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作物学报 ›› 2024, Vol. 50 ›› Issue (10): 2447-2457.doi: 10.3724/SP.J.1006.2024.44009

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

转录因子GhWRKY41促进水杨酸合成增强棉花对黄萎病菌的抗性

肖胜华1,2,**,*(), 董贤镘1,**(), 彭鑫1, 李安子1, 闭兆福1, 廖铭静1, 黄礼豪1, 管倩倩2, 胡琴1,2,*(), 朱龙付2,3,*()   

  1. 1广西大学农学院 / 亚热带农业生物资源保护与利用国家重点实验室, 广西南宁 530004
    2华中农业大学 / 作物遗传改良国家重点实验室, 湖北武汉 430070
    3华中农业大学 / 湖北洪山实验室, 湖北武汉 430070
  • 收稿日期:2024-01-15 接受日期:2024-06-20 出版日期:2024-10-12 网络出版日期:2024-07-12
  • 通讯作者: *肖胜华, E-mail: shxiao@gxu.edu.cn;朱龙付, E-mail: lfzhu@mail.hzau.edu.cn;胡琴, E-mail: huqin-0509@gxu.edu.cn
  • 作者简介:肖胜华, E-mail: shxiao@gxu.edu.cn;
    董贤镘, E-mail: 2609037845@qq.com
    **同等贡献
  • 基金资助:
    国家自然科学基金项目(32301880);国家自然科学基金项目(32230076);湖北洪山实验室基金项目(2021hszd006)

Transcription factor GhWRKY41 enhances cotton resistance to Verticillium dahliae by promoting SA synthesis

XIAO Sheng-Hua1,2,**,*(), DONG Xian-Man1,**(), PENG Xin1, LI An-Zi1, BI Zhao-Fu1, LIAO Ming-Jing1, HUANG Li-Hao1, GUAN Qian-Qian2, HU Qin1,2,*(), ZHU Long-Fu2,3,*()   

  1. 1State Key Laboratory of Conservation and Utilization of Agro-Biological Resources in Subtropical Region / College of Agriculture, Guangxi University, Nanning 530004, Guangxi, China
    2State Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, Hubei, China
    3Hubei Hongshan Laboratory, Huazhong Agricultural University, Wuhan 430070, Hubei, China
  • Received:2024-01-15 Accepted:2024-06-20 Published:2024-10-12 Published online:2024-07-12
  • Contact: *E-mail: shxiao@gxu.edu.cn;E-mail: lfzhu@mail.hzau.edu.cn; E-mail: huqin-0509@gxu.edu.cn
  • About author:**(Contributed equally to this work)
  • Supported by:
    National Natural Science Foundation of China(32301880);National Natural Science Foundation of China(32230076);Hubei Hongshan Laboratory Foundation(2021hszd006)

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摘要：

棉花是我国最重要的经济作物之一, 但其产量和品质受黄萎病菌危害而大幅度下降。挖掘棉花黄萎病抗性相关基因并解析其分子机制, 对加快棉花抗黄萎病育种进程具有重要意义。前期研究鉴定到一个在多个抗病棉花品种中均受黄萎病菌诱导显著上调表达的WRKY基因GhWRKY41, 该基因通过激活苯丙烷代谢增强棉花对黄萎病菌的抗性。本研究进一步分析了GhWRKY41在不同激素处理下的诱导表达模式, 利用病毒介导的基因沉默(VIGS)技术验证了GhWRKY41的抗病功能, 并利用前期已创制的GhWRKY41转基因棉花株系‘Jin668’测定了其内源植物抗病激素含量。 结果显示, GhWRKY41受水杨酸(SA)、Me-JA和H2O2诱导均显著上调表达; GhWRKY41被沉默后可削弱棉花对黄萎病菌的抗性; SA含量在GhWRKY41超表达棉花中显著增加, 而在GhWRKY41干涉棉花中则明显减少。RT-qPCR分析显示, SA合成基因GhSID2及SA信号转导基因GhNPR1、GhPR1、GhPR5的表达水平在超表达植株中明显上调, 而在干涉植株中则显著下降。ChIP-qPCR和双荧光素酶报告基因试验结果表明, GhWRKY41可结合并激活GhSID2、GhPR1和GhPR5的表达。此外, 外施SA可明显提高棉花的黄萎病抗性。综上表明, GhWRKY41可通过促进棉花中内源植物激素SA的合成来增强植株的黄萎病抗性, 这一结果完善了GhWRKY41在棉花抵御黄萎病菌过程中的生物学功能, 为未来利用黄萎病抗性基因创制棉花抗病材料提供了理论基础。

关键词: 棉花, 黄萎病菌, GhWRKY41, 水杨酸

Abstract:

Cotton is an important commercial crop, and its yield and quality are severely affected by Verticillium dahliae. Identifying cotton resistance genes to Verticillium dahliae and exploring the underlying molecular mechanisms is of great significance for accelerating the breeding process of cotton resistant to Verticillium wilt. In a previous study, the WRKY gene GhWRKY41 was identified as being induced by Verticillium dahliae in multiple resistant cotton varieties, enhancing cotton resistance through the activation of phenylpropanoid metabolism. This study further analyzed the expression patterns of GhWRKY41 under different hormone treatments, validated its disease resistance function in the upland cotton variety ‘Jin668’ using a virus-induced gene silencing (VIGS) assay, and measured endogenous hormone content. The results demonstrated that GhWRKY41 was significantly up-regulated by SA, Me-JA, and H2O2. Silencing GhWRKY41 weakened cotton resistance to Verticillium dahliae, while overexpression of GhWRKY41 led to a marked increase in SA content, and its RNAi lines showed a decrease in SA content. RT-qPCR results revealed that the expression levels of the SA biosynthesis gene GhSID2 and the SA signal transduction genes GhNPR1, GhPR1, and GhPR5 were significantly up-regulated in GhWRKY41-overexpression plants but decreased in GhWRKY41-RNAi plants. ChIP-qPCR and luciferase reporter gene assays showed that GhWRKY41 binds to and activates the expression of GhSID2, GhPR1, and GhPR5. Additionally, external spraying of SA significantly enhanced cotton resistance to Verticillium dahliae. In summary, GhWRKY41 enhances cotton resistance to Verticillium dahliae by promoting SA synthesis. This study elucidates the biological function of GhWRKY41 in cotton resistance to Verticillium dahliae and provides a theoretical basis for developing cotton varieties with improved resistance.

Key words: cotton, Verticillium dahliae, GhWRKY41, salicylic acid

表1

本研究所使用的引物"

引物名称
Primer name		 正向引物序列
Forward primer (5′-3′)		 反向引物序列
Reverse primer (5′-3′)
GhUB7 GAAGGCATTCCACCTGACCAAC CTTGACCTTCTTCTTCTTGTGCTTG
ChIP-qPCR-ProGhPR5-1 AGCCCCTATATTGATTTTGC AAAGCTACCACATGTCACCA
ChIP-qPCR-ProGhPR5-2 GAAGCAACTTAGGCTATCTCAT TTGGATTCGATTCCTAACTC
ChIP-qPCR-ProGhPR1-1 TGTCATCCTGTTGATTGAGCAA ACACTGTCCATTCAAACCTACCA
ChIP-qPCR-ProGhPR1-2 TAATATCCATTCCAGCCG TCTTGACTTGAAATTTGAGAGA
ChIP-qPCR-ProGhPR1-3 AATGCAGATCTTCACGAAAA AAATTGGACTCTTTTTAAAAGG
ChIP-qPCR-ProGhSID2-1 TCAACATATTAGGCAGCACTG GACGTTGTCATGCTGTTAGG
ChIP-qPCR-ProGhSID2-2 GCTTAACAATCTGCCACAA TTTGTGAGGAACCTACCTTC
ChIP-qPCR-ProGhSID2-3 CAAATGATTAGACTTAGGTTGAA TGAGTCGATCCTAACACAACA
ChIP-qPCR-ProGhSID2-4 GAATCCTCAAAACATAAACACA GGAGATTTGTAAAGTATGACTCC
TRV:GhWRKY41 CTCAAGGAAGCACGATGAGCTTGAGCAACTTCCAAGGGGTGCT TTGAACCTAAAACTTCAGACACGGACATTCCAACGATTCCTACATTGC
GhWRKY41-62-SK CGCTCTAGAACTAGTGGATCCATGGAGAACATGTGGAAG CTTGATATCGAATTCCTGCAGTTAGGAGAAAAATCCCGG
ProGhSID2-0800-LUC CTTGATATCGAATTCCTGCAGTAATGATGATGTCAACATATTAGG CGCTCTAGAACTAGTGGATCCGTTCAAGATAGGCTCATTGCTGGC
ProPR1-0800-LUC CTTGATATCGAATTCCTGCAGATGATAATAATGAAATAATGTCAT CGCTCTAGAACTAGTGGATCCAAGGTTAATGGTTAAATTGGACTC
ProPR5-0800-LUC CTTGATATCGAATTCCTGCAGTCTTCTGTCTTTTCATGGTCGG CGCTCTAGAACTAGTGGATCCTGACAACATTCAAGCATCAAACTC
GhWRKY41-qPCR GCCGTTCATGTCTCCCGA GCACCCCTTGGAAGTTGTTC
GhSID2-qPCR ATCGAGTGGCTCCATGCTCAAC CGGCGGCACCAACAAGATTATG
GhPR1-qPCR CCATGCCCAAGACTCACACCAA GTCCACTGGAGTGCACAAGGTT
GhPR5-qPCR GCAGTCAAGGCAGTTGGTGGTA ATATTCCGGCGTGTTGAAGGCA
GhNPR1-qPCR TTGTCTCTTGCCATCGCTGGTG GGTGCCTCATTCAAGTCCACTGT

图1

沉默GhWRKY41后可削弱棉花植株的黄萎病抗性 A: TRV:GhWRKY41植株接种2周后的发病症状。B: RT-PCR检测棉花植株中GhWRKY41的表达量。C: TRV:GhWRKY41植株接种黄萎病菌14 d后对子叶节部位纵切镜检观察。D: TRV:00和TRV:GhWRKY41棉花植株接种黄萎病菌后的病情指数统计。星号代表显著差异(**: P < 0.01, 采用t检验)。"

图2

GhWRKY41的表达模式分析 *、**分别代表在0.05、0.01概率水平差异显著。"

图3

GhWRKY41促进SA的合成 在正常条件与接种黄萎病菌条件下, WT、GhWRKY41超表达和GhWRKY41干涉棉花株系中SA的含量。不同字母代表显著差异(P < 0.05, 采用ANOVA检验)。"

图4

GhWRKY41转基因棉花材料中SA合成基因GhSID2及SA信号基因GhPR1、GhPR5和GhNPR1的表达量检测"

图5

GhWRKY41绑定GhSID2、GhPR1和GhPR5启动子中的W-box元件 A~C: 利用ChIP-qPCR验证GhWRKY41对GhSID2 (A)、GhPR1 (B)和GhPR5 (C)启动子的结合作用。红色、黄色和绿色线条分布表示GhSID2、GhPR1和GhPR5启动子中分布的W-box元件, P1~P4代表设计的ChIP-qPCR引物。GhUB7为棉花内参基因。"

图6

GhWRKY41激活GhSID2、GhPR1和GhPR5的表达 A: DLR试验中的载体构造示意图。EV表示空载体。B: 在烟草叶片中观察GhSID2、GhPR1和GhPR5启动子驱动LUC表达的荧光强度。C: 在棉花原生质体中验证GhWRKY41对pGhSID2-LUC、pGhPR1-LUC和pGhPR5-LUC的激活作用。星号代表显著差异(*: P < 0.05, **: P < 0.01, 采用t检验)。"

图7

SA可增强棉花对黄萎病菌的抗性 A: 在棉花中分别喷施不同浓度的SA并接种黄萎病菌V592后的发病症状。B, C: 在棉花中喷施不同浓度的SA并接种黄萎病菌后的病情指数(B)和发病级别(C)统计。D: 对棉花子叶节部位纵向剖杆镜检观察其褐化情况。E: 子叶节部位中黄萎病菌DNA含量检测。GhUB7为棉花内参基因, 真菌核糖体DNA的ITS区为qPCR的检测区域。F: 对棉花接种黄萎病菌后的子叶节部位恢复培养5 d的表型。Fluoreacence表示融合GFP的黄萎病菌V592菌落在活体成像仪下的荧光。G: 对(F)中的生物荧光进行统计。星号代表显著差异(**: P < 0.01, 采用t检验)。"

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