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作物学报 ›› 2023, Vol. 49 ›› Issue (4): 996-1005.doi: 10.3724/SP.J.1006.2023.24062

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

StEFR1正调控马铃薯对晚疫病的抗性

张卫娜(), 余慧芳, 安珍, 柳文凯, 康益晨, 石铭福, 杨昕宇, 张茹艳, 王勇, 秦舒浩*()   

  1. 甘肃农业大学园艺学院, 甘肃兰州 730070
  • 收稿日期:2022-03-21 接受日期:2022-07-21 出版日期:2023-04-12 网络出版日期:2022-08-22
  • 通讯作者: *秦舒浩, E-mail: qinsh@gsau.edu.cn
  • 作者简介:E-mail: zhangwn@gsau.edu.cn
  • 基金资助:
    甘肃农业大学科技创新基金(公招博士科研启动基金项目)(GAU-KYQD-2020-10);甘肃省自然科学基金项目(21JR7RA827);国家自然科学基金项目(32060441)

StEFR1 regulates late blight resistance positively in potato (Solanum tuberosum)

ZHANG Wei-Na(), YU Hui-Fang, AN Zhen, LIU Wen-Kai, KANG Yi-Chen, SHI Ming-Fu, YANG Xin-Yu, ZHANG Ru-Yang, WANG Yong, QIN Shu-Hao*()   

  1. College of Horticulture, Gansu Agricultural University, Lanzhou 730070, Gansu, China
  • Received:2022-03-21 Accepted:2022-07-21 Published:2023-04-12 Published online:2022-08-22
  • Contact: *E-mail: qinsh@gsau.edu.cn
  • Supported by:
    Science and Technology Innovation Fund of Gansu Agricultural University (Doctoral Research Start-up Fund Project for Public Recruitment)(GAU-KYQD-2020-10);Natural Science Foundation of Gansu Province(21JR7RA827);National Natural Science Foundation of China(32060441)

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摘要：

马铃薯晚疫病是毁灭性卵菌病害, 对我国乃至全球的农业生产造成巨大的经济损失。本试验结合生物信息学分析、表达模式和功能验证, 分析了LRR-RLK家族成员StEFR1调控晚疫病抗性的作用和潜在的调控机制。进化分析表明, StEFR1与拟南芥中功能已知的AtEFR序列相似度为53.9%。接种晚疫病菌3 d和elf18处理3 h, ‘大西洋’离体叶片中StEFR1的表达分别上调至对照的1.87倍和2.31倍。瞬时过表达StEFR1的叶片受到晚疫病菌侵染时抗性增强, 表现在叶片病斑面积较对照减小, 而叶片细胞活性较对照增强。此外, 与野生型相比, 过表达StEFR1的叶片中3个PTI标记基因、SA和JA信号通路相关基因差异表达, 且呈不同程度的显著上调, 而ET信号通路相关基因的表达量无明显变化。综上所述, StEFR1参与晚疫病菌诱导的PTI抗性, 且调控SA和JA激素信号相关基因的表达, 对晚疫病起正调控作用。本文为深入研究StEFR1调控晚疫病免疫反应的分子机制奠定了基础, 并为晚疫病分子育种研究提供重要参考。

关键词: 马铃薯晚疫病, LRR-RLK家族, 激素信号, 标记基因, 差异表达

Abstract:

Potato late blight is a devastating oomycete disease, which causes great economic losses to agricultural production. In this study, we analyzed the role and potential regulatory mechanism of StEFR1 in regulating late blight resistance by the relative expression pattern and functional verification combined with the bioinformatics methods. Evolutionary analysis showed that the sequence similarity between StEFR1 and AtEFR was 53.9%. After inoculated with Phytophthora infestans for 3 days and treatment with elf18 for 3 hours, the relative expression level of StEFR1 in isolated leaves of Atlantic was upregulated to 1.87 times and 2.31 times compared with the control, respectively. The late blight resistance significantly increased after the overexpression of StEFR1 in the isolated leaves of Atlantic by agrobacterium infiltration method. Compared with the control, the area of leaf lesion size decreased and the activity of leaf cells increased. And the marker genes of PTI, SA, and JA signaling pathways in overexpressed leaves were significantly up-regulated to varied degrees, while the relative expression levels of ET related-genes did not change significantly. In conclusion, StEFR1 was involved in the PTI resistance and regulated the relative expression levels of SA and JA hormone signaling related genes, suggesting that StEFR1 positively regulated the potato late blight. This study lays a foundation for further revealing the molecular mechanism of StEFR1 in regulating the immune response and provides important reference for the molecular breeding of late blight.

Key words: potato late blight, LRR-RLKs, hormonal signal, marker gene, differential expression

表1

实时荧光定量PCR引物"

基因名称
Gene name		 上游引物
Forward sequence (5′-3′)		 下游引物
Reverse sequence (5′-3′)
ef1α ATTGGAAACGGATATGCTCCA TCCTTACCTGAACGCCTGTCA
ChtA TTCTGGATGACAGCACAGGATAA GGCGTCCATTGCCCAAT
PR-1b GGCATCCCGAGCACAAAAT CTGCACCGGAATGAATCAAGT
PR-2 GTGAAGCTGGTTTGGGAAATG TTGCCAATCAACGTCATGTCTAC
WRKY7 CCAACTGGAAGCAACAACAA CCTGATTAGAATGATTAGCCAACA
WRKY8 CCTACTGTGACATCTCATCAATCC GGGTGCTCCCATTTCAGAC
ACRE31 CAGGATGAATCGGATCTGAAA CGGCAATCCCAATTTCTCTA
LOX CAGATCAGGCCCCGTTAATG CCTGTAAGTCCACCTTCACTTGTTG
PAL-2 GGTCACTGCCTCGGGTGAT CCTGCCAGTGAGCAAACCA
ERF3 GTGTTGACGTGAAACCAACCAT CCGGTGGAGGAAAGTTAAGGT

图1

StEFR1结构域鉴定、进化分析和序列比对 A: 对StEFR1进行结构域鉴定发现, 此基因为典型的LRR-RLK家族成员; B: StEFR1与拟南芥中功能已知的AT5G20480(AtEFR)亲缘关系相对较近; C: StEFR1与AtEFR序列相似性为53.9%。"

图2

StEFR1响应病原菌信号的表达模式 *、**分别表示在0.05和0.01概率水平差异显著。"

表2

StEFR1启动子区域顺式调控元件"

序号
Serial number		 顺式调控元件
Cis-elements		 数目
Number
1 防御和应激反应元件Defense and stress responsive elements 2
2 脱落酸响应元件Abscisic acid responsive elements 2
3 厌氧诱导相关元件Anaerobic responsive elements 7
4 干旱诱导元件Drought responsive elements 5
5 玉米醇溶蛋白代谢调节元件Regulatory element for zein metabolism 1
6 光照响应元件Light responsive elements 10

图3

瞬时超表达StEFR1后叶片对晚疫病侵染的抗性增强 A: 第2天时StEFR1的相对表达量最高; B: 第4天时WT叶片发病症状严重; C: WT和OE组叶片的病斑面积; D: WT组大量细胞呈死亡状态。WT: 接种空载体pFGC5941对照组; OE: 接种StEFR1-PFGC5941的处理组。*、**分别表示在0.05和0.01概率水平差异显著。"

图4

接种晚疫病菌后WT和OE叶片中PTI 标记基因(WRKY7、WRKY8和ACRE31)的表达模式 WT: 接种空载体pFGC5941对照组; OE: 接种StEFR1-PFGC5941的处理组。*、**分别表示在0.05和0.01概率水平差异显著。"

图5

接种晚疫病菌后WT和OE叶片中SA (ChtA、PR-1B和PR-2)、JA (LOX和PAL-2)和ET (ERF3)信号通路相关基因的表达模式 WT代表接种空载体pFGC5941对照组; OE代表接种StEFR1-PFGC5941的处理组。*、**分别表示在0.05和0.01概率水平差异显著。"

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