Crops ›› 2023, Vol. 39 ›› Issue (3): 43-50.doi: 10.16035/j.issn.1001-7283.2023.03.006

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Molecular Cloning and Functional Analysis of Resistance Gene FtABCG12 of Tartary Buckwheat to Blight

Li Guangsheng1,2(), Lu Xiang1,3, Lai Dili1,3, Zhang Kaixuan2, Wang Haihua1(), Zhou Meiliang2()   

  1. 1College of Life and Health Sciences, Hunan University of Science and Technology, Xiangtan 411201, Hunan, China
    2Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
    3College of Agriculture, Guizhou University, Guiyang 520025, Guizhou, China
  • Received:2022-06-20 Revised:2022-09-27 Online:2023-06-15 Published:2023-06-16

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Abstract:

FtABCG12 gene was cloned from tartary buckwheat Chuanqiao No.1. The FtABCG12 gene was composed of seven exons and six introns with a full length cDNA of 1734bp, encoding 577 amino acids. Phylogenetic tree showed that FtABCG12 protein was closely related to Arabidopsis thaliana AtABCG11, AtABCG12, AtABCG13 and AtABCG15 proteins. qRT-PCR results indicated that the expression of FtABCG12 gene in tartary buckwheat was induced by the infection of Rhizoctonia solani. In transgenic A.thaliana, overexpression of FtABCG12 gene significantly increased the disease resistance of A.thaliana plants. These results proved that FtABCG12 was involved in the adaptation process of tartary buckwheat under biological stress, which laid the foundation for further study on the molecular mechanism of FtABCG12 regulation of tartary buckwheat resistance to blight.

Key words: Tartary buckwheat, FtABCG12, Cloning, Arabidopsis thaliana, Resistance to blight

Table 1

Summary of primer sequence"

引物Primer 引物序列Primer sequence (5′-3′) 用途Function
FtABCG12-F ATGGGTCCTTCTGGCTGTG 基因克隆
FtABCG12-R TTACAAGGATTTTGATTTATCCCTA
FtABCG12-qPCR-F GCCTTCTTGCTAATGGG qRT-PCR
FtABCG12-qPCR-R GCTTACAACAACACCTCC
FtH3-qPCR-F GAAATTCGCAAGTACCAGAAGAG qRT-PCR
内参基因
FtH3-qPCR-R CCAACAAGGTATGCCTCAGC
1307-FtABCG12-F ACGGGGGACTCTTGACCATGGATGGGTCCTTCTGGCTGTGG 构建过表达载体
1307通用引物
1307-FtABCG12-R
 AAGTTCTTCTCCTTTACTAGTTTACAAGGATTTTGATTTATCCCTACG
CTCAAGCAATCAAGCATTCTAC

Fig.1

Gene structure of FtABCG12"

Table 2

Cis-acting elements in FtABCG12 gene promoter sequence"

元件名称
Element name		 序列
Sequence		 功能
Function
TATA-box TATA TATAA TATA框，转录起始必需元件
TATAAA TATATA
CAAT-box CAAT CAAAT CAAT框，转录增强元件
CCAAT
MYB CAACCA 转录因子结合位点
TGACG-motif TGACG MeJA反应响应元件
CGTCA-motif CGTCA
ABRE CACGTG ACGTG 脱落酸反应响应元件
AACCCGG
TCA-element TCAGAAGAGG 水杨酸反应响应元件
TATC-box TATCCCA 赤霉素反应响应元件
I-box GGATAAGGTG 光照响应元件
GATA-motif GATAGGG
G-box CACGTG
WRE3 CCACCT 损伤诱导响应元件

Fig.2

Phylogenetic tree of FtABCG12 and its homologous proteins"

Fig.3

Expression patterns of FtABCG12 under different treatments Different lowercase letters indicate significant difference (P < 0.05), the same below"

Fig.4

Phenotypic verification of disease resistance in transgenic A.thaliana Control group: no treatment; experimental group: isolated leaves infected by R.solani. WT: wild-type Arabidopsis, FtABCG12-OE: overexpression of FtABCG12 in A.thaliana. The same below"

Fig.5

DAB staining for resistance of transgenic A.thaliana"

Fig.6

Expression patterns in transgenic A.thaliana WT-V: wild type leaves in vitro were not treated; WT-RS-V: wild-type isolated leaves were infected with RS; GOE-V: overexpressing Arabidopsis thaliana FtABCG12 leaves in vitro were not treated; GOE-RS-V: isolated leaves of overexpressing A.thaliana FtABCG12 were infected with RS; WT-L: wild-type live leaves were not treated; WT-RS-L: wild-type living leaves were infected with RS; GOE-L: FtABCG12 overexpressing live leaves of A.thaliana were not infected, GOE-RS-L: FtABCG12 overexpressing live leaves of A.thaliana were infected with RS"

Fig.7

Analysis of physiological and biochemical indices related to disease resistance in transgenic A.thaliana WT-RS: wild type A.thaliana was infected with RS; GOE: FtABCG12 overexpressing A.thaliana; GOE-RS: FtABCG12 overexpressing A.thaliana was infected with RS"

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