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作物杂志,2023, 第4期: 44–51 doi: 10.16035/j.issn.1001-7283.2023.04.007

• 遗传育种·种质资源·生物技术 • 上一篇    下一篇

苦荞抗立枯病基因FtTIR的克隆及功能鉴定

陈媛媛1,2(), 李光胜2,3, 刘洋2,4, 何毓琦2, 周美亮2(), 方正武1   

  1. 1长江大学农学院，434025，湖北荆州
    2中国农业科学院作物科学研究所，100081，北京
    3湖南科技大学生命科学与健康学院，411201，湖南湘潭
    4西南大学农学与生物科技学院，400700，重庆
  • 收稿日期:2022-07-21 修回日期:2022-09-15 出版日期:2023-08-15 发布日期:2023-08-15
  • 通讯作者: 正武，从事作物遗传育种研究，E-mail：fangzhengwu88@163.com；周美亮为共同通信作者，研究方向为荞麦种质资源与品质抗逆机制解析，E-mail：zhoumeiliang@caas.cn
  • 作者简介:陈媛媛，研究方向为荞麦遗传育种与品种改良，E-mail：461980621@qq.com
  • 基金资助:
    国家重点研发计划(2019YFD1001300);国家重点研发计划(2019YFD1001303)

Molecular Cloning and Functional Identification of Resistance Gene FtTIR of Tartary Buckwheat to Blight

Chen Yuanyuan1,2(), Li Guangsheng2,3, Liu Yang2,4, He Yuqi2, Zhou Meiliang2(), Fang Zhengwu1   

  1. 1College of Agriculture, Yangtze University, Jingzhou 434025, Hubei, China
    2Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
    3College of Life Science and Health, Hunan University of Science and Technology, Xiangtan 411201, Hunan, China
    4College of Agronomy and Biotechnology, Southwest University, Chongqing 400700, China
  • Received:2022-07-21 Revised:2022-09-15 Online:2023-08-15 Published:2023-08-15

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摘要：

以苦荞“品苦1号”为材料，克隆得到FtTIR基因，研究其在立枯丝核菌侵染下的表达模式及在立枯病抗病信号通路中的作用。结果表明，FtTIR基因全长576bp，编码191个氨基酸，编码蛋白分子量为22.7kDa。进化分析显示，FtTIR蛋白与藜麦XP_021726625.1和赤豆XP_047164412.1亲缘关系较近。qRT-PCR结果表明，FtTIR基因受立枯丝核菌侵染诱导表达，且在茎中的表达量最高。转基因拟南芥中，过表达FtTIR基因显著提高了植株对立枯丝核菌的抗病性，并显著提高了叶片过氧化物酶和超氧化物歧化酶活性，提高了病程相关基因AtPR1的表达水平。

关键词: 苦荞, FtTIR, 过表达, 抗立枯病

Abstract:

Using tartary buckwheat “Pinku-1” as material, the FtTIR gene was cloned and studied for its expression pattern during the infection of Rhizoctonia solani and its role in the resistance signaling pathway against the disease. The results showed that the full length of the FtTIR gene was 576bp, encoding 191 amino acids, and the protein molecular weight was 22.7kDa. Evolutionary analysis showed that the FtTIR protein was closely related to Chenopodium quinoa XP_021726625.1 and Vigna angularis XP_047164412.1. qRT-PCR results showed that the FtTIR gene was induced by R.solani infection and the expression level was the highest in stems. In transgenic Arabidopsis, the overexpression of FtTIR gene significantly improved plant resistance to R.solani and increased the activities of peroxidase and superoxide dismutase in leaves, as well as the expression level of the disease-related gene AtPR1.

Key words: Tartary buckwheat, FtTIR, Overexpression, Resistance to blight

表1

引物序列汇总

引物Primer 引物序列（5′-3′）Primer sequence (5′-3′) 用途Function
FtTIR-F ATGCAACGTCCAGTAGCCAAA 基因克隆
FtTIR-R TTATTTGCTGTTTTCTTCAACCTCG
FtTIR-qPCR-F GCCTTCTTGCTAATGGG qRT-PCR
FtTIR-qPCR-R GCTTACAACAACACCTCC
AtPR1-qPCR-F TCATGGCTAAGTTTGCTTCC
AtPR1-qPCR-R AATACACACGATTTAGCACC
FtH3-qPCR-F GAAATTCGCAAGTACCAGAAGAG qRT-PCR内参基因
FtH3-qPCR-R CCAACAAGGTATGCCTCAGC
Atactin7-qPCR-F TCCATGAAACAACTTACAACTCCATCA
Atactin7-qPCR-R CATCGTACTCACTCTTTGAAATCCACA
121-FtTIR-F GAGAACACGGGGGACTCTAGAATGCAACGTCCAGTAGCCAAA 构建过表达载体
121-FtTIR-R CATACCTCCTCCTCCGGATCCTTTGCTGTTTTCTTCAACCTCG
121F TGACGTAAGGGATGACGCAC pBI121通用引物

图1

苦荞FtTIR基因序列扩增 M：DL2000；1：FtTIR基因的PCR扩增目的条带

图2

FtTIR基因的生物信息学分析 (a) FtTIR与其他植物TIR蛋白的多重比对；(b) FtTIR蛋白二级结构预测：α-螺旋（蓝色）、延伸链（红色）、β-转角结构（绿色）、无规则卷曲（紫色）；(c) FtTIR蛋白三级结构预测；(d) FtTIR蛋白系统进化树分析

图3

立枯丝核菌侵染下FtTIR基因在苦荞不同器官中的表达模式 “*”表示在0.05水平下有显著性差异，下同

图4

转基因拟南芥的抗病性表型验证 对照：不作处理；WT：野生型拟南芥；FtTIR-OE：FtTIR过表达拟南芥。下同

图5

转基因拟南芥的抗病性DAB染色验证

图6

转基因拟南芥中的表达模式分析 WT：野生型叶片未处理；WT-RS：野生型叶片受立枯丝核菌侵染；OE：FtTIR过表达拟南芥叶片未处理；OE-RS：FtTIR过表达拟南芥叶片受立枯丝核菌侵染。下同

图7

转基因拟南芥生理生化指标分析

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