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作物学报 ›› 2024, Vol. 50 ›› Issue (4): 1080-1090.doi: 10.3724/SP.J.1006.2024.31053

• 研究简报 • 上一篇    

基于转录组探究外源水杨酸对条锈菌侵染小麦幼苗的缓解效应及差异表达基因分析

齐学礼1(), 李莹2(), 李春盈3, 韩留鹏1, 赵明忠1, 张建周3,*()   

  1. 1河南省作物分子育种研究院, 河南郑州 450002
    2河南农业大学学报编辑部, 河南郑州 450002
    3河南省农业科学院小麦研究所, 河南郑州 450002
  • 收稿日期:2023-09-17 接受日期:2024-01-12 出版日期:2024-04-12 网络出版日期:2024-02-23
  • 通讯作者: * 张建周, E-mail: zhangjianzhou00@163.com
  • 作者简介:齐学礼, E-mail: xueliqi888@163.com;
    李莹, E-mail: liying1233@163.com
    **同等贡献
  • 基金资助:
    财政部和农业农村部国家现代农业产业技术体系建设专项(小麦, CARS-3-7);河南省农业科学院自主创新项目(2022ZC03)

Alleviative effect of salicylic acid on wheat seedlings with stripe rust based on transcriptome and differentially expressed genes

QI Xue-Li1(), LI Ying2(), LI Chun-Ying3, HAN Liu-Peng1, ZHAO Ming-Zhong1, ZHANG Jian-Zhou3,*()   

  1. 1Crops Molecular Breeding Academy of Henan, Zhengzhou 450002, Henan, China
    2Editorial Department of Journal of Henan Agricultural University, Zhengzhou 450002, Henan, China
    3Wheat Research Institute, Henan Academy of Agricultural Sciences, Zhengzhou 450002, Henan, China
  • Received:2023-09-17 Accepted:2024-01-12 Published:2024-04-12 Published online:2024-02-23
  • Contact: * E-mail: zhangjianzhou00@163.com
  • About author:**Contributed equally to this work
  • Supported by:
    China Agriculture Research System of MOF and MARA (Wheat, CARS-3-7);Independent Innovation Project of HAAS(2022ZC03)

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摘要：

为探究外源水杨酸提高小麦条锈病抗性的作用机制, 本文将周麦18分为未处理的小麦幼苗(对照)、条锈菌侵染的小麦幼苗(SR)和水杨酸处理条锈菌侵染的小麦幼苗(SA-SR), 对处理15 d后的小麦幼苗进行抗病性鉴定、氨基酸含量检测、转录组测序及荧光定量分析鉴定。结果表明: 外源水杨酸能够显著降低条锈菌对小麦幼苗的致病力; SR组叶片中氨基酸含量普遍降低, 而经过水杨酸预先处理的SA-SR组与Control无显著差异, 说明小麦条锈病造成叶片中氨基酸的含量显著降低; 通过GO富集分析发现, SR和SA-SR组的差异基因在与光合作用相关的生物过程中富集, 涉及的细胞组分包括叶绿体和类囊体等, KEGG通路富集分析显示, SR和SA-SR两个分组均在MAPK信号通路和次生代谢物的生物合成中显著富集, 在植物的抗病或抗逆中发挥重要作用。

关键词: 小麦条锈病, 水杨酸, 氨基酸, 转录组

Abstract:

In order to explore the mechanism of exogenous salicylic acid in improving the resistance of wheat stripe rust, using, the untreated wheat seedlings (control), stripe rust wheat seedlings (SR), and salicylic acid treated stripe rust wheat seedlings (SA-SR) as the experimental materials were investigated using Zhoumai 18. After 15 days of treatment, the wheat seedlings were identified for disease resistance, amino acid detection, transcriptome sequencing, and fluorescence quantification. The results indicated that the exogenous salicylic acid could significantly reduce the pathogenicity of stripe rust in wheat seedlings. The content of amino acids in leaves in the SR group was generally reduced, while there was no significant difference between the SA-SR group and the control group pretreated with salicylic acid, indicating that the content of amino acids in leaves was significantly reduced due to wheat stripe rust. GO enrichment indicated that the differentially expressed genes of SR and SA-SR groups were enriched in biological processes related to photosynthesis involving cell components such as chloroplasts and thylakoids. KEGG pathway enrichment showed that both SR and SA-SR subgroups were significantly enriched in the MAPK signaling pathway and the biosynthesis of secondary metabolites played an important role in plant disease resistance or stress resistance.

Key words: wheat stripe rust, salicylic acid, amino acid, transcriptom

图1

小麦苗期条锈病反应型鉴定标准(0~9级)"

表1

实时荧光定量PCR所用引物"

基因名称
Gene ID		 正向引物
Forward sequence (5'-3')		 反向引物
Reverse sequence (5'-3')
TraesCS2D02G433300 CGCCGTGCGTGAGTAATTC CGTCGGGTTCGACACTCTTA
TraesCS2D02G014800 CGACCTCAACTCCAACTCCATA ACGGATTCCCCTCTCCGAA
TraesCS6B02G078400 TGGGAGCAATACTTCGCCTG ATAGCCCATCACCCCCGTAT
TraesCS1B02G038100 AGTTAGACCAAATGGCCCCTC CCCCCTGAGGTATAGACCGA
TraesCS5D02G099000 AAGCCTTTGCTGTCCTACGG AATGGGCCTGTAATCGGTGG
TraesCS2A02G378900 CCAGTCGGAGCACGTTATCA GATGGTGCCGTAGATCCGTT
TraesCS2D02G375200 TCCATTGTAGGCTTGCTGGA CCGGGTGATAACCCACAAGTA
TraesCS5D02G531600 CAGCCCTTTATAGGCCCACG TAGGCCGAAAGCTAACACGG
TraesCS1D02G290100 CGACGCCTATAGCAAACGGA GCCTGAAGCCAACGAAACTC
TraesCS1B02G011100 CGTATGCGCTATTCGTTGGC GGCGAGGATTCAGGAACTGT

图2

SR组和SA-SR组对CYR32的反应情况"

表2

3个分组中小麦叶片水解氨基酸含量"

氨基酸
Amino acid		 对照Control SR SA-SR
C_1 C_2 C_3 SR_1 SR_2 SR_3 SA-SR_1 SA-SR_2 SA-SR_3
天冬氨酸Asp 3.302 3.421 3.411 2.659 2.723 2.502 3.761 3.613 3.559
谷氨酸Glu 6.076 5.078 5.295 5.492 5.333 5.880 5.878 5.295 6.492
丝氨酸Ser 1.213 1.473 1.354 1.022 0.988 0.813 1.073 1.054 1.122
组氨酸His* 0.574 0.525 0.578 0.571 0.501 0.402 0.525 0.578 0.671
甘氨酸Gly 1.500 1.621 1.548 1.447 1.413 1.500 1.521 1.548 1.747
苏氨酸Thr* 0.664 0.617 0.649 0.512 0.551 0.643 0.617 0.649 0.670
精氨酸Arg 3.233 3.701 3.623 2.612 2.813 2.233 2.791 2.820 3.609
丙氨酸Ala 1.063 1.111 1.262 1.200 0.961 1.063 1.011 0.962 1.212
酪氨酸Tyr 0.771 0.752 0.729 0.831 0.689 0.771 0.652 0.679 0.831
胱氨酸Cys-s 0.115 0.112 0.099 0.029 0.016 0.109 0.120 0.199 0.129
缬氨酸Val* 1.153 1.011 1.121 1.119 1.115 1.153 1.011 1.121 1.319
甲硫氨酸Met* 0.169 0.155 0.177 0.128 0.134 0.129 0.155 0.157 0.188
苯丙氨酸Phe* 1.378 1.216 1.313 1.209 1.008 1.018 1.216 1.113 1.209
异亮氨酸Ile* 0.984 0.851 0.894 0.727 0.696 0.683 0.851 0.894 0.827
亮氨酸Leu* 1.645 1.555 1.599 1.360 1.402 1.345 1.519 1.581 1.580
赖氨酸Lys* 0.803 0.792 0.832 0.860 0.803 0.703 0.850 0.842 1.060
脯氨酸Pro 0.912 0.902 0.927 0.878 0.820 0.790 0.960 0.938 1.268
总量 Total 24.118 24.626 24.113 21.772 19.713 20.346 24.236 22.765 26.349

图3

3个分组中小麦叶片17种水解氨基酸含量差异比较"

表3

转录组数据统计表"

样品
SA sample		 原始序列数
Raw reads		 过滤后序列数
Clean reads		 碱基错误率
Error rate (%)		 GC含量
GC content (%)		 比对率
Total mapped
C_1 96129064 95045666 0.02 55.57 92039992 (96.84%)
C_2 89832494 88505872 0.02 55.44 85551890 (96.66%)
C_3 79838786 78959024 0.02 55.61 76422986 (96.79%)
SR_1 97621350 96607070 0.02 55.67 93194776 (96.47%)
SR_2 96303100 95196330 0.02 55.72 91799894 (96.43%)
SR_3 95914068 94943432 0.02 55.72 91563514 (96.44%)
SA-SR_1 101359676 100284658 0.02 54.67 96353104 (96.08%)
SA-SR_2 109261866 108220880 0.02 54.75 104057256 (96.15%)
SA-SR_3 93098314 92227658 0.02 54.71 88651196 (96.12%)

图4

不同样本之间的转录组测序数据相关性分析 a: 组内和组间皮尔逊相关性分析; b: 组间基因表达量PCA分析。"

图5

差异表达基因火山图 a: SR与对照; b: SR与SA-SR."

图6

不同组合中DEGs分析 a: 共有差异基因聚类情况; b: SA-SR和SR、SR和对照比较组之间差异表达基因的韦恩图。"

表4

SA-SR vs SR前10个显著富集的GO term"

GO条目
GO_accession		 注释描述
Description		 P值
P-value		 上调
Up		 下调
Down		 类别
Term_type
GO:0009536 质体Plastid 2.28E-95 44 681 细胞组分Cell component
GO:0009507 叶绿体Chloroplast 2.03E-91 40 663 细胞组分Cell component
GO:0009579 类囊体Thylakoid 3.11E-61 34 362 细胞组分Cell component
GO:0042651 类囊体膜Thylakoid membrane 5.71E-56 14 222 细胞组分Cell component
GO:0015979 光合作用Photosynthesis 3.09E-51 34 263 生物过程Cell component
GO:0034357 光合作用膜Photosynthetic membrane 2.99E-49 31 265 细胞组分Cell component
GO:0009534 叶绿体类囊体Chloroplast thylakoid 3.60E-47 8 200 细胞组分Cell component
GO:0031976 质体类囊体Plastid thylakoid 3.60E-47 8 200 细胞组分Cell component
GO:0009532 质体基质Plastid stroma 1.56E-42 6 215 细胞组分Cell component
GO:0009535 叶绿体类囊体膜Chloroplast thylakoid membrane 2.67E-42 8 172 细胞组分Cell component

表5

SR与对照分组前10个显著富集的GO term"

GO条目
GO_accession		 注释描述
Description		 P值
P-value		 上调
Up		 下调
Down		 类别
Type
GO:0009800 肉桂酸生物合成过程
Cinnamic acid biosynthetic process		 1.19E-70 35 0 生物过程
Cell bioprocess
GO:0009803 肉桂酸代谢过程
Cinnamic acid metabolic process		 1.19E-70 35 0 生物过程
Cell bioprocess
GO:0045548 苯丙氨酸解氨酶活性
Phenylalanine ammonia-lyase activity		 1.19E-70 35 0 分子功能
Molecular function
GO:0016301 激酶活性
Kinase activity		 1.19E-66 1260 263 分子功能
Molecular function
GO:0006468 蛋白质磷酸化
Protein phosphorylation		 9.25E-66 1163 226 生物过程
Molecular function
GO:0004672 蛋白激酶活性
Protein kinase activity		 1.48E-65 1163 230 分子功能
Molecular function
GO:0016773 磷酸转移酶活性, 醇基作为受体 4.14E-65 1230 247 分子功能
Molecular function
GO:0016310 磷酸化
Phosphotransferase activity, alcohol group as acceptor		 1.76E-58 1208 249 生物过程
Cell bioprocess
GO:0006796 含磷酸盐化合物代谢过程
Phosphate-containing compound metabolic process		 9.77E-56 1399 361 生物过程
Cell bioprocess
GO:0006793 磷代谢过程
Phosphorus metabolic process		 2.37E-55 1401 361 生物过程
Cell bioprocess

图7

不同组合中差异表达基因的GO富集分析"

图8

两个比较组合差异基因的KEGG通路 a: SA-SR vs SR差异表达基因的KEGG通路富集分析; b: SR与对照分组差异表达基因的KEGG通路富集分析。"

图9

RNA-seq和qRT-PCR的spearman相关性分析"

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