AP2/ERF在青花菜苗期响应黑腐病的功能研究

doi:10.16420/j.issn.0513-353x.2023-0469

摘要/Abstract

摘要：

通过对黑腐病病原菌侵染后的青花菜（Brassica oleracea L. var. italica）叶片进行转录组测序分析，探究青花菜对黑腐病的响应机制，挖掘并鉴定其中的抗病相关基因。Gene Ontology（GO）分析显示，与未接种相比，接种病原菌3、5和7 d后上调的差异表达基因均显著富集在胁迫应答和植物激素信号途径；3个比较组共有的差异表达基因中，上调表达的基因编码32个AP2/ERF转录因子；AP2/ERF编码的蛋白质序列长度在132 ~ 344 aa，且启动子中含有大量应答脱落酸、生长素、茉莉酸、赤霉素和水杨酸的顺式作用元件。水杨酸（SA）处理能够显著增强青花菜对黑腐病的抗性，强烈诱导AP2/ERF以及防御相关基因的表达。此外，BolC1t04894H编码的EAR型转录抑制子ERF4定位于细胞核中。瞬时表达青花菜ERF4导致烟草叶片对黑腐病病原菌处理敏感。EMSA结果显示，青花菜ERF4可以结合WRKY33启动子中的GCC-box序列，双荧光素酶报告系统试验表明ERF4抑制WRKY33的表达。综上，黑腐病病原菌侵染后，激活了青花菜激素信号转导途径，SA强烈诱导AP2/ERF的表达，进而调控一系列抗病相关基因的转录，启动防御反应。

关键词: 青花菜, AP2/ERF转录因子, 黑腐病, 水杨酸, 抗病性

Abstract:

To explore the response mechanism of black rot and identify disease-resistance related genes through RNA-Seq analysis of broccoli（Brassica oleracea L. var. italica）leaves infected by black rot pathogen. Gene Ontology（GO）analysis showed that up-regulated differentially expressed genes（DEGs）were significantly enriched in stress response and phytohormone signaling pathways after 3，5，and 7 days infected by pathogens compared to uninoculated. At these three time points，the up-regulated genes in overlapped DEGs encoded up to 32 AP2/ERF transcription factors. Their encoded protein sequence ranged from 132 to 344 amino acids in length，and its promoter contained a large number of cis-elements in response to abscisic acid，auxin，jasmonic acid，gibberellin and salicylic acid（SA）. SA treatment significantly enhanced the resistance of broccoli to black rot，and strongly induced the expression of AP2/ERF and defense-related genes. BolC1t04894H encodes an EAR type transcriptional suppressor ERF4，which is localized in the nucleus. Transient expression of broccoli ERF4 resulted in sensitivity of tobacco leaves to Xcc treatment. Electrophoretic Mobility Shift Assay（EMSA）result indicated that ERF4 could bind the GCC-box sequence in the promoter of WRKY33. Subsequently，double luciferase reporter system experiments showed that ERF4 inhibited WRKY33 expression. Taken together，the phytohormone signalling pathway are activated in broccoli after black rot pathogen infection，and SA strongly induces the AP2/ERF expression，thereby regulating a series of defense-related genes transcription to response black rot.

Key words: broccoli, AP2/ERF transcription factor, black rot, salicylic acid, disease resistance

唐征, 陈思雀, 徐谦, 钟伟杰, 刘庆, 朱世杨. AP2/ERF在青花菜苗期响应黑腐病的功能研究[J]. 园艺学报, 2024, 51(11): 2523-2539.

TANG Zheng, CHEN Sique, XU Qian, ZHONG Weijie, LIU Qing, ZHU Shiyang. Functional Study of AP2/ERF in Response to Black Rot at Broccoli Seedling Stage[J]. Acta Horticulturae Sinica, 2024, 51(11): 2523-2539.

图/表 13

参考文献 24

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目的	引物名称		引物序列（5′-3′）
Purpose	Primer name		Primer sequence
转录水平检测	BolC9t55823H		F：GGCGAAGGGGAAGCATTAC；R：TCTCCACGGAAAGTTCAGCA
Transcriptional level detection		BolC8t48739H		F：ACGCAACACTTAATTTTCCGGA；R：CTTCAGCCATCCCTTCCTCT
		BolC7t43635H		F：GCCGTCAAAGGGAAAGCATT；R：CACGCATCCTAAAAGCAGCT
		BolC7t42203H		F：GCAGCACCGTCGAATCATC；R：ACTACGATCCATCACCACAGA
		BolC6t38421H		F：AGTGTCCGGGGAGAAGAATC；R：TCGGAGAAGTCATCAAACCCT
		BolC5t33888H		F：GGCGACTGATGGGATTGTTC；R：TCGTAGAGACCTGCATCACC
		BolC3t17033H		F：CACCCTTGACTGCGATTCTC；R：GGCGACCTAAAAGCTGACG
		BolC2t10891H		F：GGGAAGCATTACAGAGGGGT；R：CACGCATCCTAAAAGCAGCT
		BolC1t04894H		F：CTTGAGCTTAGTCTTGGCGG；R：CTCCCACAAGCCATCTTCGT
		BolC1t04025H		F：CGGTATTAGGGTTTGGCTCG；R：GAACACCCATCCTCGTAGCT
		BolC1t01080H		F：TAGAGTTTGGTTGGGGACGT；R：ACCGTTCTCCTCCGCTTC
		BolC1t00081H		F：CCACACATGAGGCACGAGTA；R：TCTTGATACTCCTCTGCTTTTGG
		PAL2		F：TAACAATCGGACAAGTCGC；R：AGAGGTCGCTCCAAACCC
		CHS		F：ACTACTACTTCCGCATCACC；R：GGGACTTCAACCACCACTA
		CHI		F：GCTAACGCCTATCCGAGCTT；R：TGTACACATGGGAACTCTGGT
		WRKY33		F：CTGTGTTCAATGCCAGCCTT；R：TTGCTTCAGGTTCACTCCCA
		Actin		F：CCAGAGGTCTTGTTCCAGCCATC；R：GTTCCACCACTGAGCACAATGTTAC
亚细胞定位和凝胶迁移 Subcellar location and EMSA	ERF4-pENTR		F：GCAGGCTCCGCGGCCATGGTTAAGATGGGTTTGAA； R：AGCTGGGTCGGCGCGGGCCTGTTCCAACGGAGGAG
双荧光素酶报告系统试验 Dual-luciferase reporter	ERF4-62SK		F：TAGAACTAGTGGATCCCCCATGGTTAAGATGGGTTTGAA； R：TATCGAATTCCTGCAGCCCTCAGGCCTGTTCCAACGGAGGAG
system		WRKY33pro-LUC		F：AGCTGGGTCGGCGCGGGCCTGTTCCAACGGAGGAG； R：TAGAACTAGTGGATCCCCCACATAAAAAAATTGAAGCTT

目的	引物名称		引物序列（5′-3′）
Purpose	Primer name		Primer sequence
转录水平检测	BolC9t55823H		F：GGCGAAGGGGAAGCATTAC；R：TCTCCACGGAAAGTTCAGCA
Transcriptional level detection		BolC8t48739H		F：ACGCAACACTTAATTTTCCGGA；R：CTTCAGCCATCCCTTCCTCT
		BolC7t43635H		F：GCCGTCAAAGGGAAAGCATT；R：CACGCATCCTAAAAGCAGCT
		BolC7t42203H		F：GCAGCACCGTCGAATCATC；R：ACTACGATCCATCACCACAGA
		BolC6t38421H		F：AGTGTCCGGGGAGAAGAATC；R：TCGGAGAAGTCATCAAACCCT
		BolC5t33888H		F：GGCGACTGATGGGATTGTTC；R：TCGTAGAGACCTGCATCACC
		BolC3t17033H		F：CACCCTTGACTGCGATTCTC；R：GGCGACCTAAAAGCTGACG
		BolC2t10891H		F：GGGAAGCATTACAGAGGGGT；R：CACGCATCCTAAAAGCAGCT
		BolC1t04894H		F：CTTGAGCTTAGTCTTGGCGG；R：CTCCCACAAGCCATCTTCGT
		BolC1t04025H		F：CGGTATTAGGGTTTGGCTCG；R：GAACACCCATCCTCGTAGCT
		BolC1t01080H		F：TAGAGTTTGGTTGGGGACGT；R：ACCGTTCTCCTCCGCTTC
		BolC1t00081H		F：CCACACATGAGGCACGAGTA；R：TCTTGATACTCCTCTGCTTTTGG
		PAL2		F：TAACAATCGGACAAGTCGC；R：AGAGGTCGCTCCAAACCC
		CHS		F：ACTACTACTTCCGCATCACC；R：GGGACTTCAACCACCACTA
		CHI		F：GCTAACGCCTATCCGAGCTT；R：TGTACACATGGGAACTCTGGT
		WRKY33		F：CTGTGTTCAATGCCAGCCTT；R：TTGCTTCAGGTTCACTCCCA
		Actin		F：CCAGAGGTCTTGTTCCAGCCATC；R：GTTCCACCACTGAGCACAATGTTAC
亚细胞定位和凝胶迁移 Subcellar location and EMSA	ERF4-pENTR		F：GCAGGCTCCGCGGCCATGGTTAAGATGGGTTTGAA； R：AGCTGGGTCGGCGCGGGCCTGTTCCAACGGAGGAG
双荧光素酶报告系统试验 Dual-luciferase reporter	ERF4-62SK		F：TAGAACTAGTGGATCCCCCATGGTTAAGATGGGTTTGAA； R：TATCGAATTCCTGCAGCCCTCAGGCCTGTTCCAACGGAGGAG
system		WRKY33pro-LUC		F：AGCTGGGTCGGCGCGGGCCTGTTCCAACGGAGGAG； R：TAGAACTAGTGGATCCCCCACATAAAAAAATTGAAGCTT

比较组	上调	下调
Comparison group	Up regulated	Down regulated
0 d vs. 1 d	蛋白泛素化 Protein ubiquitination	DNA复制起始 DNA replication initiation
	光呼吸 Photorespiration	蛋白折叠 Protein refolding
	昼夜节律 Circadian rhythm	叶绿体 Chloroplast
	还原戊糖磷酸循环 Reductive pentose phosphate cycle
0 d vs. 3 d	伤害应答 Response to wounding	翻译 Translation
	盐胁迫应答 Response to salt stress	光合作用 Photosynthesis
	高温应答 Response to heat	叶绿体 Chloroplast
	氧化胁迫应答 Response to oxidative stress	钙离子结合 Calcium ion binding
	几丁质酶应答 Response to chitin	核糖体结构成分 Structural constituent of ribosome
	Karrikin应答 Response to karrikin	rRNA结合 rRNA binding
	茉莉酸应答 Response to jasmonic acid
	脱落酸应答 Response to abscisic acid
	乙烯激活信号通路 Ethylene-activated signaling pathway
0 d vs. 5 d	伤害应答 Response to wounding	光合作用 Photosynthesis
	盐胁迫应答 Response to salt stress	生长调节 Regulation of growth
	钙离子应答 Response to calcium ion	生长素激活信号通路 Auxin-activated signaling pathway
	氧化胁迫应答 Response to oxidative stress	叶绿体 Chloroplast
	几丁质酶应答 Response to chitin	叶绿素结合 Chlorophyll binding
	Karrikin应答 Response to karrikin
	茉莉酸应答 Response to jasmonic acid
	乙烯激活信号通路 Ethylene-activated signaling pathway
	防御应答 Defense response
0 d vs. 7 d	伤害应答 Response to wounding	光和作用 Photosynthesis
	盐胁迫应答 Response to salt stress	细胞壁组织 Cell wall organization
	氧化胁迫应答 Response to oxidative stress	碳水化合物代谢 Carbohydrate metabolic process
	茉莉酸应答 Response to jasmonic acid	叶绿体 Chloroplast
	几丁质酶应答 Response to chitin
	Karrikin应答 Response to karrikin
	水杨酸应答 Response to salicylic acid
	脱落酸应答 Response to abscisic acid
	乙烯激活信号通路 Ethylene-activated signaling pathway
	防御应答 Defense response

比较组	上调	下调
Comparison group	Up regulated	Down regulated
0 d vs. 1 d	蛋白泛素化 Protein ubiquitination	DNA复制起始 DNA replication initiation
	光呼吸 Photorespiration	蛋白折叠 Protein refolding
	昼夜节律 Circadian rhythm	叶绿体 Chloroplast
	还原戊糖磷酸循环 Reductive pentose phosphate cycle
0 d vs. 3 d	伤害应答 Response to wounding	翻译 Translation
	盐胁迫应答 Response to salt stress	光合作用 Photosynthesis
	高温应答 Response to heat	叶绿体 Chloroplast
	氧化胁迫应答 Response to oxidative stress	钙离子结合 Calcium ion binding
	几丁质酶应答 Response to chitin	核糖体结构成分 Structural constituent of ribosome
	Karrikin应答 Response to karrikin	rRNA结合 rRNA binding
	茉莉酸应答 Response to jasmonic acid
	脱落酸应答 Response to abscisic acid
	乙烯激活信号通路 Ethylene-activated signaling pathway
0 d vs. 5 d	伤害应答 Response to wounding	光合作用 Photosynthesis
	盐胁迫应答 Response to salt stress	生长调节 Regulation of growth
	钙离子应答 Response to calcium ion	生长素激活信号通路 Auxin-activated signaling pathway
	氧化胁迫应答 Response to oxidative stress	叶绿体 Chloroplast
	几丁质酶应答 Response to chitin	叶绿素结合 Chlorophyll binding
	Karrikin应答 Response to karrikin
	茉莉酸应答 Response to jasmonic acid
	乙烯激活信号通路 Ethylene-activated signaling pathway
	防御应答 Defense response
0 d vs. 7 d	伤害应答 Response to wounding	光和作用 Photosynthesis
	盐胁迫应答 Response to salt stress	细胞壁组织 Cell wall organization
	氧化胁迫应答 Response to oxidative stress	碳水化合物代谢 Carbohydrate metabolic process
	茉莉酸应答 Response to jasmonic acid	叶绿体 Chloroplast
	几丁质酶应答 Response to chitin
	Karrikin应答 Response to karrikin
	水杨酸应答 Response to salicylic acid
	脱落酸应答 Response to abscisic acid
	乙烯激活信号通路 Ethylene-activated signaling pathway
	防御应答 Defense response

基因号 Gene ID	位置 Position	氨基酸数 Amino acid	定位预测 Predicted location	拟南芥同源基因（ID）
基因号 Gene ID	位置 Position	氨基酸数 Amino acid	定位预测 Predicted location	Arabidopsis thaliana homologous gene
BolC9t56886H	Chr9：37 383 176 ~ 37 383 838	220	细胞核 Nucleus	ERF105（AT5G51190）
BolC9t55823H	Chr9：23 228 516 ~ 23 229 238	240	细胞核 Nucleus	ERF2（AT5G47220）
BolC8t51758H	Chr8：44 860 010 ~ 44 860 951	313	细胞核 Nucleus	ERF053（AT2G20880）
BolC8t48739H	Chr8：24 675 340 ~ 24 676 173	277	细胞核 Nucleus	ERF054（AT4G28140）
BolC8t48489H	Chr8：22 668 610 ~ 22 669 377	255	细胞核 Nucleus	ERF1A（AT4G17500）
BolC8t48488H	Chr8：22 653 145 ~ 22 654 017	290	细胞核 Nucleus	ERF6（AT4G17490）
BolC7t43994H	Chr7：41 081 592 ~ 41 082 626	344	细胞核 Nucleus	ARF14（AT3G25730）
BolC7t43635H	Chr7：38 015 941 ~ 38 016 609	222	细胞核 Nucleus	ERF2（AT5G47220）
BolC7t42203H	Chr7：24 871 566 ~ 24 872 072	168	细胞核 Nucleus	ERF11（AT1G28370）
BolC7t41924H	Chr7：21 967 071 ~ 21 967 469	132	细胞核 Nucleus	ERF095（AT3G23220）
BolC7t41923H	Chr7：21 965 156 ~ 21 965 572	138	细胞核 Nucleus	ERF098（AT3G23230）
BolC6t38421H	Chr6：32 042 058 ~ 32 042 651	197	细胞质，细胞核Cytoplasm，nucleus	ERF018（AT1G74930）
BolC5t33888H	Chr5：46 627 650 ~ 46 628 303	217	细胞核 Nucleus	ERF4（AT3G15210）
BolC5t29217H	Chr5： 2 216 228 ~ 2 216 962	244	细胞核 Nucleus	ERF094（AT1G06160）
BolC4t28631H	Chr4：64 415 607 ~ 64 416 320	237	细胞核 Nucleus	ERF13（AT2G44840）
BolC4t23118H	Chr4：11 617 647 ~ 11 618 985	216	细胞核 Nucleus	ERF112（AT2G33710）
BolC3t20871H	Chr3：67 338 370 ~ 67 339 152	260	细胞核 Nucleus	ERF109（AT4G34410）
BolC3t19761H	Chr3：57 780 467 ~ 57 780 985	172	细胞核 Nucleus	ERF11（AT1G28370）
BolC3t17033H	Chr3：28 276 839 ~ 28 277 453	204	细胞核 Nucleus	ERF4（AT3G15210）
BolC3t14461H	Chr3：10 357 105 ~ 10 357 806	233	细胞核 Nucleus	ERF15（AT2G31230）
BolC3t13677H	Chr3： 5 792 930 ~ 5 793 562	210	细胞质，细胞核Cytoplasm，nucleus	ERF016（AT5G21960）
BolC2t10891H	Chr2：48 942 393 ~ 48 943 094	233	细胞核 Nucleus	ERF2（AT5G47220）
BolC2t10520H	Chr2：44 830 922 ~ 44 831 503	193	细胞核 Nucleus	ERF9（AT5G44210）
BolC2t09547H	Chr2：30 078 233 ~ 30 079 165	310	细胞核 Nucleus	RAP2-4（AT1G78080）
BolC2t06548H	Chr2： 3 356 340 ~ 3 358 302	254	细胞核 Nucleus	ERF113（AT5G13330）
BolC2t06200H	Chr2： 1 544 843 ~ 1 545 811	322	细胞核 Nucleus	DREB2A（AT5G05410）
BolC1t04894H	Chr1：44 469 287 ~ 44 469 943	218	细胞核 Nucleus	ERF4（AT3G15210）
BolC1t04027H	Chr1：36 457 782 ~ 36 459 026	212	细胞核 Nucleus	ERF098（AT3G23230）
BolC1t04025H	Chr1：36 421 156 ~ 36 421 788	210	细胞核 Nucleus	ERF1B（AT3G23240）
BolC1t01080H	Chr1： 6 205 090 ~ 6 205 785	231	细胞核 Nucleus	ERF1A（AT4G17500）
BolC1t01079H	Chr1： 6 195 373 ~ 6 196 152	259	细胞核 Nucleus	ERF6（AT4G17490）
BolC1t00081H	Chr1： 423 978 ~ 424 808	276	细胞核 Nucleus	ERF060（AT4G39780）