蚕豆响应交链格孢侵染的转录组分析及VfPR4的抗病功能验证

doi:10.3864/j.issn.0578-1752.2025.22.008

摘要/Abstract

摘要：

【目的】叶斑病是限制蚕豆生产的一种新型真菌病害，交链格孢（Alternaria alternata）是其主要病原菌之一。本研究通过转录组分析和抗病基因功能验证，探讨蚕豆响应交链格孢侵染的分子途径。【方法】以蚕豆品种CD-006为试验材料，对接种交链格孢后0、6、12和24 h的蚕豆叶片进行转录组测序，筛选差异表达基因（differentially expressed gene，DEG），并对其进行GO（gene ontology）功能注释和KEGG（Kyoto encyclopedia of genes and genomes）代谢通路富集分析，从而挖掘抗病相关基因。采用qRT-PCR（quantitative real-time PCR）验证转录组测序结果，从中筛选出VfPR4进行烟草的遗传转化，并通过有伤接种法完成VfPR4的抗病功能分析。【结果】侵染后6、12和24 h，蚕豆中分别有3 537、3 152和2 947个差异基因上调表达，分别有1 181、1 453和1 319个差异基因下调表达。GO分析发现差异基因主要富集于氧化还原酶活性和一些生物合成（代谢）过程，KEGG分析发现差异基因主要富集于次生代谢物的生物合成、苯丙烷生物合成、类黄酮生物合成等途径。侵染后6 h，KEGG还富集于MAPK信号途径和植物-病原菌互作途径，这2条抗病通路共同富集了32个差异基因。转录组分析发现52个PR，其中35个基因差异表达。选取6个差异基因进行qRT-PCR验证，验证结果与转录组测序结果一致，其中VfPR4在交链格孢侵染后24 h内持续上调表达。烟草遗传转化结果表明，转基因烟草的病斑症状较轻，病斑面积分别为128.94、110.57和92.92 mm²，明显小于野生型病斑面积（174.32 mm²）。【结论】通过转录组分析，发现蚕豆可能主要通过氧化爆发、次生代谢物生物合成、苯丙烷生物合成、MAPK信号途径和植物-病原菌互作等途径响应交链格孢的侵染。VfPR4能够正向调控烟草对交链格孢的抗性。

关键词: 蚕豆, 交链格孢, 转录组, VfPR4, 抗病功能

Abstract:

【Objective】Leaf spot is a new type of fungal disease that restricts the production of broad beans, and Alternaria alternata is one of its main pathogens. The objective of this study is to explore the molecular pathways of broad beans in response to the infection of A. alternata through transcriptome analysis and disease resistant gene function verification. 【Method】Broad bean cultivar CD-006 was used as the experimental material, transcriptome sequencing was performed on the leaves inoculated with A. alternata at 0, 6, 12 and 24 h to screen differentially expressed genes (DEGs). GO (gene ontology) functional annotation and KEGG (Kyoto encyclopedia of genes and genomes) metabolic pathway enrichment analysis were conducted to excavate disease resistance related genes. qRT-PCR (quantitative real-time PCR) was conducted to validate transcriptome sequencing results, and VfPR4 was screened for genetic transformation in tobacco. To complete disease resistance function analysis of VfPR4, invasive inoculation method was used. 【Result】At 6, 12 and 24 h after infection, 3 537, 3 152 and 2 947 DEGs were up-regulated in broad beans, while 1 181, 1 453 and 1 319 DEGs were down-regulated. GO enrichment analysis revealed that DEGs were mainly enriched in oxidoreductase activity and some biosynthetic (metabolic) processes, and KEGG enrichment analysis found that DEGs were mainly enriched in pathways such as biosynthesis of secondary metabolites, phenylpropanoid biosynthesis, and flavonoid biosynthesis. At 6 h after infection, KEGG was also enriched in MAPK (mitogen-activated protein kinase) signaling pathway and plant-pathogen interaction pathway, which together enriched 32 DEGs. A total of 52 PR genes were discovered in transcriptome analysis, among which 35 genes were differentially expressed. Six DEGs were selected for qRT-PCR validation, and the validation results were consistent with the transcriptome sequencing results. Among the above genes, the VfPR4 remained up-regulated within 24 h after infection with A. alternata. Tobacco genetic transformation results indicated that the lesion symptoms of transgenic lines were milder, with lesion areas of 128.94, 110.57 and 92.92 mm², respectively, which were significantly smaller than that of the wild type, with a lesion area of 174.32 mm². 【Conclusion】In this study, transcriptome analysis revealed that broad beans might mainly respond to the infection of A. alternata through pathways including oxidative burst, secondary metabolites biosynthesis, phenylpropanoid biosynthesis, MAPK signaling, and plant-pathogen interaction. VfPR4 could positively regulate tobacco’s resistance to A. alternata.

Key words: broad bean (Vicia faba), Alternaria alternata, transcriptome, VfPR4, disease resistance function

王凡, 刘陈玮, 陆红臣, 徐仁超, 卞晓春. 蚕豆响应交链格孢侵染的转录组分析及VfPR4的抗病功能验证[J]. 中国农业科学, 2025, 58(22): 4656-4672.

WANG Fan, LIU ChenWei, LU HongChen, XU RenChao, BIAN XiaoChun. Transcriptome Analysis of Vicia faba Response to Alternaria alternata Infection and Validation of the Disease Resistance Function of VfPR4[J]. Scientia Agricultura Sinica, 2025, 58(22): 4656-4672.

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基因Gene	引物序列Primer sequence (5′-3′)	扩增效率Efficiency (%)
qRT-PCR
Vfaba.Tiffany.R1.1g286840	F: GGTTTGGGGAGTGGGTCG	106.73
Vfaba.Tiffany.R1.1g286840	R: TAGAAGCCTCAGAAGCCGTAGC	106.73
Vfaba.Tiffany.R1.3g068200 (VfPR4)	F: CCTTTTGTGGACCCGTTGG	99.07
Vfaba.Tiffany.R1.3g068200 (VfPR4)	R: GTGACCCGCCTGGTAGCC	99.07
Vfaba.Tiffany.R1.4g083240	F: GTTTTGGATGACCCCACAGG	102.75
Vfaba.Tiffany.R1.4g083240	R: CGCCATTGATTATGTTGGTGAT	102.75
Vfaba.Tiffany.R1.1g351200	F: AGTTGAGAAGATTTCATTTGAGGCT	99.43
Vfaba.Tiffany.R1.1g351200	R: ATCAGGATGAGCAATACAGTAACCC	99.43
Vfaba.Tiffany.R1.1g183400	F: CATTTTGCCACTACAGACTCTAAGG	90.74
Vfaba.Tiffany.R1.1g183400	R: TGGAGGAGAAACCGTTGTCAG	90.74
Vfaba.Tiffany.R1.6g134000	F: CAGCAAAGAAACAGGTTGAAAAG	98.66
Vfaba.Tiffany.R1.6g134000	R: CACACTTGTCATCACAGTTACCTCA	98.66
VfELF1A	F: GTGAAGCCCGGTATGCTTGT	106.45
VfELF1A	R: CTTGAGATCCTTGACTGCAACATT	106.45
NtActin	F: CCTGAGGTCCTTTTCCAACCA	93.24
NtActin	R: GGATTCCGGCAGCTTCCATT	93.24
基因克隆Gene cloning
VfPR4	F: ATGGAGAGCACACAGAGAAGTTTAAC
VfPR4	R: TTAGTCACCGCAATTAACAAAGACGT
载体构建Vector construction
VfPR4	F: AGAACACGGGGGACGAGCTCATGGAGAGCACACAGAGAAGTTTAAC
VfPR4	R: ACCATGGTGTCGACTCTAGAGTCACCGCAATTAACAAAGACGTAG

样本 Sample	原始数据 Raw reads	过滤后数据 Clean reads	原始碱基 Raw bases	过滤后碱基 Clean bases	Q20 (%)	Q30 (%)	GC (%)
CK-1	44713886	44544524	6707082900	6649756373	97.21	92.37	43.04
CK-2	37108856	37009168	5566328400	5528310812	97.01	92.01	43.02
CK-3	38092376	37994204	5713856400	5674495947	97.32	92.52	43.08
T6-1	41623938	41522300	6243590700	6203365542	97.19	92.36	42.81
T6-2	50863996	50705276	7629599400	7545767138	98.54	95.56	43.20
T6-3	49319542	49134708	7397931300	7333695696	97.51	93.00	43.00
T12-1	44038582	43868258	6605787300	6563061275	97.26	92.50	42.86
T12-2	38431570	38286054	5764735500	5723593977	97.22	92.45	43.02
T12-3	45024378	44864168	6753656700	6702567067	97.27	92.43	42.88
T24-1	45809802	45609948	6871470300	6807440098	97.47	92.84	42.98
T24-2	45742880	45613266	6861432000	6807387734	98.41	95.15	43.21
T24-3	48451546	48287776	7267731900	7192462021	98.61	95.66	43.24

样本 Sample	过滤后数据 Clean reads	比对到基因组的数据及占比 Total mapped (%)	比对到基因组唯一位置的数据及占比 Unique mapped (%)
CK-1	44092194	39123402 (88.73)	36596547 (83.00)
CK-2	36492758	32535363 (89.16)	30511820 (83.61)
CK-3	37415614	33617214 (89.85)	31573700 (84.39)
T6-1	40849472	36455382 (89.24)	34177712 (83.67)
T6-2	49378582	43572672 (88.24)	40509613 (82.04)
T6-3	48266952	42929051 (88.94)	40007796 (82.89)
T12-1	43210162	38477310 (89.05)	36178498 (83.73)
T12-2	37742640	33652558 (89.16)	31555095 (83.61)
T12-3	44271762	39224804 (88.60)	36739592 (82.99)
T24-1	44969738	39722019 (88.33)	37244088 (82.82)
T24-2	45022382	39229015 (87.13)	36940861 (82.05)
T24-3	47568146	41713080 (87.69)	39012843 (82.01)

样本 Sample	比对到外显子区域的比例 Proportion of comparison to exon regions (%)	比对到内含子区域的比例 Proportion of comparison to intron regions (%)	比对到基因间区域的比例 Proportion of comparison to intergenic regions (%)
CK-1	83.46	4.44	12.10
CK-2	83.75	4.27	11.98
CK-3	84.03	4.10	11.88
T6-1	83.30	4.50	12.20
T6-2	83.35	4.45	12.20
T6-3	83.52	4.37	12.11
T12-1	82.88	4.63	12.49
T12-2	83.15	4.39	12.45
T12-3	83.12	4.58	12.31
T24-1	82.19	4.76	13.05
T24-2	82.11	4.65	13.24
T24-3	81.98	4.71	13.31

基因编号 Gene ID	基因注释 Gene annotation	表达水平Expression level (FPKM)
基因编号 Gene ID	基因注释 Gene annotation	0 h	6 h	12 h	24 h
Vfaba.Tiffany.R1.3g072280	RBOHB（MAPK信号途径MAPK signaling pathway、植物-病原菌互作Plant-pathogen interaction）	0.131	0.163	0.607	1.807
Vfaba.Tiffany.R1.1g350240	PR1（MAPK信号途径MAPK signaling pathway、植物-病原菌互作Plant-pathogen interaction）	0.663	18.979	23.724	24.035
Vfaba.Tiffany.R1.1g351200	PR10	2.033	140.937	252.342	426.230
Vfaba.Tiffany.R1.1g351320	PR10	0.077	0.850	2.324	27.403
Vfaba.Tiffany.R1.1g351400	PR10	1.868	17.391	67.293	82.358
Vfaba.Tiffany.R1.3g068200	PR4	1.363	48.051	253.160	530.807