海岛棉与枯萎病菌的互作转录组分析

doi:10.13560/j.cnki.biotech.bull.1985.2022-1316

摘要/Abstract

摘要：

棉花枯萎病是棉花生产中常见的一种病害，目前在海岛棉中发病严重，直接影响海岛棉产量和品质，对海岛棉产业发展造成巨大威胁。为解析棉花与枯萎病菌尖孢镰刀菌互作的分子机制，以尖孢镰刀菌侵染48 h的抗感棉花根部组织及致病菌体为材料，利用RNA-seq测序技术分析尖孢镰刀菌与棉花互作的基因表达特性。结果表明，在抗感棉花品种中分别检测到15 218和9 358个差异基因，在侵染抗感棉花品种的尖孢镰刀菌中分别检测到3 708和3 656个差异基因。GO功能富集分析发现，互作后棉花中主要为氧化应激反应、生长素-激活信号通路、对刺激的反应、对受伤的反应和转录因子活性等基因功能；KEGG显著富集到内吞作用、植物激素信号转导、氨基酸生物合成、碳代谢、植物-病原菌互作、苯丙烷类生物合成等代谢途径，抗病品种在调控对刺激的反应和对受伤的反应中上调显著。GO功能富集分析发现，互作后尖孢镰刀菌中的差异表达基因多参与膜的组成部分、催化活性调节、ATP结合等类别；KEGG显著富集到过氧化物酶体，丝裂原活化蛋白激酶信号通路，缬氨酸、亮氨酸和异亮氨酸降解，甘氨酸、丝氨酸和苏氨酸代谢，碳代谢，氨基酸生物合成，淀粉和蔗糖代谢等通路。本研究为棉花响应枯萎病胁迫以及尖孢镰刀菌致病性研究提供了丰富的基因资源，为深入解析尖孢镰刀菌与棉花互作的机制研究奠定基础。

关键词: 尖孢镰刀菌, 棉花, 枯萎病, 植物-病原菌互作, RNA-seq, 差异表达基因

Abstract:

Cotton Fusarium wilt is a common disease in cotton production, especially very seriously in island cotton（Gossypium barbadense）, affecting its yield and quality, which pose a great threat to the development of island cotton industry. To understand the molecular mechanism of the interaction between cotton and Fusarium oxysporum, RNA-seq sequencing technology was applied to analyze the gene expression characteristics of the interaction between Fusarium oxysporum and cotton using the resistant and susceptible cotton root tissues and pathogenic bodies infected by Fusarium oxysporum for 48 h as materials. The results showed that 15 218 and 9 358 differentially expressed genes were detected in resistant and susceptible cotton varieties respectively, 3 708 and 3 656 differentially expressed genes were identified in Fusarium oxysporum after infecting the resistant and susceptible cotton varieties. Through GO enrichment analysis, we found that the main processes in cotton after interaction were oxidative stress, auxin-activated signaling pathway, response to stimulus, response to injury and transcription factor activity. KEGG pathways were significantly enriched in endocytosis, plant hormone signal transduction, amino acid biosynthesis, carbon metabolism, plant-pathogen interactions, phenylpropane biosynthesis and other metabolic pathways. Disease-resistant cultivars were significantly up-regulated in responses to stimulation and injury. In the Fusarium oxysporum after interaction, GO enrichment analysis found that differentially expressed genes were mostly involved in membrane components, catalytic activity regulation, ATP binding etc. KEGG was significantly enriched in peroxisome, mitogen-activated protein kinase signaling pathways, valine, leucine and L-Isoleucine degradation, glycine, serine and threonine metabolism, carbon metabolism, amino acid biosynthesis, starch and sucrose metabolism and other metabolic pathways. This study provids an abundant gene resources for studying the responses of cotton to Fusarium wilt and the pathogenicity of F. oxysporum, and laid a foundation for an in-depth analysis of the mechanism of interaction between Fusarium oxysporum and cotton.

Key words: Fusarium oxysporum, cotton, Fusarium wilt, plant-pathogen interactions, RNA-seq, differentially expressed genes

杨洋, 朱金成, 娄慧, 韩泽刚, 张薇. 海岛棉与枯萎病菌的互作转录组分析[J]. 生物技术通报, 2023, 39(6): 259-273.

YANG Yang, ZHU Jin-cheng, LOU Hui, HAN Ze-gang, ZHANG Wei. Transcriptome Analysis of Interaction Between Gossypium barbadense and Fusarium oxysporum f. sp. vasinfectum[J]. Biotechnology Bulletin, 2023, 39(6): 259-273.

图/表 11

表1 RT-qPCR所用的引物

Table 1 Primers for RT-qPCR

基因名称Gene name	上游引物 Forward primer（5'-3'）	下游引物 Reverse primer（5'-3'）
Gb2763	TTCCACAGTTCCAGGGTA	GATGATCCAAGGCTTTCT
Gb1474	AAACAAGGTAAGGAGGCT	GTTGGGTAAATAATAGGC
Gb7593	CAACTTGGCATCTTATCC	CTTACATTGAGCAGCAAC
Gb8190	CATGGAGCAAAGGTTTAA	TGTTGGATTCAGGGAGAT
Gb8191	CATGGAGCAAAGGTTTAA	TGTTGGATTCAGGGAGAT
Gb9261	TTTGCCAAAGAGCGTAGA	TTAAAGAATGCGGTGTCG
Gb9263	TTTCGGCAAGTCTATGAT	TCTTTCTGGTCGTGGATG
FOTG10073	GATGCCTGCCGTGGTGGAT	GGGCGAAGAAACAGTGTAAAGC
FOTG05109	TATCGCCCAGATGTTCAA	TACCACGGTGCTGTTCCA
FOTG15482	TTTCTTCATCGCCCTGTA	ACATTGCCGACTTGGATT
FOTG11725	TTGTTGGTTTGCCCTTGT	CGAGCGGTCTGAGTGTAG
FOTG17884	GCAATCTCAGCCTCAAGTT	GACAAGCGTTCATCCATAA
FOTG17663	GCCTTCTCGGATCTTCTA	GCAATGCTGGGATGGTAT
GhUBQ7	GAAGACCTACACCAAGCCCAA	CGGACTCTACTCAATCCCCACC

图1 样品间相关性检验 A：4个棉花根系组织样品的相关性检验，其中T14p为新海14号实验组样品，T42p为新海42号实验组样品，CKp14为新海14号对照组样品，CKp42为新海42号对照组样品；B：3个枯萎病菌样品的相关性检验，CKb为枯萎病菌对照组样品，T14b为新海14号枯萎病菌样品，T42b为新海42号枯萎病菌样品；a和b为两个重复

Fig. 1 Correlation analysis among samples A: Correlation analysis of four samples from cotton root tissues. T14p indicates the treatment sample from Xinhai 14, T42p indicates the treatment sample from Xinhai 42, CKp14 indicates the control sample from Xinhai 14 and CKp42 indicates the control sample from Xinhai 42. B: Correlation analysis of three samples from Fov. CKb refers to the control sample of Fov, T14b to the Fov sample obtained from Xinhai 14, T42b to the Fov sample obtained from Xinhai 42. a and b are the duplications

图2 RT-qPCR 验证RNA-seq 结果 A-C：RNA-seq表达量图；D和E：棉花中7个基因的表达模式；F和G：尖孢镰刀菌中6个基因的表达模式；CK为对照组；T为实验组；CKb为菌对照，T14为新海14实验组，T42为新海42号实验组。*表示有差异（P<0.05），**表示差异显著（P<0.01），***表示差异极显著（P<0.001）

Fig. 2 Validation of RNA-seq data by RT-qPCR A-C : RNA-seq expression. D and E : RT-qPCR verification of 7 genes in cotton. F and G : RT-qPCR verification of 6 genes in Fusarium oxysporum. CK: Control group. T: Treatment group. CKb: Bacteria control group. T14: Xinhai 14 treatment group.T42: Xinhai 42 treatment group. * indicates difference（P<0.05）, ** indicates significant difference（P<0.01）, and *** indicates extremely significant difference（P<0.001）

图3 不同组间棉花差异表达基因火山图 A：CK14_T14；B：CK42_T42。图中绿色为下调基因，红色为上调基因。下同

Fig. 3 Volcano map of differentially expressed genes in the cotton of different treatments Green is down-regulated gene and red is up- regulated gene. The same below

图4 不同组间尖孢镰刀菌差异表达基因火山图

Fig. 4 Volcano map of differentially expressed genes of Fusarium oxysporum among different treatments A：CKb_T14；B：CKb_T42

图5 新海14接菌后差异表达基因GO分析 A-C：上调基因的GO分析；D-F：下调基因的GO分析。横坐标为每个条目富集到的基因数，纵坐标为GO条目

Fig. 5 GO analysis of differentially expressed genes in Xinhai 14 after inoculation A-C: GO analysis of up-regulated genes. D-F: GO analysis of down-regulated genes. The abscissa is number of genes enriched for each term, and the ordinate is the term of GO level

图6 新海42接菌后差异表达基因GO分析 A-C：上调基因的GO分析；D-F：下调基因的GO分析。横坐标为每个条目富集到的基因数，纵坐标为GO条目

Fig. 6 GO analysis of differentially expressed genes in Xinhai 42 after inoculation A-C: GO analysis of up-regulated genes. D-F: GO analysis of down-regulated genes. The abscissa is number of genes enriched for each term,the ordinate is the term of GO level

图7 接菌后两个棉花品种中差异表达基因KEGG分析 A：新海14号上调基因的KEGG分析；B：新海14号下调基因的KEGG分析；C：新海42号上调基因的KEGG分析；D：新海42号下调基因的KEGG分析

Fig. 7 KEGG analysis of differentially expressed genes in two cotton cultivars after inoculation A: KEGG analysis of up-regulated genes in Xinhai 14. B: KEGG analysis of down-regulated genes in Xinhai 14. C: KEGG analysis of up-regulated genes in Xinhai 42. D: KEGG analysis of down-regulated genes in Xinhai 42

图8 尖孢镰刀菌侵染新海14后差异表达基因GO分析 A-C：新海14号尖孢镰刀菌中上调基因的GO分析；D-F：新海14号尖孢镰刀菌中下调基因的GO分析。横坐标为每个条目富集到的基因数，纵坐标为GO条目

Fig. 8 GO analysis of differentially expressed genes of Fov after infecting Xinhai 14 A-C: GO analysis of up-regulated genes of Fov in Xinhai 14. D-F: GO analysis of down-regulated genes of Fov in Xinhai 14. The abscissa is number of genes enriched for each term, the ordinate is the term of GO level

图9 尖孢镰刀菌侵染新海14和新海42后差异表达基因KEGG分析 A：新海14号中尖孢镰刀菌上调基因的KEGG分析；B：新海14号中尖孢镰刀菌下调基因的KEGG分析；C：新海42号中尖孢镰刀菌上调基因的KEGG分析；D：新海42号中尖孢镰刀菌下调基因的KEGG分析

Fig. 9 KEGG analysis of differentially expressed genes of Fov after infecting Xinhai 14 and Xinhai 42 A: KEGG analysis of up-regulated genes of Fov（Fusarium oxysporum f. sp. Vasinfectum）in Xinhai 14. B: KEGG analysis of down-regulated genes of Fov in Xinhai 14. C: KEGG analysis of up-regulated genes of Fov in Xinhai 42. D: KEGG analysis of down-regulated genes of Fov in Xinhai 42

图10 尖孢镰刀菌侵染新海42后差异表达基因GO分析 A-C：新海42号尖孢镰刀菌中上调基因的GO分析；D-F：新海42号尖孢镰刀菌中下调基因的GO分析。横坐标为每个条目富集到的基因数，纵坐标为GO条目

Fig. 10 GO analysis of differentially expressed genes of Fov after infecting Xinhai 42 A-C: GO analysis of up-regulated genes of Fov in Xinhai 42. D-F: GO analysis of down-regulated genes of Fov in Xinhai 42. The abscissa is number of genes enriched for each term, the ordinate is the term of GO level

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