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作物学报 ›› 2026, Vol. 52 ›› Issue (10): 2961-2970.doi: 10.3724/SP.J.1006.2026.61021

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

转录调控因子Paf1负调控小麦对BSMV病毒的抗病反应

陈冬阳(), 赵雨佳, 辛英杰, 崔祎平, 胡译戈, 岳洁瑜, 王华忠()   

  1. 天津师范大学生命科学学院 / 天津市动植物抗性重点实验室, 天津 300387
  • 收稿日期:2026-03-11 接受日期:2026-07-15 出版日期:2026-10-12 网络出版日期:2026-07-23
  • 通讯作者: 王华忠, E-mail: skywhz@tjnu.edu.cn
  • 作者简介:陈冬阳, E-mail: 1213895318@qq.com
  • 基金资助:
    国家自然科学基金项目(31971829);天津市高校中青年骨干创新人才培养计划项目(135305JF78);天津师范大学中青年教师学术创新推进计划项目(1353P2XC1604)

Transcriptional regulator Paf1 negatively regulates wheat resistance to BSMV

Chen Dong-Yang(), Zhao Yu-Jia, Xin Ying-Jie, Cui Yi-Ping, Hu Yi-Ge, Yue Jie-Yu, Wang Hua-Zhong()   

  1. School of Life Sciences, Tianjin Normal University / Tianjin Key Laboratory of Animal and Plant Resistance, Tianjin 300387, China
  • Received:2026-03-11 Accepted:2026-07-15 Published:2026-10-12 Published online:2026-07-23
  • Contact: Wang Hua-Zhong, E-mail: skywhz@tjnu.edu.cn
  • Supported by:
    National Natural Science Foundation of China(31971829);Knowledge Innovation and Training Program of Tianjin(135305JF78);Knowledge Innovation Program of Tianjin Normal University(1353P2XC1604)

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摘要：

Paf1复合物是真核生物重要的转录调控因子, 此前尚不清楚其是否在植物的抗病毒反应中发挥作用。小麦是大麦条纹花叶病毒BSMV的天然宿主之一。本研究通过基因表达分析发现, 小麦Paf1亚基基因在表达上响应BSMV侵染。基于病毒诱导的基因沉默(VIGS)原理构建了能在侵染小麦时同步触发宿主Paf1亚基基因沉默的多个BSMV病毒克隆。在被这些病毒克隆侵染的小麦幼苗上, 各Paf1亚基基因的沉默增强了过敏性反应介导的细胞死亡。进一步选择对照病毒克隆和具有触发亚基基因TaCDC73沉默能力的病毒克隆BSMV:CDC73, 对比二者在各自侵染的小麦幼苗和成株中的增殖情况, 结果发现, BSMV:CDC73的增殖水平相对较低, 说明在BSMV:CDC73侵染的小麦中, 抗病反应因TaCDC73的沉默而得到增强。此外, 与对照病毒克隆侵染的植株相比, BSMV:CDC73侵染的植株表现出更为严重的生长抑制现象。综上所述, 本研究揭示了转录调控因子Paf1通过抑制过敏性反应细胞死亡进而负调控抗BSMV反应的作用。Paf1通过这一作用限制抗病反应的过度发生, 以保障小麦在防御BSMV侵染期间的“生长-免疫平衡”。

关键词: 小麦, BSMV, Paf1, 病毒抗性, 过敏性反应

Abstract:

The RNA polymerase II-associated factor 1 (Paf1) complex is a conserved transcriptional regulator in eukaryotes; however, whether Paf1 plays a role in plant antiviral responses remains unclear. Wheat is one of the natural hosts of barley stripe mosaic virus (BSMV). In this study, analysis of gene expression showed that wheat Paf1 subunit genes are transcriptionally responsive to BSMV infection. Based on the principle of virus-induced gene silencing (VIGS), we engineered BSMV-derived viral clones capable of inducing silencing of distinct host Paf1 subunit genes during infection of wheat. In wheat seedlings infected with these clones, silencing of each Paf1 subunit gene enhanced BSMV-triggered hypersensitive response (HR)-associated cell death. Furthermore, comparative analysis of viral proliferation in wheat seedlings and adult plants infected with either a control BSMV clone or an engineered clone designed to silence the Paf1 subunit gene TaCDC73 revealed that TaCDC73 silencing reduced viral accumulation. These results indicate that TaCDC73 silencing potentiates the antiviral response in BSMV-infected wheat plants. In addition, plants infected with the TaCDC73-silencing BSMV clone exhibited more pronounced growth inhibition than those plants infected with the control BSMV clone. Collectively, our findings demonstrate that the Paf1 complex functions as a negative regulator of resistance to BSMV by suppressing HR-associated cell death. This regulatory role of Paf1 may help maintain the growth-defense balance by preventing hyperactivation of immune responses during BSMV infection.

Key words: wheat, BSMV, Paf1, virus resistance, hypersensitive response

表1

试验所用引物"

引物名称
Primer name		 引物序列
Primer sequence (5'-3')
Primers used for construction of BSMV clones with VIGS ability
vVIP2-300-F CAAACATTTTTTTTTTTTTTTAGCTAGCGCTAAATCGCGCCCTGTCC
vVIP2-300-R GATTCTTCTTCCGTTGCTAGCCCTTACTTCCCAGTGATATTC
vVIP3-300-F CAAACATTTTTTTTTTTTTTTAGCTAGCAAGGGTGTTGCTCTGGCTG
vVIP3-300-R GATTCTTCTTCCGTTGCTAGCTCCACCGGCGAGAACACCA
vVIP4-300-F CAAACATTTTTTTTTTTTTTTAGCTAGCATTGTGCGGTGGAGAACTG
vVIP4-300-R GATTCTTCTTCCGTTGCTAGCCTTGTTCTGGGAATCAACA
vVIP5-300-F CAAACATTTTTTTTTTTTTTTAGCTAGCATGAACGACCATGTTGGGAT
vVIP5-300-R GATTCTTCTTCCGTTGCTAGCCTTCATTATCCCATACAACA
vVIP6-300-F CAAACATTTTTTTTTTTTTTTAGCTAGCGCACTTTGGCACTCAACCA
vVIP6-300-R GATTCTTCTTCCGTTGCTAGCCTCGAAGCTAGCTAAGGAAC
vCDC73-300-F CAAACATTTTTTTTTTTTTTTAGCTAGCGACCTGGACAAGATCATC
vCDC73-300-R GATTCTTCTTCCGTTGCTAGCGGCGTCCTGCGCGAAGGAGG
vGFP-300-F CAAACATTTTTTTTTTTTTTTAGCTAGCTGAGCAAGGGCGAGGAGCTG
vGFP-300-R GATTCTTCTTCCGTTGCTAGCCGTCCTTGAAGAAGATGGTG
vCDC136-F GACCTGGACAAGATCATCTTC
vCDC136-R GATTCTTCTTCCGTTGCTAGCCGAGGAAGACGGCGGCGGAGA
vGFP130-F AAGGGCATCGACTTCAAGGA
vGFP130-R GATTCTTCTTCCGTTGCTAGCGGCGGATCTTGAAGTTCACC
vCDC-PDS-F GATCTTGTCCAGGTCGATGACACCCAAAGACTGA
vGFP-PDS-F TGAAGTCGATGCCCTTGATGACACCCAAAGACTGA
vPDS-R CAAACATTTTTTTTTTTTTTTAGCTAGCCTGATCGAGTCAACGACGA
Primers used in RT-qPCR expression analyses
qAlpha-F GTTGGAACCGATTTGGAAGA
qAlpha-R CTATCAGCCACTCGTGCGTA
qGamma-F TCTTGTGTGTGTTGTGGTACCT
qGamma-R TGGACTTGCAAACACTCCCA
qVIP3-F GATTGCGGTGTATGATGCGG
qVIP3-R CCCTTCTCCTTGGCATCGTA
qVIP4-F TGAGGATGACTTGGAGGCTG
qVIP4-R ATCCAGCTCTTCCCTTCCAC
qVIP5-F GATCAGTTGGAGATGGCCCT
qVIP5-R TTCTCAGCACGGTTCTTCCT
qVIP6-F GGATGGGACTGCTGGTAGTT
qVIP6-R TGAGACGGTAAAGGAGGCTG
qCDC73-F4 TCGTGGCCGTGTTTGTGCTC
qCDC73-R4 GCCTTTTATTCTTGCTTATAG
Tubulin-F GTGGAACTGGCTCTGGC
Tubulin-R CGCTCAATGTCAAGGGA
Primers used for amplification of in vitro transcription templates
M13F GTAAAACGACGGCCAGT
BSMV-R TGGTCTTCCCTTGGGGGACCGAA

图1

小麦Paf1亚基基因响应BSMV侵染的表达分析 A: 本研究构建和使用的BSMV病毒克隆基因组结构模式图, 红色箭头示用于病毒RNA检测的RT-PCR引物位置。CP: 外壳蛋白; TGB1、2和3: 三基因区块1、2和3; B: 小麦Paf1亚基基因响应BSMV侵染的RT-qPCR表达分析。对于每种处理(Mock或BSMV:GFP侵染), 各取3个独立幼苗作为生物学重复用于所有基因的分析, 每个生物学重复的每个基因分析均设置3个技术重复。*表示差异显著(P < 0.05)。"

图2

采用BSMV-VIGS方法沉默小麦Paf1亚基基因的效率分析 采用RT-qPCR表达分析方法鉴定靶基因的沉默效率。BSMV:GFP用作对照病毒克隆。对于不同BSMV克隆, 分别取3个独立的被侵染幼苗作为生物学重复, 每个生物学重复均设置3个技术重复。*表示差异显著(P < 0.05)。"

图3

Paf1亚基基因负调控小麦响应BSMV侵染的过敏性反应细胞死亡 A-C: 在小麦幼苗上接种BSMV:GFP或BSMV:CDC73病毒克隆的感染性RNA, 通过系统叶片的症状观察(A)、台盼蓝染色(B)和离子渗漏分析(C)评估细胞死亡的发生程度。离子渗漏分析中, 对于每种处理, 各取3个独立幼苗作为生物学重复。ns表示差异不显著(P > 0.05)。D: 在小麦幼苗上接种BSMV:GFP或BSMV:CDC73病毒克隆的病毒粒子, 观察系统叶片上的细胞死亡症状(左图), 用于接种的2种病毒粒子的浓度相近(右图, 以外壳蛋白丰度表示)。CP: 外壳蛋白; RbcL: Rubisco大亚基。E: 能触发不同Paf1亚基基因沉默的BSMV病毒克隆均能诱导小麦幼苗叶片发生增强的细胞死亡。"

图4

沉默TaCDC73不影响小麦响应BSMV侵染的活性氧水平 对以感染性RNA接种的幼苗(A)和以病毒粒子接种的幼苗(B)系统叶片进行H2O2的DAB染色和O2?的NBT染色观察。"

图5

沉默TaCDC73增强小麦对BSMV的抗性 A-B: 幼苗期小麦正在生长且未展开(A)和完全展开(B)的第四叶(第二系统侵染叶)中病毒蛋白的免疫印迹检测, 1-3为3个独立的植株, TGB1: 三基因区块1。C: 成株期小麦叶片中病毒蛋白的免疫印迹检测。CP: 外壳蛋白; RbcL: Rubisco大亚基。D: 成株期小麦叶片中病毒RNA的RT-PCR检测, Tubulin为内参基因。在RNAγ的检测中, 以质粒为模板的PCR产物充当含有完整插入基因片段的PCR产物大小标记。P: 质粒; M: 分子标记。"

图6

TaCDC73沉默抑制小麦应答BSMV侵染期间的生长 A: BSMV:GFP和BSMV:CDC73侵染小麦植株的生长表型。B: 对图A中2类植株的株高和最上部完全展开叶的叶长、叶宽进行测量和统计分析。每类取6株作为生物学重复进行各指标的测量。*表示差异显著(P < 0.05)。"

图7

沉默TaCDC73削弱了BSMV介导的VIGS效率 VIGS: 病毒诱导基因沉默。TaPDS的沉默程度即叶片的漂白程度可反映VIGS效率。"

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