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

• 研究报告 • 上一篇    下一篇


俞沁含1, 焦淑珍1, 吴楠1, 张宁波2, 徐伟荣2,*()   

  1. 1宁夏大学农学院,银川 750021
    2宁夏大学食品与葡萄酒学院,银川 750021
  • 收稿日期:2020-12-21 修回日期:2021-03-21 出版日期:2021-06-25 发布日期:2021-07-08
  • 通讯作者: 徐伟荣
  • 基金资助:

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)编码1个具有E3泛素连接酶活性的蛋白,是模式植物拟南芥冷反应信号转导途径的关键负调控因子,但其在多年生葡萄属植物中参与低温胁迫应答的分子机制尚不明确。以欧洲酿酒葡萄品种‘霞多丽'的叶片为供试材料,克隆了VvHOS1基因,其全长2 931 bp,编码976个氨基酸,在第74 ~ 106个氨基酸处存在RING类型的锌指结构域(zf-C3H4_3)。系统发育分析表明,葡萄属植物的HOS1蛋白与番茄、烟草、茶树的亲缘关系最近。将VvHOS1抗原表位丰富肽段(1 ~ 320 aa)构建至原核表达载体pET28a-SUMO的BamHⅠ与Hind Ⅲ位点,转化大肠杆菌。经IPTG诱导发现,pET28a- VvHOS11-320在大肠杆菌Rosetta(DE3)中高表达,融合蛋白主要以包涵体形式存在。将纯化后的重组蛋白作为抗原免疫大耳白兔,获得anti-VvHOS1多克隆抗体,ELISA检测效价为151 200,具有较强的Western特异性。该抗血清能够特异性检测‘霞多丽'葡萄叶片中的VvHOS1蛋白。(4 ℃)低温胁迫6、36与48 h时VvHOS1上调表达显著。

关键词: 葡萄, HOS1, 原核表达, 多克隆抗体, 低温胁迫, 表达模式分析


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