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园艺学报 ›› 2021, Vol. 48 ›› Issue (1): 73-82.doi: 10.16420/j.issn.0513-353x.2020-0378

• 研究论文 • 上一篇    下一篇

芥蓝二氢黄酮醇4-还原酶基因BoaDFR的克隆与功能鉴定

郑好1, 张芬1, 简越1, 黄文莉1, 梁莎1, 江敏1, 袁巧1, 汪俏梅2, 孙勃1,**()   

  1. 1四川农业大学园艺学院,成都 611130
    2浙江大学农业与生物技术学院园艺系,杭州 310058
  • 收稿日期:2020-05-19 修回日期:2020-11-05 出版日期:2021-01-25 发布日期:2021-01-29
  • 通讯作者: 孙勃 E-mail:bsun@sicau.edu.cn
  • 基金资助:
    国家自然科学基金项目(32072586);国家自然科学基金项目(31500247);农业农村部转基因专项(2016ZX08009003-001);四川省科技厅项目(2018NZ0081);知食供应链科技有限公司科技项目(2020008)

Cloning and Function Identification of Dihydroflavonol 4-Reductase Gene BoaDFR in Chinese Kale

ZHENG Hao1, ZHANG Fen1, JIAN Yue1, HUANG Wenli1, LIANG Sha1, JIANG Min1, YUAN Qiao1, WANG Qiaomei2, SUN Bo1,**()   

  1. 1College of Horticulture,Sichuan Agricultural University,Chengdu 611130,China
    2Department of Horticulture,College of Agriculture and Biotechnology,Zhejiang University,Hangzhou 310058,China
  • Received:2020-05-19 Revised:2020-11-05 Online:2021-01-25 Published:2021-01-29
  • Contact: SUN Bo E-mail:bsun@sicau.edu.cn

摘要:

以白花芥蓝‘四季粗条’为材料克隆了BoaDFR基因,GenBank登录号为MT472647,CDS序列长为1 158 bp,编码385个氨基酸,与甘蓝型油菜、甘蓝和白菜等具有高度同源性。BoaDFR表达水平在芥蓝不同发育时期、不同器官中存在显著差异,随植株发育总体呈现先下降后上升的趋势,在幼嫩种子中表达量最高。芥蓝原生质体的亚细胞定位结果显示BoaDFR定位在细胞核上。构建了农杆菌介导的芥蓝瞬时过表达体系,将BoaDFR转入白花芥蓝‘四季粗条’和黄花芥蓝‘福州黄花’中,发现BoaDFR在两种芥蓝中均高水平表达,转基因植株叶片颜色明显变紫,花青素苷显著积累,总含量最高达461.43 μg·g-1 FW,而野生型和转空载体芥蓝叶片中几乎检测不到花青素苷。通过HPLC在转基因植株叶片中共检测到8种花青素苷,均为矢车菊花青素苷,其中矢车菊-3-阿魏酰-丙二酰-槐糖苷-5-葡萄糖苷占比最高。

关键词: 芥蓝, 二氢黄酮醇4-还原酶(DFR), 花青素苷, 基因瞬时过表达, 基因功能

Abstract:

The BoaDFR was cloned from the cultivar‘Siji Cutiao’of white flower Chinese kale,and the GenBank accession number was MT472647. BoaDFR is 1 158 bp-long CDS sequence,encoding 385 amino acids. BoaDFR has high homology with DFR of Brassica napus,B. oleracea and B. rapa. The expression level of BoaDFR differed significantly at different stages and organs,and generally showed a trend of decreasing first and then rising during the plant growth. The highest expression level of BoaDFR was in young seeds,which was significantly higher than other organs. The subcellular localization showed that BoaDFR was localized in the nucleus. The Agrobacterium-mediated transient overexpression system in Chinese kale was constructed,and BoaDFR was transferred into‘Siji Cutiao’and‘Fuzhou Huanghua’. It was found that BoaDFR can be expressed at high levels in both two cultivars and the leaves of transgenic plants obviously turned purple,the anthocyanin accumulated significantly and the highest content was up to 461.43 μg·g -1FW,whereas the anthocyanin almost could not be detected in both wild-type and empty vector plants. Furthermore,a total of eight anthocyanins were detected in the transgenic plant leaves by HPLC,all of which were cyanidins,and cyanidin 3-feruloylmalonylso- phoroside-5- glucoside was predominant.

Key words: Chinese kale, dihydroflavonol 4-reductase(DFR), anthocyanin, gene transient overexpression, gene function

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