Crops ›› 2023, Vol. 39 ›› Issue (4): 44-51.doi: 10.16035/j.issn.1001-7283.2023.04.007

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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
  • Contact: Zhou Meiliang E-mail:461980621@qq.com;fangzhengwu88@163.com

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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

Table 1

Summary of primer sequence"

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

Fig.1

Sequence amplification of FtTIR gene intartary buckwheat M: DL2000; 1: the PCR amplification target band of FtTIR gene"

Fig.2

Bioinformatics analysis of FtTIR gene (a) Multiple alignment of FtTIR with other plant TIR proteins; (b) The secondary structure prediction of FtTIR protein:α-helix (blue), extended strand (red), β-turn (green), random coil (purple); (c) Thetertiary structure prediction of FtTIR protein; (d) Phylogenetic tree analysis of FtTIR protein"

Fig.3

Expression patterns of FtTIR genes in different organs of tartary buckwheat under infestation with R.solani “*”indicates significantly different at the 0.05 level, the same below"

Fig.4

Phenotypic verification of disease resistance in transgenic Arabidopsis Control: no treatment; WT: wild-type Arabidopsis; FtTIR-OE: overexpression of FtTIR in Arabidopsis. The same below"

Fig.5

DAB staining for resistance of transgenic Arabidopsis"

Fig.6

Analysis of expression patterns in transgenic Arabidopsis WT: Untreated wild type leaves; WT-RS: Wild-type leaves infected by R.solani; OE: Untreated FtTIR overexpressed Arabidopsis leaves; OE-RS: Overexpressed Arabidopsis leaves infected by R.solani. The same below"

Fig.7

Analysis of biochemical indices in transgenic Arabidopsis"

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