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作物学报 ›› 2026, Vol. 52 ›› Issue (4): 1103-1115.doi: 10.3724/SP.J.1006.2026.54106

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

乙烯响应因子CsERF9调控茶树炭疽病抗性的分子机制研究

杨影1,2(), 郝豫皖2, 张学宁2, 方佳璐2, 马月华2, 杨伟龙3, 孙文清1,2, 王新超2, 王玉春1,*(), 黄建燕2,*()   

  1. 1浙江农林大学茶学与茶文化学院 / 浙江省植物种质资源保育与利用国际科技合作基地, 浙江杭州 311300
    2中国农业科学院茶叶研究所 / 国家茶树改良中心 / 农业农村部特种经济动植物生物学与遗传育种重点实验室, 浙江杭州 310008
    3河南农业大学园艺学院, 河南郑州 450046
  • 收稿日期:2025-09-02 接受日期:2026-01-22 出版日期:2026-04-12 网络出版日期:2026-02-05
  • 通讯作者: *王玉春, E-mail:ycwang0201@126.com; 黄建燕, E-mail: huangjianyan@caas.cn
  • 作者简介:E-mail: 15608383473@163.com
  • 基金资助:
    国家重点研发计划项目“园艺作物优质多抗新种质创制与应用”(2024YFD1200504-1);国家自然科学基金项目(32202552);中国农业科学院优秀青年英才计划项目资助

Molecular mechanism of CsERF9-mediated regulation of anthracnose resistance in tea plant

Yang Ying1,2(), Hao Yu-Wan2, Zhang Xue-Ning2, Fang Jia-Lu2, Ma Yue-Hua2, Yang Wei-Long3, Sun Wen-Qing1,2, Wang Xin-Chao2, Wang Yu-Chun1,*(), Huang Jian-Yan2,*()   

  1. 1College of Tea Science and Tea Culture, Zhejiang A & F University / Zhejiang International Science and Technology Cooperation Base for Plant Germplasm Resources Conservation and Utilization, Hangzhou 311300, Zhejiang, China
    2Tea Research Institute, Chinese Academy of Agricultural Sciences / National Center for Tea Improvement / Key Laboratory of Biology, Genetics and Breeding of Special Economic Animals and Plants, Ministry of Agriculture and Rural Affairs, Hangzhou 310008, Zhejiang, China
    3College of Horticulture, Henan Agricultural University, Zhengzhou 450046, Henan, China
  • Received:2025-09-02 Accepted:2026-01-22 Published:2026-04-12 Published online:2026-02-05
  • Contact: *E-mail:ycwang0201@126.com; E-mail: huangjianyan@caas.cn
  • Supported by:
    National Key Research and Development Program of China “Creation and Application of High-Quality, Multi-Resistant New Germplasm for Horticultural Crops”(2024YFD1200504-1);National Natural Science Foundation of China(32202552);Elite Youth Program of Chinese Academy of Agricultural Sciences

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摘要：

茶树是全球重要的经济作物之一, 炭疽病作为茶树的主要真菌病害, 显著影响茶树生长及茶叶品质。乙烯响应因子(ERF)家族成员是植物抗病信号通路的核心调控因子, 在病害防御中发挥关键作用。为挖掘茶树抗病调控关键基因, 本研究基于前期茶树接种山茶炭疽菌(C. camelliae LS_19)后的转录组数据, 从差异表达基因中筛选出候选基因CsERF9并开展系统研究。亚细胞定位结果显示, CsERF9定位于细胞核, 符合转录因子的功能定位特征。启动子顺式元件分析发现, CsERF9启动子区富含多种与防御反应和激素响应相关的顺式作用元件, 表明其可能参与病害诱导的激素信号通路调控。RT-qPCR结果表明, CsERF9的表达受炭疽菌显著诱导, 在侵染后期(24 h后)持续上调。功能验证结果显示, 茶树叶片瞬时过表达CsERF9后显著降低了对炭疽菌的抗性。与空载体对照组相比, 接种24、48和72 h后, 过表达CsERF9叶片的病斑面积分别增加了170.6%、48.9%、40.7%, 证实CsERF9在茶树抗炭疽病过程中发挥负调控作用。进一步分析发现, CsERF9过表达可显著抑制SA通路的标志性基因CsPR1的表达, 同时显著上调了JA/ET信号通路的标志基因CsPR3的表达水平。上述结果表明, CsERF9可能通过抑制SA通路防御反应、激活JA/ET通路信号, 打破不同抗病信号通路的平衡, 进而负调控茶树对炭疽病的免疫响应。本研究为深入解析茶树抗炭疽病的分子网络提供了新的见解, 并为抗病育种提供了潜在的候选基因靶点。

关键词: 茶树, 炭疽病, 乙烯响应转录因子, CsERF9, 分子机制

Abstract:

Tea plant (Camellia sinensis) is a globally important economic crop. Anthracnose, a major fungal disease caused by Colletotrichum camelliae, seriously threatens tea plant growth and leaf quality. Members of the ethylene response factor (ERF) family are key regulators in plant disease resistance signaling pathways. To identify critical genes involved in tea plant defense, this study screened differentially expressed genes based on previously obtained transcriptome data from tea plants inoculated with C. camelliae strain LS_19. From these, the candidate gene CsERF9 was selected for systematic investigation. Subcellular localization analysis confirmed that CsERF9 is localized in the nucleus, consistent with its predicted function as a transcription factor. Promoter cis-element analysis revealed that the CsERF9 promoter region is enriched with various defense- and hormone-response related elements, suggesting its potential involvement in pathogen-induced hormonal signaling. RT-qPCR results showed that CsERF9 expression was significantly induced upon C. camelliae infection and remained upregulated during later stages (24 h post-inoculation and beyond). Functional validation demonstrated that transient overexpression of CsERF9 in tea leaves significantly reduced resistance to anthracnose. Compared with the empty vector control, lesion areas in CsERF9-overexpressing leaves increased by 170.6%, 48.9%, and 40.7% at 24, 48, and 72 h post-inoculation, respectively, indicating that CsERF9 acts as a negative regulator of tea plant resistance to anthracnose. Further analysis revealed that CsERF9 overexpression significantly suppressed the expression of the salicylic acid (SA) pathway marker gene CsPR1, while upregulating the jasmonic acid/ethylene (JA/ET) pathway marker gene CsPR3. These findings suggest that CsERF9 may negatively regulate the tea plant’s immune response to C. camelliae by suppressing SA-mediated defenses and activating JA/ET signaling, thereby disrupting the balance between distinct disease-resistance pathways. This study provides novel insights into the molecular network of tea plant resistance to anthracnose and identifies CsERF9 as a potential candidate gene target for breeding disease-resistant tea cultivars.

Key words: tea plant, C. camelliae, ethylene-responsive transcription factors, CsERF9, molecular mechanism

表1

本研究中所使用的引物"

引物名称
Primer name		 引物序列
Primer sequence (5′-3′)		 目的
Purpose
p2300-CsERF9-F ATTTGGAGAGGACAGGGTACCATGGCGCCCAAGGAAAAA 过表达载体的构建
Construction of overexpression vectors
p2300-CsERF9-R TCCTCCTCCTCTAGAGGATCCGGCGGTTTCAGGCGGCGG
pGFP4-CsERF9-F ATTTACGAACGATAGGGTACCATGGCGCCCAAGGAAAAA 亚细胞定位载体构建
Construction of subcellular localization vectors
pGFP4-CsERF9-R GCTCACCATGGATCCGTCGACGGCGGTTTCAGGCGGCGG
qPCR-ERF9-F AGAGCAGTACCGTCGAGTCT 实时荧光定量PCR
RT-qPCR
qPCR-ERF9-R AGACTGAGATCGAGCGGAGG
qPCR-CsPR1-F CCTCCTATGCCCAAAACTCA 实时荧光定量PCR
RT-qPCR
qPCR-CsPR1-R CAGCAAGGTTCTCCCCATAA
qPCR-CsPR3-F GATTGCTGCTTTCTTGGCCC 实时荧光定量PCR
RT-qPCR
qPCR-CsPR3-R AGTAGTCAGGAGGGTTGCCT
qPCR-CsPR4-F TGTGGGACAACCGATGACTA 实时荧光定量PCR
RT-qPCR
qPCR-CsPR4-R GTCCACAAAAGGCAGTCCAT
qPCR-CsPR5-F ACCAGCCAAAAAGCATCATC 实时荧光定量PCR
RT-qPCR
qPCR-CsPR5-R TGAGCTGACATAGCCACCAC
qPCR-CsPR10-F CTCATTTGCCACATTACACC 实时荧光定量PCR
RT-qPCR
qPCR-CsPR10-R ACATTCTGTTGGCTGCTACT
qPCR-CsPTB-F TGACCAAGCACACTCCACACTATCG 实时荧光定量PCR
RT-qPCR
qPCR-CsPTB-R TGCCCCCTTATCATCATCCACAA

图1

TEA007038表达量分析 A: RNA-seq数据中的FPKM值, 图中数据为平均值±标准误(n = 3); B: RT-qPCR测定TEA007038在LJ43受山茶炭疽菌LS_19不同侵染阶段的相对表达量, 图中数据为平均值±标准误(n = 3); C: RT-qPCR测定TEA007038在ZN12受山茶炭疽菌LS_19不同侵染阶段的相对表达, 图中数据为平均值±标准误(n = 6); D: TEA007038在茶树不同组织中的转录水平, 图中数据为平均值±标准误(n = 3); E: TEA007038在低温胁迫下的转录水平, 图中数据为平均值±标准误(n = 3); F: TEA007038在干旱胁迫下的转录水平, 图中数据为平均值±标准误(n = 3)。星号表示与对照组相比存在显著差异, 该差异通过t检验确定, *: P < 0.05; **: P < 0.01。"

图2

CsERF9与其他植物ERFs的系统发育分析和序列比对 A: 茶树CsERF9与拟南芥ERF家族蛋白的系统进化树; B: CsERF9与不同植物物种中同源蛋白的序列比对。保守的AP2/ERF结构域用红线标示。物种及GenBank登录号信息如下, 拟南芥: AtERF9 (NP_199234.1); 枳: PtERF9 (Pt2g011090); 烟草: NtERF9 (NP_001311661.1); 梨: PpERF9 (Pbr000398.1)。A图中星号标识的蛋白为本研究中的TEA007038。"

图3

CsERF9启动子区域顺式作用元件分析"

表2

CsERF9启动子区域顺式作用元件分析的具体信息"

顺式作用元件
Cis-element		 序列
Sequence		 预测功能
Predictive function		 数量
Quantity
TC-rich repeats GTTTTCTTAC 防御和应激响应调控元件Regulatory element of defense and stress responsiveness 1
TGACG-motif TGACG 茉莉酸甲酯响应元件 Response element for methyl jasmonate 2
TCT-motif TCTTAC 光响应元件 Response element for light 3
Box4 ATTAAT 光响应元件 Response element for light 8
GATA-motif GATAGGA 光响应元件 Response element for light 1
ARE AAACCA 厌氧诱导响应元件 Anaerobic induced response element 2
O2-site GATGACATGG 玉米蛋白质代谢调控元件 Regulatory element of zein metabolism regulation 1
GCN4_motif TGAGTCA 胚乳表达调控元件 Regulatory element of endosperm expression 1
AT-rich element ATAGAAATCAA AT富集DNA结合蛋白(ATBP-1)的结合位点 Binding site of AT-rich DNA binding protein (ATBP-1) 1
EBS ATGTAT EIN3结合位点 EIN3-binding site 3
GCC-box GCCGCC ERF结合位点 ERF-binding site 1

图4

CsERF9在烟草叶片中的亚细胞定位 标尺= 20 μm。"

图5

CsERF9负调控茶树对炭疽菌的抗性 A: 瞬时过表达CsERF9 (OE-CsERF9)及空载体对照(CK)的茶树叶片, 在接种炭疽菌株C. camelliae LS_19后的病害表型。照片拍摄于接种后24、48和72 h (hpi)。比例尺= 1 cm。B: RT-qPCR检测各处理组叶片中CsERF9的相对表达水平。图中数据为平均值±标准误(n = 3)。C: 接种后叶片病斑直径的定量统计。图中数据为平均值±标准误(n = 6)。hpi表示接种山茶炭疽菌后的时间。星号表示与对照组相比存在显著差异, 该差异通过t检验确定, *: P < 0.05; **: P < 0.01。"

图6

CsERF9对病程相关基因(PRs)表达的调控 A和B: RT-qPCR分析茶树叶片在接种山茶炭疽菌(C. camelliae LS_19)后不同时期CsPRs的相对表达量; C和D: 接种山茶炭疽菌后, 对照组(CK)和过表达(OE-CsERF9)叶片中CsPRs的相对表达量。图中数据为平均值±标准误(n = 3)。hpi表示接种山茶炭疽菌后的时间。*: P < 0.05; **: P < 0.01。"

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