bZIP转录因子CsFcr3调控暹罗炭疽菌对DMI和QoI类杀菌剂敏感性的功能研究

doi:10.13560/j.cnki.biotech.bull.1985.2025-0543

生物技术通报 ›› 2025, Vol. 41 ›› Issue (12): 342-350.doi: 10.13560/j.cnki.biotech.bull.1985.2025-0543

bZIP转录因子CsFcr3调控暹罗炭疽菌对DMI和QoI类杀菌剂敏感性的功能研究

杨洪¹^,³(), 张超², 薛雯轩¹, 黄韦媛¹, 林春花³(), 王立丰¹()

^1.农业农村部橡胶树生物学与遗传资源利用重点实验室省部共建国家重点实验室培育基地－海南省热带作物栽培生理学重点实验室中国热带农业科学院橡胶研究所，海口 571101
^2.宜春市科学院，宜春 336028
^3.海南大学热带农林学院（农业农村学院、乡村振兴学院），儋州 571737

收稿日期:2025-05-26 出版日期:2025-12-26 发布日期:2026-01-06
通讯作者: 林春花，女，博士，教授，研究方向：植物病理学；E-mail: lin3286320@hainanu.edu.com
王立丰，男，博士，研究员，研究方向：橡胶树生理学；E-mail: lfwang@catas.cn
作者简介:杨洪，男，副研究员，研究方向：橡胶树病理学；E-mail: yang_hong0317@126.com
基金资助:
海南省“南海新星”科技创新人才平台项目(NHXXRCXM202329);海南省自然科学基金面上项目(323MS078)

Functional Study of bZIP Transcription Factor CsFcr3 in Regulating the Sensitivity of Colletotrichum siamense to DMI and QoI Fungicides

YANG Hong¹^,³(), ZHANG Chao², XUE Wen-xuan¹, HUANG Wei-yuan¹, LIN Chun-hua³(), WANG Li-feng¹()

^1.Key Laboratory of Biology and Genetic Resources of Rubber Tree, Ministry of Agriculture and Rural Affairs/State Key Laboratory Incubation Base for Cultivation & Physiology of Tropical Crops/Rubber Research Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101
^2.Yichun Academy of Sciences, Yichun 336028
^3.College of Tropical Agriculture and Forestry, Hainan University, Danzhou 571737

Received:2025-05-26 Published:2025-12-26 Online:2026-01-06

摘要/Abstract

摘要：

目的从我国橡胶树炭疽病的田间优势菌暹罗炭疽菌（Colletotrichum siamense）中克隆bZIP转录因子CsFcr3，明确其序列和结构特征，分析CsFcr3在暹罗炭疽菌对6种不同类型杀菌剂敏感性中的生物学功能，为深入研究病原真菌耐药性调控机制奠定基础。方法采用同源克隆从C. siamense中克隆了bZIP转录因子CsFcr3，并进行序列及结构特征分析，利用实时荧光定量PCR（RT-qPCR）分析杀菌剂胁迫下CsFcr3的表达模式；利用同源重组和PEG介导的原生质体转化技术对CsFcr3进行基因敲除，分析敲除突变体对不同杀菌剂敏感性的变化。结果 CsFcr3开放阅读框长度为1 101 bp，编码336个氨基酸残基，具有bZIP_Yap保守结构域；RT-qPCR结果表明，DMI类杀菌剂苯醚甲环唑、戊唑醇、咪鲜胺及甲氧基丙烯酸酯（QoI）类杀菌剂吡唑醚菌酯胁迫下CsFcr3均显著上调表达；与野生型菌株相比，CsFcr3基因缺失突变体对氟康唑、戊唑醇和咪鲜胺的敏感性均降低，对吡唑醚菌酯的敏感性增加，而对苯醚甲环唑和咯菌腈的敏感性无显著变化。结论从暹罗炭疽菌中克隆得到具有bZIP转录因子典型基序和保守结构域的CsFcr3，且与酿酒酵母Yap3p高度同源。CsFcr3主要参与调控暹罗炭疽菌对DMI类和QoI类杀菌剂的敏感性而不参与菌株对吡咯类杀菌剂敏感性的调控。

关键词: 炭疽病, 暹罗炭疽, 巴西橡胶树, bZIP转录因子, 杀菌剂, 敏感性

Abstract:

Objective The bZIP transcription factor CsFcr3 was cloned from Colletotrichum siamense, the predominant species responsible for Colletotrichum leaf disease (CLD) in rubber trees. This study characterized its sequence and structural features and elucidated its biological role in regulating the sensitivity of C. siamense to six different fungicides. These findings establish a foundational understanding of resistance regulatory mechanisms in pathogenic fungi and provide a theoretical framework for the scientific and efficient management of fungal diseases in rubber tree cultivation. Method The bZIP transcription factor CsFcr3, having high sequence homology with the fluconazole resistance protein Fcr3 from Candida albicans, was cloned from Colletotrichum siamense using homology-based cloning. Comprehensive sequence and structural analyses were conducted to characterize this gene. Real-time quantitative PCR (RT-qPCR) was employed to profile the expression patterns of CsFcr3 under fungicide stress. Additionally, the gene knockout of CsFcr3 was performed by homologous recombination and PEG-mediated protoplast transformation, and the changes in sensitivity of the knockout mutants to different fungicides were analyzed. Result The open reading frame (ORF) of CsFcr3 encompassed 1 101 base pairs, encoding a protein of 336 amino acids with a conserved bZIP_Yap domain. RT-qPCR results demonstrated significant upregulation of CsFcr3 expression under stress induced by demethylation inhibitor (DMI) fungicides (difenoconazole, tebuconazole, prochloraz) and quinone outside inhibitor (QoI) fungicides (pyraclostrobin). Three CsFcr3 knockout mutants (ΔCsFcr3-3, ΔCsFcr3-15, and ΔCsFcr3-16) were successfully generated. Compared to the wild-type strain, the ΔCsFcr3s presented significantly reduced sensitivity to fluconazole, tebuconazole, and prochloraz, increased sensitivity to pyraclostrobin, and no significant differences in sensitivity to difenoconazole and fludioxonil. Conclusion The bZIP transcription factor CsFcr3, which contains conserved motifs and structural domains typical of the bZIP family, is cloned from C. siamense. Phylogenetic analysis reveals its close homology to the Saccharomyces cerevisiae Yap3p protein. Functional characterization demonstrates that CsFcr3 primarily regulates C. siamense sensitivity to DMI and QoI fungicides, exhibiting no significant regulatory role in response to pyrrolnitrin-class fungicides.

Key words: anthracnose, Colletotrichum siamense, Hevea brasiliensis, bZIP transcription factor, fungicide, sensitivity

杨洪, 张超, 薛雯轩, 黄韦媛, 林春花, 王立丰. bZIP转录因子CsFcr3调控暹罗炭疽菌对DMI和QoI类杀菌剂敏感性的功能研究[J]. 生物技术通报, 2025, 41(12): 342-350.

YANG Hong, ZHANG Chao, XUE Wen-xuan, HUANG Wei-yuan, LIN Chun-hua, WANG Li-feng. Functional Study of bZIP Transcription Factor CsFcr3 in Regulating the Sensitivity of Colletotrichum siamense to DMI and QoI Fungicides[J]. Biotechnology Bulletin, 2025, 41(12): 342-350.

图/表 7

表1 本研究所用引物信息

Table 1 Primers used in this study

引物名称 Primer name	引物序列 Primer sequence （5′-3′）	用途 Usage
UF	ACAACAAGCGTTTAGCGTCTC	扩增目的基因上臂
UR	AGATGTGGGGCACTGTGGCGTTGGCACGGTCGCGCGATGTTGTCCTGGAAGAG	扩增目的基因上臂
DF	TATTGCACGGGAATTGCATGCTCTCACCAGCCCCCTCTCTCCCCAGTG	扩增目的基因下臂
DR	GATGCCTAAGCCTGCAAAC	扩增目的基因下臂
InF	TCACTGGTGGATTGCCGCTTAG	转化子验证
InR	TGTGCTGTGGTGGTTGCTGAG	转化子验证
OUF	TGTCTTGTCTTGCCTGCAAC	转化子验证
OUR	AAAGAACTCGGCAGTAAGCTTG	转化子验证
S2F	GGCGGTGCTATCCTTCCCGTGTT	扩增氯嘧磺隆抗性基因
S2R	GTGAGAGCATGCAATTCCCGTGCAATA	扩增氯嘧磺隆抗性基因
qCsFcr3_F	ATGGACTTCTCCCACCAGTA	RT-qPCR
qCsFcr3_R	CGGCAGCGACGAGCTCCTGTAG	RT-qPCR
qCsActin_F	GATTTGGCACCACACCTTCTACA	内参
qCsActin_R	TCTCTGTTGGACTTGGGGTTGAT	内参

图1 CsFcr3基因的全长克隆A：CsFcr3基因PCR扩增；B：CsFcr3核苷酸序列及推导氨基酸。M：DL5000 DNA Marker；Fcr3：CsFcr3基因PCR扩增产物；CK：阴性对照

Fig. 1 Full-length cloning of the CsFcr3 geneA: PCR amplification of the CsFcr3 gene. B: Nucleotide sequence and derived amino acids of CsFcr3. M: DL5000 DNA Marker. Fcr3: PCR amplification product of CsFcr3 gene. CK: Negative control

图2 CsFcr3序列特征分析

Fig. 2 Sequence characterization analysis of CsFcr3

图3 杀菌剂处理下HN08表型（A）及抑制率（B）不同字母代表差异显著（P < 0.05），下同

Fig. 3 Phenotype (A) and inhibition rate (B) of HN08 in fungicide treatmentDifferent letters indicate significant differences at P < 0.05, the same below

图4 杀菌剂胁迫下CsFcr3表达分析

Fig. 4 Expression analysis of CsFcr3 under fungicide stress

图5 CsFcr3基因敲除突变体构建流程（A）及PCR鉴定（B）

Fig. 5 Construction process (A) and PCR verification (B) of the CsFcr3 gene knockout mutant

图6 ΔCsFcr3s对6种杀菌剂的敏感性分析A：杀菌剂胁迫下ΔCsFcr3s的表型；B：杀菌剂胁迫下ΔCsFcr3s的抑制率。*P < 0.05，** P < 0.01

Fig. 6 Sensitivity analysis of ΔCsFcr3s to 6 fungicidesA: Phenotype of ΔCsFcr3s under fungicide stress. B: Inhibition rate of ΔCsFcr3s under fungicide stress, *P < 0.05, **P < 0.01

参考文献 34

[1]	Talhinhas P. Hosts of Colletotrichum [J]. Mycosphere, 2023, 14(si2): 158-261.
[2]	李博勋, 刘先宝, 陈丽琼, 等. 橡胶树主要病害研究现状与展望 [J]. 中国科学: 生命科学, 2024, 54(10): 1798-1813.
	Li BX, Liu XB, Chen LQ, et al. Current status and prospects of research on main diseases of rubber trees [J]. Sci Sin Vitae, 2024, 54(10): 1798-1813.
[3]	林春花, 张宇, 刘文波, 等. 我国巴西橡胶树炭疽病的研究进展 [J]. 热带生物学报, 2021, 12(3): 393-402, 268.
	Lin CH, Zhang Y, Liu WB, et al. Research advances on Colletotrichum leaf fall disease of rubber trees in China [J]. J Trop Biol, 2021, 12(3): 393-402, 268.
[4]	Liu XB, Li BX, Yang Y, et al. Pathogenic adaptations revealed by comparative genome analyses of two Colletotrichum spp., the causal agent of anthracnose in rubber tree [J]. Front Microbiol, 2020, 11: 1484.
[5]	Liu XB, Li BX, Cai JM, et al. Colletotrichum species causing anthracnose of rubber trees in China [J]. Sci Rep, 2018, 8: 10435.
[6]	Cao XR, Xu XM, Che HY, et al. Three Colletotrichum species, including a new species, are associated to leaf anthracnose of rubber tree in Hainan, China [J]. Plant Dis, 2019, 103(1): 117-124.
[7]	贺春萍, 樊兰艳, 吴贺, 等. 枯草芽孢杆菌Czk1脂肽物质对橡胶树炭疽病和白粉病的抑制效果研究 [J]. 中国农业科技导报, 2023, 25(6): 126-134.
	He CP, Fan LY, Wu H, et al. Study on inhibitory effect of lipopeptides produced by Bacillus subtilis Czk1 on anthracnose and powdery mildew of rubber tree [J]. J Agric Sci Technol, 2023, 25(6): 126-134.
[8]	刘凤华, 马迪成, 张晓敏, 等. 植物病原真菌对甾醇脱甲基抑制剂类杀菌剂抗性分子机制研究进展 [J]. 农药学学报, 2022, 24(3): 452-464.
	Liu FH, Ma DC, Zhang XM, et al. Research progress on molecular resistance mechanism of plant pathogenic fungi to sterol demethylase inhibitors [J]. Chin J Pestic Sci, 2022, 24(3): 452-464.
[9]	叶滔, 马志强, 毕秋艳, 等. 植物病原真菌对甾醇生物合成抑制剂类(SBIs)杀菌剂的抗药性研究进展 [J]. 农药学学报, 2012, 14(1): 1-16.
	Ye T, Ma ZQ, Bi QY, et al. Research advances on the resistance of plant pathogenic fungi to SBIs fungicides [J]. Chin J Pestic Sci, 2012, 14(1): 1-16.
[10]	Han YC, Zeng XG, Guo C, et al. Reproduction response of Colletotrichum fungi under the fungicide stress reveals new aspects of chemical control of fungal diseases [J]. Microb Biotechnol, 2022, 15(2): 431-441.
[11]	Egner U, Gerbling KP, Hoyer GA, et al. Design of inhibitors of photosystem II using a model of the D1 protein [J]. Pestic Sci, 1996, 47(2): 145-158.
[12]	Furukawa K, Randhawa A, Kaur H, et al. Fungal fludioxonil sensitivity is diminished by a constitutively active form of the group III histidine kinase [J]. FEBS Lett, 2012, 586(16): 2417-2422.
[13]	曹学仁, 车海彦, 杨毅, 等. 2014年海南省橡胶炭疽病菌对多菌灵和咪鲜胺的敏感性测定 [J]. 植物病理学报, 2015, 45(6): 626-631.
	Cao XR, Che HY, Yang Y, et al. Sensitivity of Colletotrichum spp. from Hevea brasiliensis to carbendazim and prochloraz in Hainan Province in China in 2014 [J]. Acta Phytopathol Sin, 2015, 45(6): 626-631.
[14]	蔡志英, 李加智, 王进强, 等. 橡胶胶孢炭疽菌和尖孢炭疽菌对杀菌剂的敏感性测定 [J]. 云南农业大学学报, 2008, 23(6): 787-790.
	Cai ZY, Li JZ, Wang JQ, et al. Sensitivity test of Colletotrichum gloeosporioides and Colletotrichum acutatum isolated from rubber to the fungicides [J]. J Yunnan Agric Univ, 2008, 23(6): 787-790.
[15]	林春花, 徐轩, 戈玉琪, 等. 中国2种橡胶树炭疽病菌对咪鲜胺敏感性比较 [J]. 热带作物学报, 2017, 38(1): 111-115.
	Lin CH, Xu X, Ge YQ, et al. Comparison of sensitivity between two Colletotrichum species from Hevea brasiliensis to prochloraz in China [J]. Chin J Trop Crops, 2017, 38(1): 111-115.
[16]	Shelest E. Transcription factors in fungi [J]. FEMS Microbiol Lett, 2008, 286(2): 145-151.
[17]	Shelest E. Transcription factors in fungi: TFome dynamics, three major families, and dual-specificity TFs [J]. Front Genet, 2017, 8: 53.
[18]	Leiter É, Emri T, Pákozdi K, et al. The impact of bZIP Atf1 ortholog global regulators in fungi [J]. Appl Microbiol Biotechnol, 2021, 105(14): 5769-5783.
[19]	Takeda T, Toda T, Kominami K, et al. Schizosaccharomyces pombe atf1+ encodes a transcription factor required for sexual development and entry into stationary phase [J]. EMBO J, 1995, 14(24): 6193-6208.
[20]	Li YF, Li YS, Lu HH, et al. The bZIP transcription factor ATF1 regulates blue light and oxidative stress responses in Trichoderma guizhouense [J]. mLife, 2023, 2(4): 365-377.
[21]	Tang C, Li TY, Klosterman SJ, et al. The bZIP transcription factor VdAtf1 regulates virulence by mediating nitrogen metabolism in Verticillium dahliae [J]. New Phytol, 2020, 226(5): 1461-1479.
[22]	Song M, Fang SQ, Li ZG, et al. CsAtf1, a bZIP transcription factor, is involved in fludioxonil sensitivity and virulence in the rubber tree anthracnose fungus Colletotrichum siamense [J]. Fungal Genet Biol, 2022, 158: 103649.
[23]	Tang W, Ru YY, Hong L, et al. System-wide characterization of bZIP transcription factor proteins involved in infection-related morphogenesis of Magnaporthe oryzae [J]. Environ Microbiol, 2015, 17(4): 1377-1396.
[24]	Hussain S, Tai BW, Hussain A, et al. Genome-wide identification and expression analysis of the basic leucine zipper (bZIP) transcription factor gene family in Fusarium graminearum [J]. Genes, 2022, 13(4): 607.
[25]	Yin WB, Reinke AW, Szilágyi M, et al. bZIP transcription factors affecting secondary metabolism, sexual development and stress responses in Aspergillus nidulans [J]. Microbiology, 2013, 159(Pt 1): 77-88.
[26]	Zuriegat Q, Zheng YR, Liu H, et al. Current progress on pathogenicity-related transcription factors in Fusarium oxysporum [J]. Mol Plant Pathol, 2021, 22(7): 882-895.
[27]	Fernandes L, Rodrigues-Pousada C, Struhl K. Yap, a novel family of eight bZIP proteins in Saccharomyces cerevisiae with distinct biological functions [J]. Mol Cell Biol, 1997, 17(12): 6982-6993.
[28]	Chen KH, Miyazaki T, Tsai HF, et al. The bZip transcription factor Cgap1p is involved in multidrug resistance and required for activation of multidrug transporter gene CgFLR1 in Candida glabrata. [J]. Gene, 2007, 386(1/2): 63-72.
[29]	Yang XS, Talibi D, Weber S, et al. Functional isolation of the Candida albicans FCR3 gene encoding a bZip transcription factor homologous to Saccharomyces cerevisiae Yap3p [J]. Yeast, 2001, 18(13): 1217-1225.
[30]	Yang H, Huang WY, Fan SL, et al. Systematic characterization of the bZIP gene family in Colletotrichum siamense and functional analysis of three family members [J]. Int J Biol Macromol, 2025, 286: 138463.
[31]	Guan XL, Song M, Lu JW, et al. The transcription factor CsAtf1 negatively regulates the cytochrome P450 gene CsCyp51G1 to increase fludioxonil sensitivity in Colletotrichum siamense [J]. JoF, 2022, 8(10): 1032.
[32]	Zhao QQ, Pei H, Zhou XL, et al. Systematic characterization of bZIP transcription factors required for development and aflatoxin generation by high-throughput gene knockout in Aspergillus flavus [J]. J Fungi, 2022, 8(4): 356.
[33]	张一宾. 甲氧基丙烯酸酯类杀菌剂的全球市场概况及进展 [J]. 世界农药, 2016, 38(4): 30-34.
	Zhang YB. Development and global market of strobilurin fungicides [J]. World Pestic, 2016, 38(4): 30-34.
[34]	赵健钦, 金京, 陈杰. QoI类杀菌剂应用与抗性机制研究进展 [J]. 世界农药, 2023, 45(7): 19-30.
	Zhao JQ, Jin J, Chen J. Research progress on application and resistance of QoI fungicides [J]. World Pestic, 2023, 45(7): 19-30.

bZIP转录因子CsFcr3调控暹罗炭疽菌对DMI和QoI类杀菌剂敏感性的功能研究

Functional Study of bZIP Transcription Factor CsFcr3 in Regulating the Sensitivity of Colletotrichum siamense to DMI and QoI Fungicides

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 7

参考文献 34

相关文章 15

编辑推荐

Metrics

本文评价

[1]	张吉昌, 许云凤, 蒋凌雁. 柱花草内生细菌ZW21发酵条件优化及其抑菌物质稳定性测定[J]. 生物技术通报, 2025, 41(5): 280-289.
[2]	牛若宇, 高瞻, 熊显鹏, 祝德, 罗皓天, 马学远, 胡冠菁. 棉花野生种质资源的育种应用研究与前景[J]. 生物技术通报, 2025, 41(4): 21-32.
[3]	刘倩, 马连杰, 张慧, 王冬, 范茂, 廖敦秀, 赵正武, 卢文才. 辣椒炭疽病生防菌株TN2的筛选鉴定与抑菌效果[J]. 生物技术通报, 2025, 41(1): 287-297.
[4]	徐伟芳, 李贺宇, 张慧, 何仔昂, 高文恒, 谢紫洋, 王传文, 尹登科. 生防细菌HX0037对栝楼炭疽病的防病能力及其机制[J]. 生物技术通报, 2024, 40(4): 228-241.
[5]	杨艳, 胡洋, 刘霓如, 殷璐, 杨锐, 王鹏飞, 穆霄鹏, 张帅, 程春振, 张建成. ‘红满堂’苹果MbbZIP43基因的克隆与功能研究[J]. 生物技术通报, 2024, 40(2): 146-159.
[6]	李白雪, 李金玲, 陈春林, 杜清洁, 李猛, 王吉庆, 马勇斌, 肖怀娟. 辣椒CabZIP42的克隆及表达分析[J]. 生物技术通报, 2024, 40(12): 93-101.
[7]	章乐乐, 王冠, 柳凤, 胡汉桥, 任磊. 芒果炭疽病拮抗菌分离、鉴定及生防机制研究[J]. 生物技术通报, 2023, 39(4): 277-287.
[8]	周家燕, 邹建, 陈卫英, 吴一超, 陈奚潼, 王倩, 曾文静, 胡楠, 杨军. 植物多基因干扰载体体系构建与效用分析[J]. 生物技术通报, 2023, 39(1): 115-126.
[9]	时雅倩, 申亚茹, 陈漫影, 何淑敏, 刘予涵, 何天楠, 陈清西, 文志丰. 黄毛草莓F-box蛋白基因FnFBOX1及其启动子的克隆和表达分析[J]. 生物技术通报, 2022, 38(2): 44-56.
[10]	李峰, 陈雯雯, 邓江丽, 毛清黎, 权文利, 毛雅慧. 白藜芦醇对核桃细菌性黑斑病菌的抑制作用[J]. 生物技术通报, 2021, 37(6): 58-65.
[11]	杨洪, 岳镒繁, 胡燕玲, 邓治, 代龙军, 李德军. 巴西橡胶树HbAIH基因的克隆及表达分析[J]. 生物技术通报, 2020, 36(5): 120-129.
[12]	陶尚琨, 王国梁, 刘文德. 稻瘟病菌机械敏感性离子通道的鉴定及功能分析[J]. 生物技术通报, 2017, 33(6): 97-103.
[13]	李杨, 李河, 周国英, 刘君昂. 油茶新炭疽病原Colletotrichum camelliae鉴定及致病性测定[J]. 生物技术通报, 2016, 32(6): 96-102.
[14]	张文华. 香菇多糖增强黄瓜幼苗对炭疽病菌抗性机理[J]. 生物技术通报, 2016, 32(3): 93-97.
[15]	田媛媛, 周国英, 左杰, 刘倩丽, 杨权. 檀香炭疽病拮抗细菌的分离筛选及鉴定[J]. 生物技术通报, 2015, 31(9): 158-162.