Acta Horticulturae Sinica ›› 2021, Vol. 48 ›› Issue (6): 1173-1180.doi: 10.16420/j.issn.0513-353x.2020-0557

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Molecular Cloning,Expression and Polyclonal Antibody Preparation of E3 Ubiquitin Ligase Gene HOS1 from Vitis vinifera

YU Qinhan1, JIAO Shuzhen1, WU Nan1, ZHANG Ningbo2, XU Weirong2,*()   

  1. 1College of Agronomy,Ningxia University,Yinchuan 750021,China
    2School of Food and Wine,Ningxia University,Yinchuan 750021,China
  • Received:2020-12-21 Revised:2021-03-21 Online:2021-06-25 Published:2021-07-08
  • Contact: XU Weirong


HOS1High expression of osmotically responsive genes 1)encodes a protein with E3 ubiquitin ligase activity,and is a key negative regulator of cold response signal transduction pathway of model plant Arabidopsis,but its molecular mechanisms involved in the low temperature stress response in perennial Vitis vinifera are unclear. The VvHOS1 gene was cloned from the leaves of Vitis vinifera ‘Chardonnay'. The full length of VvHOS1 was 2 931 bp,coding 976 aa,and the presence of a RING-type zinc finger domain was located at 74-106 aa(zf-C3H4_3). Phylogenetic tree analysis showed that VvHOS1 protein in Vitis vinifera is most closely related to tomato,tobacco,and tea tree. Furthermore, the VvHOS1 antigen epitope rich fragment(1-320 aa)was constructed into the prokaryotic expression vector pET28a-SUMO at the BamHⅠandHind Ⅲ restriction sites. The pET28a- VvHOS11-320was found to be highly expressed inEscherichia coliRosetta(DE3)with isopropyl-β-D-thiogalactoside(IPTG)induction. The fusion proteins were mainly in the form of inclusion body proteins. The purified recombinant protein was used as antigen to immunize white rabbits,and the resulting anti-VvHOS1 polyclonal antibody was obtained and detected by ELISA. The potency was 151 200 with a strong Western specificity. The antiserum specifically detects VvHOS1 protein in grapevine leaves after cold stress(4 ℃),and Western blot analysis indicated that the expression of VvHOS1 was significantly up-regulated at 6,36 and 48 h.

Key words: grape, HOS1, prokaryotic expression, polyclonal antibody, cold stress, expression pattern analysis

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