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"

[1] 唐宇, 邵继荣, 周美亮. 中国荞麦属植物分类学的修订. 植物遗传资源学报, 2019, 20(3):646-653.
doi: 10.13430/j.cnki.jpgr.20181210001
[2] Zhang K X, He M, Fan Y, et al. Resequencing of global Tartary buckwheat accessions reveals multiple domestication events and key loci associated with agronomic traits. Genome Biology, 2021, 22:23.
doi: 10.1186/s13059-020-02217-7 pmid: 33430931
[3] 田秀红, 刘鑫峰, 闫峰, 等. 苦荞麦的药理作用与食疗. 农产品加工(学刊), 2008(8):31-33.
[4] 卢文洁, 王莉花, 周洪友, 等. 荞麦立枯病的发病规律与综合防治措施. 江苏农业科学, 2013, 41(8):138-139.
[5] 齐杨菊, 陈振江, 李振霞, 等. 荞麦病害研究进展. 草业科学, 2020, 37(1):75-86.
[6] 宁晓雪, 苏跃, 马玥, 等. 立枯丝核菌研究进展. 黑龙江农业科学, 2019(2):140-143.
[7] 罗吉. 荞麦立枯病生防菌的筛选鉴定及生防效果初探. 成都:成都大学, 2021.
[8] 卢文洁, 王艳青, 李春花, 等. 不同杀菌剂防治荞麦立枯病田间药效试验. 山东农业科学, 2014, 46(8):121-122.
[9] 刘玉晴, 燕高伟, 张彤, 等. 超表达OsPR1A增强了Xa21介导的水稻对白叶枯病的抗性反应. 中国农业科学, 2021, 54(23):4933-4942.
doi: 10.3864/j.issn.0578-1752.2021.23.001
[10] 王显宗. 水稻立枯病抗性资源筛选及抗病基因初步定位. 哈尔滨:东北农业大学, 2018.
[11] Cui H M, Qu S, Lamboro A, et al. Bioinformatics analysis of disease resistance gene PR1 and its genetic transformation in soybeans and cultivation of multi-resistant materials. Phyton- International Journal of Experimental Botany, 2022, 91(7):1445- 1464.
[12] 彭再慧. 毛白杨PtoABCG40基因的克隆与功能分析. 重庆:西南大学, 2019.
[13] 孟泓君. 拟南芥ABCG36转运蛋白在木质素生物合成过程中的功能研究. 重庆:西南大学, 2019.
[14] 李冰飞. AtABCG14调控拟南芥抗旱反应的作用机理研究. 金华:浙江师范大学, 2019.
[15] Clough S J, Bent A F. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. The Plant Journal, 1998, 16(6):735-743.
doi: 10.1046/j.1365-313x.1998.00343.x
[16] Ji N N, Wang J, Zuo X X, et al. PpWRKY45 is involved in methyl jasmonate primed disease resistance by enhancing the expression of jasmonate acid biosynthetic and pathogenesis- related genes of peach fruit. Postharvest Biology and Technology, 2021, 172:111390.
doi: 10.1016/j.postharvbio.2020.111390
[17] Saputra R, Hamzah A, Puspita F, et al. Study of salicylic acid concentration in suppressing the development of Ganoderma spp. causes of stem rot disease in vitro and its effect on oil palm seedling growth. IOP Conference Series: Earth and Environmental Science, 2022, 978(1):12-24.
[18] 马钰娇. 番茄脱落酸缺失突变体抗灰叶斑病机制的探究与应用. 泰安:山东农业大学, 2021.
[19] Hunjan, M S, Kumar S, Lore J S, et al. Efficiency of different Rhizoctonia solani inoculum source against sheath blight screening in rice under field conditions. Tropical Plant Pathology, 2022, 47:309-313.
doi: 10.1007/s40858-021-00489-3
[20] Imad K, Mohammed A, Abdellatif B, et al. Morphological, pathogenic and molecular characterisation of Rhizoctonia solani strains isolated from potato. Annual Research & Review in Biology, 2018, 29(4):1-16.
[21] 刘博, 孔婷婷, 刘限, 等. 玉米纹枯病病菌γ-谷氨酰转肽酶基因克隆与表达分析. 江苏农业科学, 2015, 43(5):39-41.
[22] Olga K A, Anna V S, Elena Z K, et al. Pathogenesis-related genes of PR1, PR2, PR4, and PR5 families are involved in the response to Fusarium infection in garlic (Allium sativum L.). International Journal of Molecular Sciences, 2021, 22(13):6688.
doi: 10.3390/ijms22136688
[23] 刘银萍. ‘洛阳红’花期花瓣抗氧化酶SOD、POD和CAT活性的研究. 河南林业科技, 2020, 40(3):13-15,41.
[24] 李易初, 石凤梅, 马立功, 等. 核盘菌菌丝内POD和SOD活性与其对大豆致病力关系初探. 黑龙江八一农垦大学学报, 2021, 33(1):1-6,14.
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