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小豆VaWRKY70基因克隆及其抗锈病功能的验证

引用本文：姚砚文,孙伟娜,丁欣,殷丽华,柯希望,左豫虎.小豆VaWRKY70基因克隆及其抗锈病功能的验证.植物保护学报,2024,51(6):1317-1326

DOI：10.13802/j.cnki.zwbhxb.2024.2023061

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作者	单位	E-mail
姚砚文	黑龙江八一农垦大学, 黑龙江省作物有害生物互作生物学及生态防控重点实验室, 国家杂粮工程技术研究中心, 大庆 163319
孙伟娜	黑龙江八一农垦大学, 黑龙江省作物有害生物互作生物学及生态防控重点实验室, 国家杂粮工程技术研究中心, 大庆 163319
丁欣	黑龙江八一农垦大学, 黑龙江省作物有害生物互作生物学及生态防控重点实验室, 国家杂粮工程技术研究中心, 大庆 163319
殷丽华	黑龙江八一农垦大学, 黑龙江省作物有害生物互作生物学及生态防控重点实验室, 国家杂粮工程技术研究中心, 大庆 163319
柯希望	黑龙江八一农垦大学, 黑龙江省作物有害生物互作生物学及生态防控重点实验室, 国家杂粮工程技术研究中心, 大庆 163319
左豫虎	黑龙江八一农垦大学, 黑龙江省作物有害生物互作生物学及生态防控重点实验室, 国家杂粮工程技术研究中心, 大庆 163319	zuoyuhu@byau.edu.cn

中文摘要:为明确VaWRKY70基因在小豆Vigna angularis应答豇豆单胞锈菌Uromyces vignae侵染中的作用，采用PCR技术克隆VaWRKY70基因，分析该基因编码蛋白的序列结构及系统进化关系等特征，采用实时荧光定量PCR（quantitative real-time PCR，qPCR）技术比较抗、感不同小豆品种接种豇豆单胞锈菌后VaWRKY70基因的表达情况；构建VaWRKY70基因的植物表达载体，应用烟草瞬时表达体系明确VaWRKY70蛋白的亚细胞定位和VaWRKY70基因在抗病过程中的作用。结果显示：VaWRKY70基因序列全长1 997 bp，包含2个内含子，编码区长858 bp，编码285个氨基酸，具有一个保守的WRKY蛋白结构域，启动子区顺式作用元件丰富，包含8个激素和1个防御应答元件。接种豇豆单胞锈菌后12、120和192 h，抗病品种中VaWRKY70基因的相对表达量显著升高，分别是对照的2.35倍、48.08倍和12.44倍，而感病品种仅在接种后24 h显著高于对照，其余时间均与对照差异不显著。VaWRKY70基因编码的蛋白定位于细胞核，瞬时表达该基因能显著提高烟草对灰霉病的抗性。表明VaWRKY70作为正调控因子在植物抗病中发挥着重要作用。

中文关键词:小豆小豆锈病瞬时表达功能细胞定位基因抗性

Coning and functional verification for rust resistance of the VaWRKY70 gene in adzuki bean

Author Name	Affiliation	E-mail
Yao Yanwen	National Coarse Cereals Engineering Research Center, Heilongjiang Provincial Key Laboratory of Crop-Pest Interaction Biology and Ecological Control, Heilongjiang Bayi Agricultural University, Daqing 163319, Heilongjiang Province, China
Sun Weina	National Coarse Cereals Engineering Research Center, Heilongjiang Provincial Key Laboratory of Crop-Pest Interaction Biology and Ecological Control, Heilongjiang Bayi Agricultural University, Daqing 163319, Heilongjiang Province, China
Ding Xin	National Coarse Cereals Engineering Research Center, Heilongjiang Provincial Key Laboratory of Crop-Pest Interaction Biology and Ecological Control, Heilongjiang Bayi Agricultural University, Daqing 163319, Heilongjiang Province, China
Yin Lihua	National Coarse Cereals Engineering Research Center, Heilongjiang Provincial Key Laboratory of Crop-Pest Interaction Biology and Ecological Control, Heilongjiang Bayi Agricultural University, Daqing 163319, Heilongjiang Province, China
Ke Xiwang	National Coarse Cereals Engineering Research Center, Heilongjiang Provincial Key Laboratory of Crop-Pest Interaction Biology and Ecological Control, Heilongjiang Bayi Agricultural University, Daqing 163319, Heilongjiang Province, China
Zuo Yuhu	National Coarse Cereals Engineering Research Center, Heilongjiang Provincial Key Laboratory of Crop-Pest Interaction Biology and Ecological Control, Heilongjiang Bayi Agricultural University, Daqing 163319, Heilongjiang Province, China	zuoyuhu@byau.edu.cn

Abstract:To reveal the function of the VaWRKY70 gene in adzuki bean in response to Uromyces vignae infection, the gene was cloned by using PCR. The deduced amino acid sequence and the phylogenetic features of the protein encoded by VaWRKY70 were analyzed. Subsequently, the relative expression of VaWRKY70 in different rust resistant cultivars responsive to U. vignae infection in adzuki bean were conducted by quantitative real-time PCR (qPCR). Thereafter, a plant expression vector of VaWRKY70 was constructed to determine the subcellular localization of VaWRKY70 protein and its effect on tobacco resistance to Botrytis cinerea infection by the tobacco transient expression system. The results showed that the full-length of VaWRKY70 was 1 997 bp with two introns, and the coding sequence was 858 bp that encoded 285 amino acids with a conserved WRKY domain. The promoter region was rich in cis-acting elements, which contains eight phytohormone and one defense responsive elements. The relative expression of VaWRKY70 in the resistant cultivar was significantly up regulated at 12, 120 and 192 h after inoculation, which were 2.35, 48.08 and 12.44 times higher than the non-inoculated control, respectively. While in the susceptible cultivar, the relative expression of VaWRKY70 was only increased at 24 h after inoculation, and no significant differences were detected at other stages of U. vignae infection. By using the tobacco transient expression system, we found that the VaWRKY70 protein was localized in the nucleus and transient expression of the VaWRKY70 gene could significantly enhance the resistance of tobacco to gray mold. The results indicated that VaWRKY70 plays an important role in plant disease resistance as a positive regulator.

keywords:Vigna angularis adzuki bean rust transient expression function cellular localization gene resistance

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