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作物杂志,2020, 第5期: 33–40 doi: 10.16035/j.issn.1001-7283.2020.05.005

• 遗传育种·种质资源·生物技术 • 上一篇    下一篇

苦荞鼠李糖基转移酶FtF3GT1基因的克隆与转化毛状根研究

卢晓玲1,2(), 何铭2, 张凯旋2, 廖志勇1(), 周美亮2()   

  1. 1温州大学生命与环境科学学院,325035,浙江温州
    2中国农业科学院作物科学研究所,100081,北京
  • 收稿日期:2020-01-19 修回日期:2020-03-07 出版日期:2020-10-15 发布日期:2020-10-12
  • 通讯作者: 廖志勇,周美亮
  • 作者简介:卢晓玲,主要从事荞麦品质代谢调控方面研究,E-mail： luxiaoling07@163.com
  • 基金资助:
    国家重点研发计划中国-欧盟国际合作重点项目(2017YFE0117600);国家自然科学基金面上项目(31871536)

Study on the Cloning and Transformation of Rhamnose Transferase FtF3GT1 Gene in Tartary Buckwheat

Lu Xiaoling1,2(), He Ming2, Zhang Kaixuan2, Liao Zhiyong1(), Zhou Meiliang2()   

  1. 1College of Life and Environmental Science, Wenzhou University, Wenzhou 325035, Zhejiang, China
    2Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
  • Received:2020-01-19 Revised:2020-03-07 Online:2020-10-15 Published:2020-10-12
  • Contact: Liao Zhiyong,Zhou Meiliang

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摘要：

苦荞中的黄酮类化合物具有很高的药用价值和保健功能。以苦荞川荞1号为材料,克隆出FtF3GT1基因,其CDS全长为1 401bp,编码467个氨基酸。系统发育分析表明,FtF3GT1与甜荞中的FeUF7GT蛋白亲缘关系最近。组织特异性表达分析结果表明,FtF3GT1基因在各个组织中均有表达,在根部最低,在茎部最高;MeJA处理苦荞和转pCAMBIA3301:FtF3GT1pro::GUS毛状根中发现,FtF3GT1基因的表达量以及GUS的活性明显受MeJA诱导;转过表达FtF3GT1基因毛状根中,总黄酮含量明显升高,推测FtF3GT1基因可促进黄酮类物质的合成。

关键词: 克隆, 苦荞, 黄酮合成

Abstract:

Flavonoids in tartary buckwheat have high medicinal value and health function. Based on the tartary buckwheat Chuanqiao No.1, FtF3GT1 gene was cloned from tartary buckwheat and its CDS length was 1 401bp, encoding 467 amino acids. Phylogenetic analysis showed that FtF3GT1 was closely related to FeUF7GT protein in common tartary buckwheat. Specific expression analysis results showed that FtF3GT1 gene was expressed in all tissues, but the lowest in roots and the highest in stems. Treated with MeJA, expression of FtF3GT1 gene and GUS activity in pCAMBIA3301:FtF3GT1pro::GUS in transgenic hairy roots of tartary buckwheat were significantly induced by MeJA, it is speculated that FtF3GT1 gene was induced by MeJA and promoted the biosynthesis of flavonoids synthesis.

Key words: Cloning, Tartary buckwheat, Flavonoids synthesis

表1

引物序列

引物名称Primer name 引物序列(5'-3') Primer sequence (5'-3')
FtF3GT1-F(CDS) 5'-ATGGGAACCCAATCAAGC-3'
FtF3GT1-R(CDS) 5'-CTACTGCTTACCAACCAAAC-3'
pCAMBIA1307-FtF3GT1-F 5'-GGGGGCGGCCGCTCTAGAATGGGAACCCAATCAAGC-3'
pCAMBIA1307-FtF3GT1-R 5'-AAGCTTGATATCGAATTCCTACTGCTTACCAACCAAAC-3'
pCAMBIA3301-FtF3GT1-F 5'-GAATCCTTAAATGGTTCATATTACTATGCAATTAG-3'
pCAMBIA3301-FtF3GT1-R 5'-TCTAGATTTGTAAAATGCTTGATTTGTTTCTTG-3'
FtH3-QF 5'-GAAATTCGCAAGTACCAGAAGAG-3'
FtH3-QR 5'-CCAACAAGGTATGCCTCAGC-3'
FtF3GT1-QF 5'-TCAAATAAGCTCGCCTC-3'
FtF3GT1-QR 5'-GCTGCATTTTGTCAAGAGCG-3'
FtActin-QF 5'-GAAATTCGCAAGTACCAGAAGAG-3'
FtActin-QR 5'-CCAACAAGGTATGCCTCAGC-3'

图1

FtF3GT1基因CDS和启动子的克隆 A：M：DL 2 000 DNA标记;1：FtF3GT1基因CDS扩增产物;B：M：DL 2 000 DNA标记;1：FtF3GT1基因启动子的扩增产物

图2

FtF3GT1与其他植物GTs蛋白序列多重比对

图3

FtF3GT1与其他植物GTs蛋白氨基酸序列的系统发育分析

表2

FtF3GT1基因启动子序列中的顺式作用元件

位点名称Site name 序列Sequence 位点功能Function of site
TGACG-motif TGACG 参与MeJA反应的顺式作用调控元件cis-acting regulatory element involved in the MeJA-responsiveness
CGTCA-motif CGTCA 参与MeJA反应的顺式作用调控元件cis-acting regulatory element involved in the MeJA-responsiveness
ABRE ACGTG 参与ABA反应的顺式作用调控元件cis-acting element involved in the abscisic acid responsiveness
GARE-motif TCTGTTG 响应GA元素gibberellin-responsiveness
P-box CCTTTTG 响应GA元素gibberellin-responsiveness
TCA-element CCATCTTTTT 参与SA反应的顺式作用调控元件cis-acting element involved in salicylic acid responsiveness
G-box CACGTC 光调控元件cis-acting regulatory element involved in light responsiveness
ARE AAACCA 厌氧诱导所必需的顺式调节元件cis-acting regulatory element essential for the anaerobic induction
TATA-box TATATA 转录起始-3-O核心启动子元件core promoter element of 3-O transcription start

图4

FtF3GT1基因的组织特异性表达 FtH3作为内参基因,每组数据代表3次重复试验的平均值±SD。*表示t检验有显著性差异（P<0.05）,下同

图5

MeJA诱导下FtF3GT1基因的表达情况

图6

转基因根系的鉴定及GUS染色 A：FtF3GT1pro::GUS转基因毛状根鉴定图;M：DL 2 000 DNA标记;1：pCAMBIA3301:FtF3GT1pro::GUS质粒;2~4：转A4根系DNA;5~7：转pCAMBIA3301-35S::GUS根系DNA;8~11：转FtF3GT1pro::GUS根系DNA;B：MeJA处理FtF3GT1pro::GUS毛状根的GUS染色情况

图7

苦荞毛状根的诱导过程和转基因根系的鉴定 A：苦荞毛状根的诱导过程;B：转基因根系的鉴定;M：DL 2 000 DNA标记;1：pCAMBIA1307-FtRT1质粒;2：天然的苦荞根系（WT）;3：转pCAMBIA1307空载体根系;4~8：转pCAMBIA1307-FtRT1根系DNA检测

图8

A4、pCAMBIA1307空载体和pCAMBIA1307-FtF3GT1过表达根系中总黄酮的测定

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