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作物学报 ›› 2022, Vol. 48 ›› Issue (5): 1129-1140.doi: 10.3724/SP.J.1006.2022.12010

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

水稻转录因子WRKY68在Xa21介导的抗白叶枯病反应中发挥正调控作用

朱峥1,2(), 王田幸子1,2, 陈悦1,2, 刘玉晴1,2, 燕高伟1,2, 徐珊1,2, 马金姣1,2, 窦世娟1,2, 李莉云1,2,*(), 刘国振1,2,*()   

  1. 1河北农业大学生命科学学院, 河北保定 071001
    2河北省植物生理与分子病理学重点实验室, 河北保定 071001
  • 收稿日期:2021-02-10 接受日期:2021-09-09 出版日期:2022-05-12 网络出版日期:2021-10-19
  • 通讯作者: 李莉云,刘国振
  • 作者简介:E-mail: zhuzheng_mbb@163.com
  • 基金资助:
    国家自然科学基金项目资助(31171528);国家自然科学基金项目资助(31400700)

Rice transcription factor WRKY68 plays a positive role in Xa21-mediated resistance to Xanthomonas oryzae pv. oryzae

ZHU Zheng1,2(), WANG Tian-Xing-Zi1,2, CHEN Yue1,2, LIU Yu-Qing1,2, YAN Gao-Wei1,2, XU Shan1,2, MA Jin-Jiao1,2, DOU Shi-Juan1,2, LI Li-Yun1,2,*(), LIU Guo-Zhen1,2,*()   

  1. 1College of Life Sciences, Hebei Agricultural University, Baoding 071001, Hebei, China
    2Hebei Key Laboratory of Plant Physiology and Molecular Pathology, Baoding 071001, Hebei, China
  • Received:2021-02-10 Accepted:2021-09-09 Published:2022-05-12 Published online:2021-10-19
  • Contact: LI Li-Yun,LIU Guo-Zhen
  • Supported by:
    National Natural Science Foundation of China(31171528);National Natural Science Foundation of China(31400700)

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摘要：

白叶枯病是一种严重影响水稻产量的细菌性病害。Xa21是第一个克隆的抗白叶枯病基因, 具有广谱抗性, 在水稻抗病育种中被广泛应用。转录因子的鉴定对解析Xa21介导的水稻抗白叶枯病分子机制具有重要意义。本研究构建了Xa21背景下的WRKY68-RNAi转基因水稻。与受体材料4021相比, 转基因水稻中WRKY68蛋白质丰度下调, 接种白叶枯病菌(Xanthomonas oryzae pv. oryzae, Xoo)后抗病性下降, 证明WRKY68基因在Xa21介导的抗白叶枯病反应中发挥正调控作用。此外, 转基因受体材料4021和感病对照TP309接种后不同时期和部位叶片中WRKY68的蛋白质丰度没有显著差异, 表明WRKY68蛋白质的表达不受抗病基因Xa21和病原物Xoo诱导, 推测其功能主要是调控下游基因的表达。WRKY68-RNAi转基因水稻接种后, PR1A、PR5、PR10A、PR-pha和PAL1等病程相关蛋白质表达丰度发生变化, 表明相关基因可能在WRKY68基因的调控下参与下游抗病反应。

关键词: 水稻, 白叶枯病, 抗病基因Xa21, WRKY转录因子, 病程相关蛋白质

Abstract:

Leaf blight is a bacterial disease of rice which leads to heavy yield losses. Xa21 which harbors broad spectrum resistance to leaf blight is the first resistant gene cloned in rice and is widely used in rice breeding program. The identification of transcription factor involved in Xa21-mediated resistance to bacterial leaf blight is of great significance. In this study, transgenic WRKY68-RNAi lines with reduced expression level of WRKY68 protein in the background of Xa21 were generated. The WRKY68-RNAi transgenic lines showed reduced resistance to Xanthomonas oryzae pv. oryzae (Xoo), indicating that WRKY68 played a positive role in Xa21-mediated resistance in rice. No detectable changes of WRKY68 protein abundance in leaves at different stages and positions after inoculation were identified by Western blot, indicating that the expression of WRKY68 protein were independent of Xa21 and Xoo, and thus WRKY68 gene may function as a transcription factor to regulate downstream genes. It was further found that the abundance of a number of pathogenesis-related proteins, including PR1A, PR5, PR10A, PR-pha, and PAL1 were dramatically changed in WRKY68-RNAi transgenic lines when inoculated with Xoo, suggested that these pathogenesis-related proteins were regulated by WRKY68 and the corresponding genes were involved in the downstream defenses responses.

Key words: rice, bacterial leaf blight, resistance gene Xa21, WRKY transcription factor, pathogenesis-related protein

图1

WRKY68-RNAi转基因株系的鉴定 R#1、R#2和R#3: 独立的WRKY68-RNAi转基因株系; CK: 4021 (转基因受体); 1~8: T2代不同的转基因植株; WRKY68: 抗WRKY68抗体识别条带; HSP: 抗HSP82抗体识别条带作为上样量标志。"

图2

WRKY68-RNAi转基因株系的农艺性状分析 A: 株高; B: 分蘖数; C: 千粒重。CK: 4021 (转基因受体); R#1、R#2和R#3: 独立的WRKY68-RNAi转基因株系。用Excel软件的数据统计分析对数据进行t-检验(Student’s t-test), **表示平均值间差异极显著(P < 0.01), 误差线标示标准差(SD)。"

图3

WRKY68-RNAi转基因株系对接种Xoo的反应 A: 接种Xoo 12 d的叶片照片。B: 叶片病斑长度进展图。C: 接种Xoo 12 d叶片病斑长度柱形图。箭头所指为病斑位置。**代表极显著差异(P < 0.01)。标尺为1 cm。R#1、R#2和R#3: 独立的WRKY68-RNAi转基因株系; TP309: 野生型; 4021: 表达Xa21的转基因TP309植株。"

附图1

水稻接种叶片中Xoo蛋白质丰度 采集水稻叶片, 提取总蛋白质后, 通过SDS-PAGE分离后转膜, 一抗用抗-Xoo抗体检测。R#2、R#3: WRKY68-RNAi转基因株系; TP309: 野生型; 4021: 表达Xa21的转基因TP309植株。"

附图2

水稻叶片接种Xoo后病程相关蛋白质的表达特征 以接种5 d、6 d的叶片(病斑线±1 cm)为材料, 通过SDS-PAGE或者Tricine-SDS-PAGE分离总蛋白质, 红框内为正文(图6)展示的部分。PR1A: 抗PR1a抗体识别条带; PR2: 抗PR2抗体识别条带; PR5: 抗PR5抗体识别条带; PR10A: 抗PR10a抗体识别条带; PR-pha: 抗PR-pha抗体识别条带; PAL1: 抗PAL1抗体识别条带; HSP: 抗HSP82抗体识别条带。R#1、R#2和R#3: WRKY68-RNAi转基因株系; TP309: 野生型; 4021: 表达Xa21的转基因TP309植株。"

图4

水稻接种叶片中Xoo蛋白质丰度 采集接种后水稻全叶片, 提取总蛋白质后用SDS-PAGE分离, 转膜后用抗-Xoo抗体检测(附图1), 用化学发光成像仪和Sage Capture软件检测WB信号, 用Lane1D软件采集WB信号, 计算3次实验的平均值和方差, 误差线表示标准差SD。以OD600为0.5的Xoo菌液蛋白质做为共同内参CR (common reference)归一化不同WB间的信号强度。R#2、R#3: 独立的WRKY68-RNAi转基因株系; TP309: 野生型; 4021: 表达Xa21的转基因TP309植株。"

图5

水稻WRKY68蛋白质接种Xoo后的表达特征 A: WRKY68蛋白质在接种后不同时间点的表达特征; B: 接种后不同部位取材模式图; C: WRKY68蛋白质在接种后不同部位的表达特征。WRKY68: 抗WRKY68抗体识别的条带; HSP: 抗HSP82抗体识别的条带。TP309: 野生型; 4021: 表达Xa21的转基因TP309植株。"

图6

水稻叶片接种Xoo后病程相关蛋白质的表达特征 PR1A: 抗PR1A抗体识别条带; PR2: 抗PR2抗体识别条带; PR5: 抗PR5抗体识别条带; PR10A: 抗PR10A抗体识别条带; PR-pha: 抗PR-pha抗体识别条带; PAL1: 抗PAL1抗体识别条带; HSP: 抗HSP82抗体识别条带作为上样量标志。R#1、R#2和R#3: 独立的WRKY68- RNAi转基因株系; TP309: 野生型; 4021: 表达Xa21的转基因TP309植株。"

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