞, 等. 杂交构树UDP-葡萄糖脱氢酶基因编码蛋白的亚细胞定位及其启动子5′端缺失片段的功能分析[J]. 植物研究, 2020, 40(6): 932−942. doi: 10.7525/j.issn.1673-5102.2020.06.016

      Ji R H, Zhang W B, Lin X F, et al. Subcellular localization of the protein coded by the UDP-glucose dehydrogenase gene from paper mulberry and functional of its promoter 5′-end deletion fragment[J]. Bulletin of Botanical Research, 2020, 40(6): 932−942. doi: 10.7525/j.issn.1673-5102.2020.06.016
      [4]
      殷金瑶, 王义, 徐良向, 等. 橡胶树白粉菌(HO-73)启动子WY172不同长度片段的克隆及表达活性分析[J]. 生物技术通报, 2020, 36(1): 29−36. doi: 10.13560/j.cnki.biotech.bull.1985.2019-0728

      Yin J Y, Wang Y, Xu L X, et al. Cloning and expression analysis of different-length fragments of Oidium heveae (HO-73) promoter WY172[J]. Biotechnology Bulletin, 2020, 36(1): 29−36. doi: 10.13560/j.cnki.biotech.bull.1985.2019-0728
      [5]
      肖前林. 转录因子ZmMYB14、ZmNAC126参与玉米淀粉合成调控机制研究[D]. 雅安: 四川农业大学, 2017.

      Xiao Q L. The mechanism of transcription factors ZmMYB14 and ZmNAC126 participated in the regulation of maize starch synthesis[D]. Ya’an: Sichuan Agricultural University, 2017.
      [6]
      杨桂燕, 郭宇聪, 张凤娇, 等. 不同长度ThVHAc1基因启动子片段分离及活性分析[J]. 林业科学, 2016, 52(1): 55−61.

      Yang G Y, Guo Y C, Zhang F J, et al. Isolation and activity analysis of different length ThVHAc1 promoters[J]. Scientia Silvae Sinicae, 2016, 52(1): 55−61.
      [7]
      Appelhagen I, Weisshaar B, Sagasser M, et al. TRANSPARENT TESTA1 interacts with R2R3-MYB factors and affects early and late steps of flavonoid biosynthesis in the endothelium of Arabidopsis thaliana seeds[J]. The Plant Journal, 2011, 67(3): 406−419. doi: 10.1111/j.1365-313X.2011.04603.x
      [8]
      Sagasser M, Lu G, Hahlbrock K, et al. A. thaliana TRANSPARENT TESTA 1 is involved in seed coat development and defines the WIP subfamily of plant zinc finger proteins[J]. Genes & Development, 2002, 16(1): 138−149.
      [9]
      王艳花, 荐红举, 邱晓, 等. 白菜型油菜粒色主效基因BrTT1的调控机制分析[J]. 作物学报, 2020, 46(11): 1678−1689.

      Wang Y H, Jian H J, Qiu X, et al. Regulatory mechanism of the seed coat color gene BrTT1 in Brassica rapa L.[J]. Acta Agronomica Sinica, 2020, 46(11): 1678−1689.
      [10]
      Li X M, Chao D Y, Wu Y, et al. Natural alleles of a proteasome α2 subunit gene contribute to thermotolerance and adaptation of African rice[J]. Nature Genetics, 2015, 47(7): 827−833. doi: 10.1038/ng.3305
      [11]
      杨毅. TT1基因在提高棉花抗逆性中的用途: 201110022652.1[P]. 2012−07−25.

      Yang Y. The use of TT1 gene in improving cotton resistance: 201110022652.1[P]. 2012−07−25.
      [12]
      Yang G, Chen S, Li D, et al. Multiple transcriptional regulation of walnut JrGSTTau1 gene in response to osmotic stress[J]. Physiologia Plantarum, 2019, 166(3): 748−761. doi: 10.1111/ppl.12833
      [13]
      王艺, 张尚昆, 赵翔, 等. 核桃TT1类转录因子的筛选及干旱响应分析[J]. 西北林学院学报, 2020, 35(1): 86−93. doi: 10.3969/j.issn.1001-7461.2020.01.13

      Wang Y, Zhang S K, Zhao X, et al. Identification and expression of the TT1-like transcription factor from Juglans regia under drought stress[J]. Journal of Northwest Forestry University, 2020, 35(1): 86−93. doi: 10.3969/j.issn.1001-7461.2020.01.13
      [14]
      Yang G Y, Zhang W H, Liu Z X, et al. Both JrWRKY2 and JrWRKY7 of Juglans regia mediate responses to abiotic stresses and abscisic acid through formation of homodimers and interaction[J]. Plant Biology, 2017, 19(2): 268−278. doi: 10.1111/plb.12524
      [15]
      Yang G Y, Zhang T T, Zhai M Z, et al. Two novel WRKY genes from Juglans regia, JrWRKY6 and JrWRKY53, are involved in abscisic acid-dependent stress responses[J]. Biologia Plantarum, 2017, 61(4): 611−621. doi: 10.1007/s10535-017-0723-x
      [16]
      Clough S J, Bent A F. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana[J]. The Plant Journal, 1998, 16(6): 735−743. doi: 10.1046/j.1365-313x.1998.00343.x
      [17]
      Zheng L, Liu G, Meng X, et al. A WRKY gene from Tamarix hispida, ThWRKY4, mediates abiotic stress responses by modulating reactive oxygen species and expression of stress-responsive genes[J]. Plant Molecular Biology, 2013, 82(4−5): 303−320. doi: 10.1007/s11103-013-0063-y
      [18]
      吴艳菊, 冷彦儒, 景思荦, 等. 利用真空侵染法在紫花苜蓿中瞬时表达GUS基因[J]. 分子植物育种, 2022, 20(3): 859−864.

      Wu Y J, Leng Y R, Jing S L, et al. Expression of GUS gene in Medicago sativa L. by vacuum infiltration[J]. Molecular Plant Breeding, 2022, 20(3): 859−864.
      [19]
      Yang G, Gao X, Ma K, et al. The walnut transcription factor JrGRAS2 contributes to high temperature stress tolerance involving in Dof transcriptional regulation and HSP protein expression[J]. BMC Plant Biology, 2018, 18(1): 367. doi: 10.1186/s12870-018-1568-y
      [20]
      Yang G, Wang C, Wang Y, et al. Overexpression of ThVHAc1 and its potential upstream regulator, ThWRKY7, improved plant tolerance of Cadmium stress[J]. Scientific Reports, 2016, 6: 18752. doi: 10.1038/srep18752
      [21]
      Lippok B, Birkenbihl R P, Rivory G, et al. Expression of AtWRKY33 encoding a pathogen- or PAMP-responsive WRKY transcription factor is regulated by a composite DNA motif containing W box elements[J]. Molecular Plant Microbe Interactions, 2007, 20(4): 420−429. doi: 10.1094/MPMI-20-4-0420
      [22]
      郭丹华, 彭倩, 胡敏伦, 等. 紫心甘薯IbCHS基因启动子的克隆及功能分析[J]. 分子植物育种, 2019, 17(3): 700−705.

      Guo D H, Peng Q, Hu M L, et al. Cloning and functional analysis of the promoter of IbCHS gene from purple sweet potato[J]. Molecular Plant Breeding, 2019, 17(3): 700−705.
      [23]
      Tounsi S, Saidi M N, Abdelhedi R, et al. Functional analysis of TmHKT1;4-A2 promoter through deletion analysis provides new insight into the regulatory mechanism underlying abiotic stress adaptation[J]. Planta, 2021, 253(1): 18. doi: 10.1007/s00425-020-03533-9
      [24]
      秦琳琳, 张曦, 姜骋, 等. 白桦BpZFP4基因启动子克隆和逆境响应元件功能分析[J]. 植物研究, 2019, 39(6): 917−926.

      Qin L L, Zhang X, Jiang C, et al. Cloning and functional analysis of BpZFP4 promoter from birch (Betula platyphylla)[J]. Bulletin of Botanical Research, 2019, 39(6): 917−926.
      [25]
      Feng Y, Cui R, Wang S, et al. Transcription factor BnaA9. WRKY47 contributes to the adaptation of Brassica napus to low boron stress by up-regulating the boric acid channel gene BnaA3. NIP5;1[J]. Plant Biotechnology Journal, 2020, 18(5): 1241−1254. doi: 10.1111/pbi.13288

    Catalog

      Article views (695) PDF downloads (70) Cited by()

      /

      DownLoad:  Full-Size Img  PowerPoint
      Return
      Return