Scientia Agricultura Sinica ›› 2017, Vol. 50 ›› Issue (13): 2411-2422.doi: 10.3864/j.issn.0578-1752.2017.13.001

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS •     Next Articles

Response of Wheat Zinc-Finger Transcription Factor TaDi19A to Cold and Its Screening of Interacting Proteins

RU JingNa1,2, YU TaiFei2, CHEN Jun 2, CHEN Ming2, ZHOU YongBin2, MA YouZhi2XU ZhaoShi2, MIN DongHong1   

  1. 1 Northwest A & F University/State Key Laboratory of Crop Stress Biology for Arid Areas, Yangling 712100, Shaanxi; 2 Institute of Crop Science, Chinese Academy of Agricultural Sciences/National Key Facility for Crop Gene Resources and Genetic Improvement/ Key Laboratory of Biology and Genetic Improvement of Triticeae Crop, Ministry of Agriculture, Beijing 100081
  • Received:2017-01-23 Online:2017-07-01 Published:2017-07-01

Abstract: 【Objective】Zinc-finger transcription factors play an important role in stress signal transduction and abiotic stress response in plants. In this study, the function of TaDi19A was identified under low temperature stress and its interacting proteins were screened by yeast two-hybrid system to explore the regulation mechanism of TaDi19A.【Method】TaDi19A gene was isolated from the cold-treated wheat transcriptome profile. Bioinformatics method was used to analyze the molecular properties of the TaDi19A gene, SMART online tools were used for protein structure analysis; GSDS and PHYRE2 online tools were used to analyze gene structure and tertiary structure of TaDi19A protein; NetPhos 2.0 Server database was used to predict phosphorylation sites of TaDi19A protein. The quantitative real-time PCR (qRT-PCR), conducted using the cold-treated wheat cDNA based on SYBR Green technology, was used to analyze the expression pattern of TaDi19A under cold temperature stress treatment in different time periods. TaDi19A was fused with PBI121 to transform into wild-type (WT) Arabidopsis plants (Columbia-0) mediated by the floral dip method, homozygous T3 seeds of transgenic lines and WT were used for cold tolerance analysis which the root length, fresh weight, and survival rate were measured before and after cold treatment. The expressions of four stress-response genes were investigated in transgenic lines and WT under normal and low temperature conditions to analyze the cold-resistant regulation mechanism of TaDi19A. Bait plasmid pGBKT7-TaDi19A was constructed and the self-activation was detected. The pGBKT7-TaDi19A and wheat cDNA library was co-transformed into yeast cell AH109 by two-hybrid system, and the positive clones were screened via SD/-Trp/-Leu/-His/-Ade and SD/-Trp/-Leu/-His/-Ade /X-α-gal plate and these single clones were sequenced and analyzed by BLAST to obtain the interaction candidate proteins.【Result】The full length of TaDi19A gene was 747 bp with 4 exons, encoding 248 amino acids, and the protein molecular weight and isoelectric point of were 28.03 kDa and 4.74, respectively. TaDi19A protein included Zinc-finger binding domain, Di19 domain and the predicted tertiary structure contained 2 alpha helix. Phosphorylation site analysis showed that there were 12 serine, 9 threonine, and 3 tyrosine phosphorylation sites in TaDi19A protein. qRT-PCR analysis showed that TaDi19A was induced by low temperature. The root length and cold tolerance assays revealed that TaDi19A transgenic Arabidopsis increased the cold tolerance. The expression of several cold-stress-responsive genes was monitored through PCR analysis, the expression of genes CBL1, CBL2, CBL3 and KIN1 showed elevated levels in both WT and transgenic Arabidopsis plants under cold-stress condition, and the expression levels in transgenic plants were significantly higher than those in WT. Analysis of candidate proteins screened by yeast two-hybrid system revealed that those proteins mainly affected the signal transduction and abiotic stress response, which demonstrated that TaDi19A is critical to stress signal transduction and abiotic stress response in plants. 【Conclusion】Cold-inducible TaDi19A improved cold tolerance in transgenic Arabidopsis; TaDi19A might work via interacting with other proteins.

Key words: Triticum aestivum, zinc-finger transcription factor, cold tolerance, yeast two-hybrid, protein interaction

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