花生 NBS-LRR 类抗病基因 <em>AhRRLLS1</em> 及其启动子的克隆与表达分析

doi:10.3969/j.issn.1674-7968.2023.06.004

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摘要 NBS-LRR (nucleotide binding site-leucine rich repeats)类基因是抗病基因(resistance genes, R)中最大的类群, 在花生 (Arachis hypogaea) 抗病过程中起重要的作用。本研究团队利用花生抗晚斑病病菌 (Phaeoisariopsis personata) QTL-seq 定位,筛选到 1 个花生抗晚斑病候选基因 1 (Arachis hypogaea resistance rust and late leaf spot, AhRRLLS1),为研究该基因的表达特征,本研究以花生品种'粤油 92'的 cDNA 和基因组 DNA 为模板,克隆得到 AhRRLLS1 (GenBank No. OQ675414)基因 CDS 和启动子序列,对其进行生物信息学分析 ,采用 qPCR 检测 AhRRLLS1 基因在外源激素诱导及抗、感晚斑病花生品种内的表达特征 ,构建 PAhRRLLS1 启动子的 GUS 融合表达载体并遗传转化拟南芥(Arabidopsis thaliana),进行 GUS 染色组织化学检测。结果显示,AhRRLLS1 基因 CDS 全长 3 129 bp,编码 1 042 个氨基酸,含有 TIR、NB-ARC 和 LRR 保守结构域 ,系统进化分析显示 ,该基因归为 TIR- NBS-LRR (TNLs)类抗性基因。qPCR 检测结果表明 , AhRRLLS1 基因能响应脱落酸、茉莉酸甲酯、乙烯、水杨酸和晚斑病菌的诱导表达 ,在高抗晚斑病花生品种'粤油 92'和易感晚斑病花生品种'白沙 1016'内 ,叶片中 AhRRLLS1 基因表达差异显著(P<0.05) ,前3 d'粤油 92'内 AhRRLLS1 基因表达量低于'白沙 1016'。转基因拟南芥植株组织 GUS 染色结果显示 ,根、茎、叶、花和荚果都能被染色。研究结果表明 ,AhRRLLS1 基因属于TIR-NBS-LRR 类抗性基因 ,推测在花生受晚斑病菌侵染时发挥调控作用 ,在各个组织中均能够表达。本研究为 AhRRLLS1 基因的功能研究及花生抗病机制提供了理论参考。

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	陈湘瑜
	孙涛
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	杨强
	蔡铁城
	储文婷
	庄宇慧

关键词 ：抗病, 晚斑病, 花生抗晚斑病候选基因 1 (AhRRLLS1), NBS-LRR

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.

Key words： Resistance disease Late leaf spot Arachis hypogaea resistance rust and late leaf spot (AhRRLLS1) nucleotide binding site-leucine rich repeats (NBS-LRR)

收稿日期: 2022-06-13

基金资助:福建省属公益类科研院所基本科研专项(2021R10310012); 福建省科技厅高校产学合作项目(2021N5007); 福建省农业科学院科技创新团队(CXTD2021008-3); 福建省农科院对外合作项目(DWHZ2021-20)

通讯作者: **000q821035@fafu.edu.cn。*同等贡献作者

引用本文:

陈湘瑜, 孙涛, 王珊珊, 谢文萍, 杨强, 蔡铁城, 储文婷, 庄宇慧. 花生 NBS-LRR 类抗病基因 AhRRLLS1 及其启动子的克隆与表达分析[J]. 农业生物技术学报, 2023, 31(6): 1147-1158.
CHEN Xiang-Yu , SUN Tao , WANG Shan-Shan , XIE Wen-Ping , YANG Qiang , CAI Tie-Cheng , CHU Wen-Ting, ZHUANG Yu-Hui. Cloning and Expression Analysis of NBS-LRR Resistant Gene AhRRLLS1 and Its Promoter in Arachis hypogaea. 农业生物技术学报, 2023, 31(6): 1147-1158.

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http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2023.06.004 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2023/V31/I6/1147

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