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园艺学报 ›› 2020, Vol. 47 ›› Issue (2): 205-219.doi: 10.16420/j.issn.0513-353x.2019-0329

• 研究论文 •    下一篇

中国野生毛葡萄芪合酶基因抗白粉病功能分析

吴凤颖,刘梦琦,王跃进   

  1. 西北农林科技大学园艺学院,旱区作物逆境生物学国家重点实验室,农业部西北地区园艺作物生物与种质创制重点实验室,陕西杨凌 712100
  • 出版日期:2020-02-25 发布日期:2020-02-25
  • 基金资助:
    国家自然科学基金项目(31672129)

Function Analysis of the Stilbene Synthase VqSTS12 and VqSTS25 Genes of the Resistance to Powdery Mildew in Vitis quinquangularis

WU Fengying,LIU Mengqi,and WANG Yuejin   

  1. College of Horticulture,Northwest Agriculture and Forestry University/State Key Laboratory of Crop Stress Biology in Arid Areas/Key Laboratory of Horticultural Plant Germplasm Resource Utilization in Northwest China,Ministry of Agriculture,Yangling,Shaanxi 712100,China
  • Online:2020-02-25 Published:2020-02-25

摘要: 中国野生毛葡萄(Vitis quinquangularis)‘丹凤–2’白藜芦醇含量高,并抗白粉病。以此为材料,利用同源序列克隆技术获得两个芪合酶基因 VqSTS12 和 VqSTS25。以欧洲葡萄‘无核白’分生愈伤组织为受体材料,通过农杆菌介导法进行遗传转化,分别获得 7 个 VqSTS12 和 5 个 VqSTS25‘无核白’葡萄转基因植株。实时定量 PCR 分析发现,与野生型植株相比,转基因植株中芪合酶基因 STS 与下游白藜芦醇糖基转移酶基因 RSGT 表达量升高,与 STS 有底物竞争关系的查尔酮合酶基因 CHS 表达量降低。STS 的表达产物中主要的芪类物质反式云杉新苷含量增加。选择芪类物质含量高的转基因植株接种白粉菌发现,转基因植株对白粉菌的敏感性降低,发病较晚,病情较轻,在转基因植株上部分孢子萌发受抑制,菌丝发育迟缓。转基因植株中 VqSTS12 和 VqSTS25 受白粉菌诱导表达:在接菌后 1 ~ 3 d 内表达量显著升高,随后略有下降,在 6 ~ 7 d 表达量再次上升。同时高效液相色谱(HPLC)检测到 STS 的表达产物白藜芦醇、云杉新苷、葡萄素与紫檀芪含量增加。研究结果表明‘丹凤–2’毛葡萄拥有的 VqSTS12 和VqSTS25 及其表达的芪类物质具有抑制白粉菌的作用。

关键词: 毛葡萄, 芪合酶基因, 白藜芦醇, 抗病性, 白粉病

Abstract: Vitis quinquangularis‘Danfeng-2’is an important research material with high resistance to powdery mildew due to its high resveratrol content. In the present study,two stilbene synthase genes VqSTS12 and VqSTS25 were obtained from‘Danfeng-2’leaves using homologous sequence cloning. The overexpression vector was constructed and the transgenic seedlings were obtained by Agrobacteriummediated transformation with meristematic bulks of Vitis vinifera‘Thompson Seedless’was used as the receptor material. Through genetic transformation,we finally obtained 7 and 5 transgenic seedlings of VqSTS12 and VqSTS25 respectively,which were confirmed by PCR at DNA level and Western Blot at protein level. Afterwards,the expression of genes related to resveratrol synthesis in transgenic‘Thompson Seedless’seedlings was analyzed by real-time quantitative PCR,which revealed that the expression of stilbene synthase(STS)gene and downstream resveratrol glycosyltransferase(RSGT)gene were up-regulated,while that of chalcone synthase(CHS)gene was down-regulated due to the substrate competition with STS. Meanwhile,the content of stilbene components was higher in transgenic‘Thompson Seedless’seedlings compared to that in wild-type seedlings by HPLC,mainly existing in the form of trans-piceid. The transgenic plants with high content of stilbene components were subsequently selected for the functional study. As a result,the transgenic‘Thompson Seedless’seedlings were obviously less sensitive to powdery mildew than wild-type seedlings. The occurrence of powdery mildew on the transgenic‘Thompson Seedless’seedlings was later and milder compared with non-transgenic seedlin