Cloning and Expression Analysis of NBS-LRR Resistant Gene AhRRLLS1 and Its Promoter in Arachis hypogaea
CHEN Xiang-Yu1,2* , SUN Tao1* , WANG Shan-Shan1 , XIE Wen-Ping1 , YANG Qiang1 , CAI Tie-Cheng1 , CHU Wen-Ting1 , ZHUANG Yu-Hui3**
1 College of Agronomy, Fujian Agriculture and Forestry University, Fuzhou 350002, China; 2 Institute of Crop Research, Fujian Academy of Agricultural Sciences/Fujian Engineering Research Center for Characteristic Upland Crops Breeding, Fuzhou 350013, China; 3 College of Life Science, Fujian Agriculture and Forestry University, Fuzhou 350002, China
Abstract Nucleotide binding site-leucine rich repeats (NBS-LRR) class of resistant gene takes the most part of resistance genes (R) and plays an important role in peanut (Arachis hypogaea) disease resistance. Our group discovered a candidate gene Arachis hypogaea resistance rust and late leaf spot 1 (AhRRLLS1) conferring resistance to late leaf spot in peanut by the QTL-seq approach. In order to explore the gene expression regulation, the AhRRLLS1 gene and its promoter were cloned using cDNA and gDNA of 'Yueyou 92' as templates. Sequence analysis of the AhRRLLS1 gene and its promoter was conducted via bioinformatic techniques. The qPCR detected the gene expression of AhRRLLS1 in the peanut varieties induced by exogenous hormone, the resistant and susceptible peanut varieties for late leaf spot disease. The GUS gene driven by AhRRLLS1 promoter was genetically transformed into Arabidopsis thaliana for histochemical GUS staining. The results showed that the AhRRLLS1 gene encompassed the coding region of 3 129 bp, and encoded the protein of 1 042 amino acids, containing TIR, NB-ARC and LRR conserved domains. Phylogenetic analysis showed that the AhRRLLS1 gene belonged to a TIR-NBS-LRR (TNLs) class of R genes. The qPCR analysis showed that the AhRRLLS1 gene could respond to abscisic acid, methyl jasmonate, ethylene, salicylic acid and Phaeoisariopsis personata. The gene expression level of AhRRLLS1 revealed a significant difference between the resistant variety of 'Yueyou 92' and the susceptible variety of 'Baisha 1016' (P＜0.05). The expression of AhRRLLS1 gene in 'Yueyou 92' was lower than that in 'Baisha 1016' in the first 3 days. The GUS staining for transgenic A. thaliana showed that roots, stems, leaves, flowers and pods could be stained. The results indicated that AhRRLLS1 belonged to a TIR-NBS-LRR class of resistance gene, playing a regulatory role in resistance against infection with Phaeoisariopsis personata in peanut, and the AhRRLLS1 promoter might drive the expression of the gene in different tissue of peanut. This study provides a theoretical guidance for characterization of AhRRLLS1 and the mechanism of disease resistance in peanut.
CHEN Xiang-Yu,SUN Tao,WANG Shan-Shan, et al. Cloning and Expression Analysis of NBS-LRR Resistant Gene AhRRLLS1 and Its Promoter in Arachis hypogaea[J]. 农业生物技术学报, 2023, 31(6): 1147-1158.
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