花椰菜抗感品种响应黑腐病侵染的转录组分析及抗病基因挖掘

doi:10.7668/hbnxb.20195488

摘要/Abstract

摘要：

为探究花椰菜抗病品种与感病品种在接种黑腐病菌后的转录组差异,挖掘与花椰菜抗黑腐病相关的基因,以花椰菜感病品种Y1-2和抗病品种EC-247为研究对象,分别提取接种黑腐病菌0,1,3,5 d的花椰菜叶片总RNA,利用Illumina RNA-Seq测序平台进行高通量无参转录组测序,采用实时荧光定量PCR技术对部分差异表达基因进行验证,筛选抗病相关的差异基因并对其表达量进行分析。结果显示,4个时间点与感病品种相比,抗病品种共有6 355个基因表现出显著差异;KEGG富集分析发现,在这些差异表达基因富集通路中,与植物抗病性紧密相关的通路受到关注,其中47个基因与植物-病原菌互作相关,61个基因参与植物激素信号传导途径;对这些相关基因的表达量进行聚类分析发现,植物-病原菌互作途径中1个CDPK基因、4个CML基因、1个PTK基因、1个CaM基因、1个RLK基因和1个SGT1基因,植物激素信号传导途径中3个生长素响应蛋白基因、1个TIFY基因、1个吲哚-3-乙酸酰胺合成酶基因、2个油菜素唑抗性蛋白基因和1个Shaggy相关的蛋白激酶zeta基因,在抗病品种中的表达量显著高于感病品种,并且抗感品种不同时间点的表达模式表明其积极响应病原菌侵染。综上,这些差异基因可能与花椰菜抗病相关,为花椰菜抗病分子育种提供了重要的基因资源和科学依据。

关键词: 花椰菜, 黑腐病, 转录组, 差异表达基因, 抗病型, 敏感型

Abstract:

To investigate the transcriptome differences between resistant and susceptible varieties of cauliflower after inoculation with Xanthomonas campestris pv.campestris (Xcc),and to identify genes associated with cauliflower resistance to black rot disease,the susceptible variety Y1-2 and the resistant variety EC-247 of cauliflower were selected as the research subjects.Total RNA was extracted from cauliflower leaves at 0,1,3,and 5 days post-inoculation with Xcc,respectively.High-throughput parametric transcriptome sequencing was then conducted utilizing the Illumina RNA-Seq platform,followed by Real-time Quantitative PCR for validation of selected differentially expressed genes(DEGs).DEGs associated with disease resistance were screened and analyzed.The findings revealed that 6 355 genes exhibited significant differential expression between resistant and susceptible cultivars across the four time points.KEGG enrichment analysis focused on plant disease resistance pathways,identifying 47 genes involved in plant-pathogen interactions and 61 genes related to plant hormone signaling.Cluster analysis of these gene expression levels disclosed specific genes,including one CDPK,four CMLs,one PTK,one CaM,one RLK,and one SGT1 in the plant-pathogen interaction pathway,and three auxin-responsive protein genes,a TIFY gene,an indole-3-acetic acid-amido synthetase gene,two brassinazole-resistant protein genes,and a Shaggy-associated protein kinase zeta gene in the plant hormone signaling pathway.Notably,the expression of these genes was significantly higher in resistant varieties compared to susceptible ones,indicating their active response to pathogen infection at various time points.The results indicated that these differential genes might be related to disease resistance in cauliflower,which provided important genetic resources and scientific basis for molecular breeding of disease resistance in cauliflower.

Key words: Brassica oleracea L.var.botrytis, Xanthomonas campestris pv.campestris, Transcriptome, Differentially expressed genes, Disease-resistant type, Sensitive type

李子雨, 姚玉荣, 郝永娟, 霍建飞, 贲海燕, 王旭东, 王万立, 姚星伟, 李二峰. 花椰菜抗感品种响应黑腐病侵染的转录组分析及抗病基因挖掘[J]. 华北农学报, 2025, 40(2): 161-172. doi: 10.7668/hbnxb.20195488.

LI Ziyu, YAO Yurong, HAO Yongjuan, HUO Jianfei, BEN Haiyan, WANG Xudong, WANG Wanli, YAO Xingwei, LI Erfeng. Transcriptome Analysis and Resistance Gene Mining of Resistant and Susceptible Cauliflower Cultivars in Response to Black Rot Infection[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(2): 161-172. doi: 10.7668/hbnxb.20195488.

http://www.hbnxb.net/CN/Y2025/V40/I2/161

图/表 13

表1 qRT-PCR引物序列及片段大小

Tab.1 Primer sequence and fragment size of qRT-PCR

基因ID Gene ID	引物序列(5'—3') Primer sequence (5'—3')	退火温度/℃ Annealing temperature	片段大小/bp Product size
TRINITY_DN16231_c1_g2	F: AGATCGTTGGCTGGTTTGGT	60.0	166
	R: GGAGGGTGCTGCTGTTTGAA	62.0
TRINITY_DN16447_c0_g1	F: TGGACGAGTAGCCTTGAGAG	62.0	96
	R: GCTCCTATCTAGCCGCAC	58.0
TRINITY_DN11917_c3_g2	F: CTTTAGCCATCAAAGGCGGC	62.0	139
	R: GACTCCGGGGCTTTCCATTC	64.0
TRINITY_DN12966_c1_g11	F: GTTAGCACAAGCGCCTTCAC	62.0	131
	R: TCTTGTTCTGTGATTCTCTCCTT	54.8
TRINITY_DN10243_c0_g2	F: TAGCGTCGAAGGGAAACTGC	62.0	121
	R: GTCCGAATGAACAATACCGGAG	58.1
TRINITY_DN12323_c1_g1	F: AACCTGTGTGGGGAAAGAAGA	62.0	103
	R: AGAGTGAACCAACCATCTCCG	57.8
TRINITY_DN15781_c4_g7	F: CTCGTTTTTCTTCTCTCTCGCATC	58.6	132
	R: GAGGAGGTTGTTGTCGTTGGA	57.8
TRINITY_DN16687_c1_g5	F: CTCACATGCCCAGTTCCTCG	64.0	181
	R: CCCTAACTTTGCTTCGCCCG	64.0
TRINITY_DN15781_c4_g1	F: GATCACGATGCTCCAGTCGC	64.0	187
	R: TTGGTGCTCCTTCACTGCTC	62.0
ACTIN	F: GAGAGATAGATTTGTGTGTTGT	52.5	122
	R: AGATTAGTTGGAGTAGCAGAA	51.9
GAPDH	F: TCTTCACTCACTTGACTATTCT	52.5	136
	R: GATAACTCTCGCCACCAA	54.0

图1 花椰菜叶片接种表现

Fig.1 The appearance of cauliflower leaves inoculated with Xanthomonas campestris pv.campestris

表2 测序数据统计与质量评估

Tab.2 Sequencing data statistics and quality assessment

样本 Sample	过滤后数据 Clean reads	过滤后碱基数 Clean bases	Q30碱基质量/% Q30 bases quality	GC含量/% GC content	比对率/% Mapped ratio
EC-247-0 d A	43 154 426	6 456 548 603	93.77	47.57	77.66
EC-247-0 d B	43 974 850	6 577 996 221	94.29	47.40	77.31
EC-247-0 d C	50 699 770	7 592 056 814	94.35	47.64	78.14
EC-247-1 d A	42 988 736	6 413 315 635	94.96	47.20	77.34
EC-247-1 d B	52 500 002	7 835 972 173	94.77	47.65	78.11
EC-247-1 d C	48 391 930	7 234 185 005	95.00	47.70	77.97
EC-247-3 d A	51 191 174	7 646 822 724	94.78	47.54	77.79
EC-247-3 d B	45 770 458	6 842 511 433	95.15	47.70	78.39
EC-247-3 d C	49 606 438	7 407 208 299	95.20	47.55	77.32
EC-247-5 d A	53 245 644	7 954 157 908	94.78	47.73	78.35
EC-247-5 d B	55 391 982	8 273 179 902	95.15	47.80	78.92
EC-247-5 d C	49 013 112	7 290 224 189	93.63	47.73	76.77
Y1-2-0 d A	47 286 472	7 063 573 512	93.51	47.93	77.51
Y1-2-0 d B	47 117 450	7 050 441 980	94.41	47.75	78.20
Y1-2-0 d C	52 893 978	7 918 048 749	94.43	47.79	77.68
Y1-2-1 d A	53 443 280	8 005 561 235	94.68	47.73	78.00
Y1-2-1 d B	48 226 630	7 223 351 270	94.30	47.85	78.03
Y1-2-1 d C	56 712 154	8 467 526 725	95.16	47.73	78.29
Y1-2-3 d A	50 350 874	7 526 585 702	95.00	47.59	78.79
Y1-2-3 d B	46 535 650	6 955 289 009	94.72	47.73	78.26
Y1-2-3 d C	45 238 288	6 763 421 485	95.25	47.54	77.68
Y1-2-5 d A	49 279 840	7 359 206 999	95.16	47.25	78.63
Y1-2-5 d B	50 816 334	7 595 400 207	95.16	47.15	78.94
Y1-2-5 d C	49 402 268	7 370 757 559	94.93	47.45	78.76

图2 抗感花椰菜样本间聚类分析

Fig.2 The cluster analysis among resistant and susceptible cauliflower samples

图3 样品间PCA分析

Fig.3 PCA analysis between samples

图4 样本间表达量韦恩图

Fig.4 The Venn diagram of expression levels among samples

图5 4个时间点抗感品种差异表达基因韦恩图

Fig.5 Venn diagram of differentially expressed genes between resistant and susceptible varieties at four time points

图6 抗感品种4个时间点差异表达基因GO富集 1.细胞壁;2.外部包裹结构;3.膜的内在成分;4.膜的整合成分;5.细胞外区域;6.膜部分;7.果胶分解过程;8.ADP结合;9.多糖代谢过程;10.药物分解过程;11.细胞壁组织;12.细胞壁组织或生物合成;13.半乳糖醛酸代谢过程;14.果胶代谢过程;15.跨膜转运活性;16.作用于糖苷键的水解酶活性;17.多糖分解过程;18.外部包裹结构组织;19.水解O-糖苷化合物的水解酶活性;20.转运活性。

Fig.6 GO enrichment of differentially expressed genes between resistant and susceptible varieties at four time points 1.Cell wall;2.External encapsulating structure;3.Intrinsic component of membrane;4.Integral component of membrane;5.Extracellular region;6.Membrane part;7.Pectin catabolic process;8.ADP binding;9.Polysaccharide metabolic process;10.Drug catabolic process;11.Cell wall organization;12.Cell wall organization or biogenesis;13.Galacturonan metabolic process;14.Pectin metabolic process;15.Transmembrane transporter activity;16.Hydrolase activity,acting on glycosyl bonds;17.Polysaccharide catabolic process;18.External encapsulating structure organization;19.Hydrolase activity,hydrolyzing O-glycosyl compounds;20.Transporter activity.

图7 抗感品种4个时间点差异表达基因KEGG功能富集 1.苯丙烷类生物合成;2.戊糖和葡糖醛酸相互转化;3.二苯乙烯类、二芳基庚烷类和姜酚类生物合成;4.DNA复制;5.柠檬烯和萜品烯降解;6.α-亚麻酸代谢;7.淀粉和蔗糖代谢;8.角质、软木脂和蜡质生物合成;9.苯丙氨酸代谢;10.葡糖异硫氰酸盐生物合成;11.泛醌及其他萜类醌生物合成;12.同源重组;13.错配修复;14.亚油酸代谢。

Fig.7 KEGG functional enrichment of differentially expressed genes between resistant and susceptible varieties at four time point 1.Phenylpropanoid biosynthesis;2.Pentose and glucuronate interconversions;3.Stilbenoid,diarylheptanoid and gingerol biosynthesis;4.DNA replication;5.Limonene and pinene degradation;6.Alpha-linolenic acid metabolism;7.Starch and sucrose metabolism;8.Cutin,suberine and wax biosynthesis;9.Phenylalanine metabolism;10.Glucosinolate biosynthesis;11.Ubiquinone and other terpenoid-quinone biosynthesis;12.Homologous recombination;13.Mismatch repair;14.Linoleic acid metabolism.

图8 4个时间点抗感品种间共表达上下调差异基因KEGG功能富集 A:1.柠檬烯和萜品烯降解,2.芥子油苷生物合成,3.二苯乙烯类、二芳基庚烷类和姜酚类生物合成;B:1.苯丙烷类生物合成,2.戊糖和葡糖醛酸相互转化,3.DNA复制,4.淀粉和蔗糖代谢,5.α-亚麻酸代谢,6.同源重组,7.错配修复,8.氰基氨基酸代谢,9.黄酮类生物合成,10.角质、软木脂和蜡质生物合成,11.苯丙氨酸代谢。

Fig.8 KEGG functional enrichment of co-expressed up-and down-regulated differentially expressed genes between resistant and susceptible varieties at four time points A:1.Limonene and pinene degradation,2.Glucosinolate biosynthesis,3.Stilbenoid,diarylheptanoid and gingerol biosynthesis;B:1.Phenylpropanoid biosynthesis,2.Pentose and glucuronate interconversions,3.DNA replication,4.Starch and sucrose metabolism,5.Alpha-linolenic acid metabolism,6.Homologous recombination,7.Mismatch repair,8.Cyanoamino acid metabolism,9.Flavonoid biosynthesis,10.Cutin,suberine and wax biosynthesis,11.Phenylalanine metabolism.

图9 与植物-病原菌互作途径相关基因的表达分析

Fig.9 Expression analysis of genes related to plant-pathogen interaction pathways

图10 与植物激素信号传导途径相关基因的表达分析

Fig.10 Expression analysis of genes related to plant hormone signal transduction pathways

图11 9个差异表达基因的qRT-PCR 验证

Fig.11 qRT-PCR validation of nine differentially expressed genes

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