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作物学报 ›› 2025, Vol. 51 ›› Issue (3): 621-631.doi: 10.3724/SP.J.1006.2025.44120

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

芥菜型油菜响应菌核病侵染表达特性与高抗性关联基因分析

张金泽1(), 周庆国2, 杨旭1, 王倩1, 肖莉晶1, 金海润1, 欧阳青静1, 余坤江1, 田恩堂1,*()   

  1. 1贵州大学农学院, 贵州贵阳 550025
    2安徽鼎科种业有限责任公司, 安徽合肥 230001
  • 收稿日期:2024-07-25 接受日期:2024-10-25 出版日期:2025-03-12 网络出版日期:2024-11-13
  • 通讯作者: *田恩堂, E-mail: erictian121@163.com
  • 作者简介:E-mail: a3184349049@163.com
  • 基金资助:
    贵州省科技支撑计划项目(Guizhou Kehe Support [2022] Key 031);国家自然科学基金项目(32160483);国家自然科学基金项目(32360497);贵州大学国家自然科学基金项目([2023] 093);贵州省粮油作物分子育种重点实验室项目(Guizhou Kehezhong Yindi [2023] 008);贵州省高等学校功能农业重点实验室项目(Guizhou Jiaoji [2023] 007)

Analysis of genes associated with expression characteristics and high resistance in response to Sclerotinia sclerotiorum infection in Brassica juncea

ZHANG Jin-Ze1(), ZHOU Qing-Guo2, YANG Xu1, WANG Qian1, XIAO Li-Jing1, JIN Hai-Run1, OU-YANG Qing-Jing1, YU Kun-Jiang1, TIAN En-Tang1,*()   

  1. 1College of Agriculture, Guizhou University, Guiyang 550025, Guizhou, China
    2Anhui Dingke Seed Industry Co., Ltd., Hefei 230001, Anhui, China
  • Received:2024-07-25 Accepted:2024-10-25 Published:2025-03-12 Published online:2024-11-13
  • Contact: *E-mail: erictian121@163.com
  • Supported by:
    Guizhou Provincial Science and Technology Support Program(Guizhou Kehe Support [2022] Key 031);National Natural Science Foundation of China(32160483);National Natural Science Foundation of China(32360497);National Natural Science Foundation of Guizhou University([2023] 093);Guizhou Provincial Key Laboratory of Molecular Breeding of Grain and Oil Crops(Guizhou Kehezhong Yindi [2023] 008);Guizhou Provincial Key Laboratory of Functional Agriculture in Colleges and Universities(Guizhou Jiaoji [2023] 007)

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摘要：

菌核病是油菜的主要病害, 侵染后能导致油菜大幅减产。本研究对200份芥菜型油菜株系进行了菌核病抗性鉴定, 并从中筛选出部分高抗性育种材料。然后选取高抗株系G21-243-1 (HR)和感病株系G21-149-2 (LR)作为研究材料, 通过实验室模拟核盘菌侵染12 h、24 h、36 h后的叶片为材料进行转录组分析(RNA-Seq), 共获得138.16 Gb的Clean Data。以菌核病感病株系为对照, 以接种菌核病的同时期抗病株系为处理组, 进行差异基因表达分析后, 共检测到1899个上调表达基因, 1330个下调基因表达, 在2个时期同时检测到445个差异表达基因, 在3个时期同时检测到90个差异表达基因。对全部差异表达基因进行GO和KEGG功能分析, 在KEGG分析中显著富集的通路为Plant-pathogen interactions、Plant hormone signaling、MAPK signaling pathways-plants。结合转录组分析检测到的DEG及功能分析结果, 初步筛选出20个菌核病抗性相关的候选基因, 并随机选取其中6个候选基因进行了qRT-PCR分析。本研究的开展可为解析菌核病侵染寄生植物基因表达特性、菌核病抗性基因筛选及油菜的菌核病抗性育种奠定基础。

关键词: 芥菜型油菜, 菌核病, 转录组分析, 差异表达基因

Abstract:

Sclerotinia sclerotiorum is a major disease affecting rapeseed, often causing significant yield losses after infection. In this study, we evaluated the resistance of 200 Brassica juncea lines to S. sclerotiorum and selected one highly resistant line (G21-243-1, HR) and one susceptible line (G21-149-2, LR) for transcriptome analysis. A total of 138.16 Gb of clean data was generated by simulating leaf responses at 12, 24, and 36 hours post-infection (hpi) with S. sclerotiorum. The analysis identified 1899 up-regulated genes and 1330 down-regulated genes, with 445 differentially expressed genes (DEGs) detected across two time points and 90 DEGs across all three time points. Subsequent Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional analyses revealed significant enrichment in pathways related to plant-pathogen interactions, plant hormone signaling, and the MAPK signaling pathway in plants. By integrating transcriptome and functional analysis results, we preliminarily identified 20 candidate genes associated with resistance to S. sclerotiorum. Six of these genes were randomly selected for validation via qRT-PCR analysis. The findings of this study provide a foundation for future research on S. sclerotiorum, including the characterization of gene expression, the identification of resistance genes, and the development of resistant rapeseed varieties.

Key words: Brassica juncea, Sclerotinia sclerotiorum, transcriptome analysis, differentially expressed genes

表1

候选基因的qRT-PCR引物序列"

基因名称
Gene name		 正向引物
Forward primer (5'-3')		 反向引物
Reverse primer (5'-3')
BjuB043567 AAGGCTTCGACTTCTGGTGG GGCGCAAACGAGCATCTAAA
BjuB043588 GGTTGAGCAGAAAAGGTACGC AGCTGGAACTCTGCCTTTAAGA
BjuA023180 CGGAAACCCATACCCAACCA AGACGAGGCTCTGAGTCCAT
BjuA027474 TCCTAGAGACGGCGGAGAAA ATGCGACCGTCGTCTTCTAG
BjuA040356 CCTTCCGACCAACAGAACCA GCAAAACCAAACGGGCTCAT
BjuA024994 TCAGCTGCTGACAGTGACAG GCTCGAACAGGCCAAGTACT
Actin1 CCCTGGAATTGCTGACCGTA TGGAAAGTGCTGAGGGATGC

图1

200份芥菜型油菜菌核病抗性频率分布(A)及HR和LR的叶片菌核病抗性差异图(B和C) *** 表示在0.001水平差异显著。"

表2

测序数据统计与质量检测"

样本
Sample		 过滤后读长
Clean reads		 过滤后碱基
Clean bases		 匹配读长
Mapped reads		 匹配比率
Mapping rate (%)		 Q30碱基比例
Q30 bases rate (%)
HR12 21,049,953 6,304,265,677 39,172,284 93.04 93.56
HR24 20,890,365 6,255,809,943 38,776,834 93.02 93.50
HR36 21,801,040 6,529,517,940 40,687,767 93.31 94.05
LR12 21,522,196 6,444,737,219 39,803,989 92.48 93.88
LR24 20,142,620 6,032,972,157 37,585,078 92.98 93.69
LR36 20,359,340 6,097,753,385 37,899,609 93.08 93.68

图2

不同时间点DEG统计(A)和DEG韦恩图(B)"

图3

不同时期DEG的GO富集通路统计 Biological process、Molecular function富集数量前5的条目, Cellular component富集数量前3的条目。"

图4

不同时期DEG的KEGG显著富集图 DEG富集前10且3个时期共有的KEGG通路。"

表3

油菜菌核病抗性候选基因"

候选基因ID
Candidate
gene ID		 拟南芥同源基因信息
Information of Arabidopsis homologous genes		 基因功能
Gene function
BjuB043567 AT4G31800, 转录因子WRKY18
AT4G31800, transcription factor WRKY18		 调节防御相关基因的表达
Regulates the expression of defense-related genes
BjuB043588 AT4G30530, γ-谷氨酰肽酶GGP1
AT4G30530, γ-glutamyl peptidase GGP1		 硫苷和植保素的合成
Synthesis of glucosinolate and phytoprophylaxis
BjuA037008 AT1G12220, 抗病蛋白家族基因RPS5
AT1G12220, disease resistance protein family gene RPS5		 芥子油苷相关合成
Glucosinolin synthesis
BjuB032919 AT1G32640, 转录因子MYC2
AT1G32640, transcription factor MYC2		 茉莉酸介导的防御反应
Jasmonic acid-mediated defense responses
BjuB043577 AT4G31550, 转录因子WRKY11
AT4G31550, transcription factor WRKY11		 茉莉酸(JA)信号通路的激活
Activation of the jasmonic acid (JA) signaling pathways
BjuA024994 AT5G45510, (LRR)家族蛋白
AT5G45510, (LRR) family proteins		 细胞黏附和信号传递
Cell adhesion and signaling
BjuB023180 AT3G54030, 蛋白激酶蛋白BSK6
AT3G54030, protein kinase protein BSK6		 调节低R:FR反应基因的表达
Regulates the expression of low R:FR response genes
BjuB027474 AT2G01640, 核仁蛋白SAHY1
AT2G01640, nucleolar protein SAHY1		 核质之间穿梭转运
Shuttle transport between nucleoplasms
BjuB040356 AT4G25690, HLS家族 AT4G25690, HLS family 赤霉素相关合成 Gibberellin-related synthesis
BjuA017705 AT2G47140, 短链脱氢酶还原酶SDR5
AT2G47140, short-chain dehydrogenase reductase SDR5		 植物防御反应
Plant defense responses
BjuA028613 AT4G17390, 核糖体蛋白家族基因EL15Y
AT4G17390, ribosomal protein family gene EL15Y		 核糖体生物合成和蛋白质翻译
Ribosomal biosynthesis and protein translation
BjuA024292 AT5G66900, 抗病蛋白基因NRG1A
AT5G66900, the disease resistance protein gene NRG1A		 植物防御反应和细胞凋亡
Plant defense response and apoptosis
BjuB011722 AT5G59070, UDP-糖基转移酶超家族蛋白
AT5G59070, UDP-glycosyltransferase superfamily proteins		 转移酶活性, 转移糖基
Transferase activity, transfer glycans
BjuB041379 AT2G46450, 环核苷酸门控通道CNGC12
AT2G46450, cyclic nucleotide-gated channel CNGC12		 触发Ca2+信号转导
Triggers Ca2+ signaling
BjuA014448 AT3G62600, 结构域蛋白基因ERDJ3B
AT3G62600, domain protein gene ERDJ3B		 病原体受体信号通路
Pathogen receptor signaling pathways
BjuB033192 AT1G13060, 蛋白酶体基因PBE1
AT1G13060, the proteasome gene PBE1		 降解受损蛋白
Degradation of damaged proteins
BjuA022756 AT3G48000, 醛脱氢酶基因ALDH2
AT3G48000, aldehyde dehydrogenase gene ALDH2		 编码线粒体醛脱氢酶
Encodes mitochondrial aldehyde dehydrogenase
BjuA039403 AT3G01640, 葡萄糖醛酸激酶基因GLCAK
AT3G01640, glucuronide kinase gene GLCAK		 D-GlcA磷酸化为D-GlcA-1-磷酸
D-GlcA phosphorylation to D-GlcA-1-phosphate
BjuA022423 AT3G48950, 果胶裂解酶样超家族蛋白基因PGF7
AT3G48950, pectin lyase-like superfamily protein gene PGF7		 碳水化合物代谢过程
Carbohydrate metabolism processes
BjuO000727 AT1G65930, 异柠檬酸脱氢酶基因CICDH
AT1G65930, isocitrate dehydrogenase gene CICDH		 与防御反应相关的氧化还原信号传导
Redox signaling associated with defense responses

图5

qRT-PCR验证(A)和RNA-Seq分析结果(B) H1、H2和H3为抗病材料接种12、24和36 h, L1、L2和L3为感病材料接种12、24和36 h。"

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