Scientia Agricultura Sinica ›› 2014, Vol. 47 ›› Issue (21): 4318-4327.doi: 10.3864/j.issn.0578-1752.2014.21.017

• HORTICULTURE • Previous Articles     Next Articles

Expression and Protein Interaction Analysis of Light Responsive bZIP Transcription Factor MdHY5

LI Hui-feng1, WANG Xiao-fei2, RAN Kun1, HE Ping1, WANG Hai-bo1, LI Lin-guang1   

  1. 1Shandong Institute of Pomology, Tai’an 271000, Shandong
    2College of Horticulture Science and Engineering, Shandong Agricultural University, Tai’an 271018, Shandong
  • Received:2014-03-31 Revised:2014-06-30 Online:2014-11-01 Published:2014-11-01

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Abstract: 【Objective】Cloning of the key light responsive bZIP (Basic-leucine zipper) transcription factors from Malus domestica Borkh, gene expression and protein interaction analysis, which were determined to identify the function and molecular mechanism of MdHY5.【Method】First, semi-quantitative primers used for detecting expression levels were designed from the apple in order to identify the light-responsive bZIP genes. Differentially screened and identified a light responsive gene named as MdHY5 based on comparative analysis of homologous. Subsequently, the primers were designed to clone the MdHY5 gene. Phylogenetic relationship including the MdHY5 protein with Arabidopsis bZIP proteins was analyzed using MEGA5 and the multiple alignment of bZIP domain was constructed, while the conserved protein domain was predicted, spatio expression analysis was made to detect the MdHY5 in different organs. The expression of MdHY5 in response to various light qualities was detected by semi-quantitative RT-PCR based on promoter prediction analysis, combined with the Arabidopsis【Result】A bZIP transcription factor was isolated in apple through the light response analysis. Phylogenetic relationship showed that MdHY5 is a homolog of Arabidopsis AtHY5. This gene is located on chromosome 12 of apple genome, with the gene number MDP0000586302, MdHY5 gene contains four exons and three introns in genome structure, which is similar to its homolog. The length of the cDNA is 1 112 bp, with 214 bp in the 5′ untranslated region, 403 bp in 3′ untranslated region, and 495 bp of open reading frame, which encode a 164 amino acid residues. MdHY5 contains a leucine zipper structure typical (bZIP domain) in the C-terminal of the protein, with a nuclear localization signal domain (NLS domain) included. Expression analysis showed that MdHY5 is expressed in various tissues, with the highest level in leaves. Promoter analysis showed that multiple light responsive cis-acting element exists in the promoters of MdHY5, such as G-box, GT-1-box, I-box and as so on. Quantitative expression analysis showed that MdHY5 gene could be induced by white, blue and UV light. The prokaryotic expression vector was transformed into BL21 and the fusion protein presented mainly in the supernatant after sonication. Pull down assay showed that MdHY5 could interact with MdCOP1 protein in vitro. 【Conclusion】 MdHY5 is a light responsive bZIP transcription factor and homologous to AtHY5, which is the key transcription factor in photomorphogenesis of Arabidopsis, MdHY5 has similar genome structure and conserved protein domain to AtHY5, and can be interacted with MdCOP1. microarray database. Finally, MdHY5 gene was inserted into the prokaryotic vector pGEX-4T-1, and IPTG was used to induce the fusion protein expression in E.coli BL21. Then the fusion protein was purified and detected by Western blot. The interaction between MdHY5 and MdCOP1 was detected by pull down assay.

Key words: apple, bZIP transcription factor, MdHY5, light responsive, protein interaction

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