拟轮枝镰孢与玉米籽粒互作的差异基因筛选及代谢通路分析

doi:10.3864/j.issn.0578-1752.2023.06.006

摘要/Abstract

摘要：

【目的】拟轮枝镰孢（Fusarium verticillioides）引起的玉米穗腐病是我国玉米产区发生严重的病害之一，论文旨在了解病原菌与玉米籽粒互作过程中的基因表达差异，为揭示病原菌的致病机制和玉米抗病机制提供依据。【方法】对拟轮枝镰孢侵染玉米籽粒0、4、12和72 h的样品进行转录组测序，之后采用生物信息学分析，分别以玉米和拟轮枝镰孢基因组为参考，以|log₂FC|≥1，P-adjust<0.05为阈值筛选互作过程中玉米和拟轮枝镰孢的差异表达基因，利用GO和KEGG对其进行功能注释及富集分析。Goatools软件分析植物-病原互作、MAPK途径和植物激素信号转导通路相关差异基因的表达变化，采用实时荧光定量PCR（qRT-PCR）方法对测序差异基因进行验证。【结果】在互作4、12和72 h后拟轮枝镰孢分别有140、400和1 945个基因上调表达，有9、302和1 784个基因下调表达；玉米分别有293、692 和1 426个基因上调表达，320、482和153个基因下调表达。GO和KEGG富集分析显示，侵染早期拟轮枝镰孢在细胞间隙生长，差异基因主要富集在RNA生物合成、细胞壁结构成分、脂肪酸生物合成、蛋白质代谢、碳水化合物代谢、生物过程和代谢过程等通路中。拟轮枝镰孢早期侵染触发了玉米活性氧（ROS）爆发，差异基因主要富集在对活性氧、过氧化氢的反应，几丁质酶、单加氧酶活性，木质素代谢过程等相关通路中。侵染后期拟轮枝镰孢继续在籽粒中定殖及扩展，差异基因主要富集在碳水化合物和细胞壁多糖分解代谢过程、跨膜转运、氧化还原酶活性等功能通路中。玉米主要通过苯丙素、木质素、类黄酮生物合成，MAPK 信号通路、植物-病原互作和植物激素信号转导等途径差异基因大量表达响应病原菌侵染。随机选取6个玉米和6个拟轮枝镰孢的差异表达基因进行qRT-PCR分析，基因表达规律与转录组测序结果一致，证实了RNA-seq的准确性。【结论】在病原菌侵染早期，拟轮枝镰孢在细胞间隙生长，触发玉米活性氧爆发，相关通路差异基因表达；侵染中后期，病原菌以淀粉为营养素，继续在籽粒中定殖及扩展，玉米通过苯丙素、木质素及几丁质酶的生物合成等方面的相关基因表达响应拟轮枝镰孢侵染，同时植物-病原互作、MAPK途径和激素信号转导等途径参与抗拟轮枝镰孢侵染。

关键词: 拟轮枝镰孢, 玉米穗腐病, 转录组, 植物-病原互作, 基因表达, 差异表达基因

Abstract:

【Objective】 Maize ear rot caused by Fusarium verticillioides is one of the most serious diseases in maize producing areas in China. The objective of this study is to understand the differences in gene expression during the plant-pathogen interaction at different stages, and to provide a basis for pathogenic mechanism of the pathogen infection and resistance mechanism of maize. 【Method】 Illumina platform was used to sequence the transcriptome of maize kernels infected with F. verticillioides at 0, 4, 12, and 72 h. The differentially expressed genes (DEGs) of maize and F. verticillioides were screened with |log₂FC|≥1, P-adjust<0.05 as threshold and clean reads were compared with genome of maize and F. verticillioides, separately. Functional annotation and enrichment analysis of DEGs were carried out by using GO and KEGG databases. Goatools software was used to analyze the expression changes of genes related to plant-pathogen interaction, MAPK signaling pathway and plant hormone signal transduction pathway. Sequencing results were verified by quantitative real-time PCR (qRT-PCR). 【Result】 A total of 140, 400 and 1 945 DEGs were up-regulated and 9, 302, and 1 784 DEGs were down-regulated in F. verticillioides after 4, 12 and 72 h interaction, respectively. A total of 293, 692, and 1 426 DEGs were up-regulated and 320, 482, and 153 DEGs were down-regulated in maize after 4, 12 and 72 h interaction, respectively. GO and KEGG enrichment analysis of DEGs showed that F. verticillioides grew in intercellular space at the early stage of pathogen infection. The DEGs were enriched in RNA biosynthesis, cell wall structural component, fatty acid biosynthesis, protein metabolism, carbohydrate metabolism, biological process, and metabolic process. Reactive oxygen species (ROS) was triggered in maize at the early stage of infection. The DEGs were enriched in response to ROS, hydrogen peroxide, chitinase activity, monooxygenase activity, lignin metabolism. At the later stage of infection, F. verticillioides colonized and expanded in maize, and the DEGs were enriched in carbohydrate and cell wall polysaccharide catabolic process, transmembrane transport and oxidoreductase activity. Maize responded to pathogen infection through phenylpropanoid, lignin, flavonoid biosynthesis, MAPK signaling pathway, plant-pathogen interaction and plant hormone signal transduction. Six DEGs of maize and six DEGs of F. verticillioides were randomly selected for qRT-PCR. The results were consistent with those of transcriptome sequencing, which confirmed the accuracy of RNA-seq. 【Conclusion】 At the early stage of infection, F. verticillioides grew in the intercellular space, triggering ROS outbreak in maize and the expression of related pathway differential genes. At the middle and late stages of infection, the pathogen further colonized and expanded in maize with starch as nutrient. Maize responded to the infection of F. verticillioides through biosynthesis of phenylpropanoid, lignin and chitinase. Meanwhile, plant-pathogen interaction, MAPK signaling pathway, and plant hormone signal transduction were involved in the resistance to the infection of F. verticillioides.

Key words: Fusarium verticillioides, maize ear rot, transcriptome, plant-pathogen interaction, gene expression, differentially expressed gene (DEG)

渠清, 刘宁, 邹金鹏, 张雅璇, 贾慧, 孙蔓莉, 曹志艳, 董金皋. 拟轮枝镰孢与玉米籽粒互作的差异基因筛选及代谢通路分析[J]. 中国农业科学, 2023, 56(6): 1086-1101.

QU Qing, LIU Ning, ZOU JinPeng, ZHANG YaXuan, JIA Hui, SUN ManLi, CAO ZhiYan, DONG JinGao. Screening of Differential Genes and Analysis of Metabolic Pathways in the Interaction Between Fusarium verticillioides and Maize Kernels[J]. Scientia Agricultura Sinica, 2023, 56(6): 1086-1101.

图/表 12

表1

表2

图1

图2

表3

拟轮枝镰孢侵染玉米不同时间点差异表达基因的 GO富集分析"

侵染时间Infection time	GO ID	GO功能描述 GO description	功能类别 Term type	基因数量 Gene number	P value
4 h	GO:0032774	RNA生物合成过程RNA biosynthetic process	BP	9	0.042121302
	GO:0034248	细胞酰胺代谢过程的调控Regulation of cellular amide metabolic process	BP	2	0.017806275
	GO:0006417	调控翻译Regulation of translation	BP	2	0.016413092
	GO:0005198	结构分子活性Structural molecule activity	MF	5	0.023663705
	GO:0004553	水解酶活性，水解 O-糖基化合物 Hydrolase activity, hydrolyzing O-glycosyl compounds	MF	6	0.030782616
	GO:0016798	水解酶活性，作用于糖基键Hydrolase activity, acting on glycosyl bonds	MF	6	0.042898824
	GO:0005618	细胞壁Cell wall	CC	4	1.60E-06
	GO:0009277	真菌类型的细胞壁Fungal-type cell wall	CC	3	1.20E-05
	GO:0000786	核小体Nucleosome	CC	2	0.003814409
12 h	GO:0043043	肽生物合成过程Peptide biosynthetic process	BP	75	4.61E-07
	GO:0006518	肽代谢过程Peptide metabolic process	BP	75	5.47E-07
	GO:0019538	蛋白质代谢过程Protein metabolic process	BP	105	2.42E-06
	GO:0005975	碳水化合物代谢过程Carbohydrate metabolic process	BP	44	2.92E-06
	GO:0044238	初级代谢过程Primary metabolic process	BP	205	4.42E-06
	GO:0008150	生物过程Biological process	BP	359	4.94E-06
	GO:0008152	代谢过程Metabolic process	BP	263	5.73E-06
	GO:0005198	结构分子活性Structural molecule activity	MF	77	7.92E-07
	GO:0003735	核糖体的结构成分Structural constituent of ribosome	MF	71	9.56E-07
	GO:0016798	水解酶活性Hydrolase activity	MF	27	0.002644
	GO:0003723	RNA结合RNA binding	MF	35	0.000165
	GO:0032991	蛋白质复合物Protein-containing complex	CC	108	0.000515
	GO:0044464	细胞部分Cell part	CC	183	5.33E-05
72 h	GO:0005975	碳水化合物代谢过程Carbohydrate metabolic process	BP	177	2.74E-10
	GO:0044347	细胞壁多糖分解代谢过程Cell wall polysaccharide catabolic process	BP	13	0.000117
	GO:0055085	跨膜运输Transmembrane transport	BP	366	4.16E-10
	GO:0003824	催化活性Catalytic activity	MF	1430	4.38E-05
	GO:0022857	跨膜转运蛋白活性Transmembrane transporter activity	MF	239	2.10E-08
	GO:0016491	氧化还原酶活性Oxidoreductase activity	MF	466	5.60E-08
	GO:0005215	转运蛋白活性Transporter activity	MF	243	7.30E-08
	GO:0016021	膜的组成部分Integral component of membrane	CC	1080	1.25E-07
	GO:0044425	膜部分Membrane part	CC	1090	4.33E-08

表3

图3

表4

图4

图5

图6

图7

图8

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引物名称 Primer name	引物序列 Primer sequence (5′- 3′)
Zm00001d026018-F	AGTTTTCACCGATGTTGTTGG
Zm00001d 026018-R	TTCCTTGTTGTGTTTCTGCCC
Zm00001d 002540-F	TGCGGGGTTGAAGGAAATGT
Zm00001d 002540-R	TGCGGGGTTGAAGGAAATGT
Zm00001d 043025-F	ATGAAAAGGCTGCCACGAGA
Zm00001d 043025-R	GCTCCAGTATCTTCCCCTC
Zm00001d 022017-F	TGACCTCTGTACGCCTCTT
Zm00001d 022017-R	GGCTCTCCATTTCCTCTTTGTC
Zm00001d 043350-F	GCGGTTCTTGAAGTGGTTGCTC
Zm00001d 043350-R	CGAGGACAAGACCAGGAACT
Zm00001d 014250-F	GATGGGGTGGGAGGAGATGA
Zm00001d 014250-R	CCAGAAGAGGAAGGAGAGGAT
UBQ9-F	TACAGTTCTACAAGGTGGACGAC
UBQ9-R	GCAGTAGTGGCGGTCGAAGT
FVEG_06768-F	GTTATTTCCGCCGCCCTTTCA
FVEG_06768-R	GCCACCACCAGCCATACTCGT
FVEG_06181-F	GAGTCGCACAAAGAACAAGGA
FVEG_06181-R	TACAGAAGGATAGGGAAAGCA
FVEG_04041-F	CTTCACTCCCGACGACCCCAA
FVEG_04041-R	TATGCCCACATCAGAAACCAG
FVEG_06896-F	CTGACGCTTCTACTCCTCTCG
FVEG_06896-R	GGGTCTCAATGCCCTCAACTC
FVEG_07280-F	CCTGTTCTGATTGGTGGTAAG
FVEG_07280-R	CCATCGTTTTTGAGTGGTTCC
FVEG_09248-F	CTCAGCGGAACCACCACCCCT
FVEG_09248-R	ACCTCGGACAGCAACAGCACG
Actin-F	TGCTCCTGAGGCTCTCTTCCA
Actin-R	AAGCAAGAATAGAACCACCGA

文库 Library	原始测序数据的总条目数 Raw reads	质控后测序数据的总条目数 Clean reads	比对到玉米基因组的百分比 Total mapped rate (maize) (%)	比对到拟轮枝镰孢基因组的百分比 Total mapped rate (Fv) (%)	准确率在99.9%以上的碱基所占百分比 Q30 (%)
Mock_1	51587734	51110882	94.63	-	95.41
Mock_2	50799662	50311356	95.17	-	95.44
Mock_3	44878654	44387490	94.91	-	95.12
Fv_1	46302536	45922668	-	95.19	95.29
Fv_2	50383740	49966126	-	95.28	95.45
Fv_3	41064824	40694544	-	95.28	95.42
Fv4_1	102436676	101474514	95.21	0.05	95.06
Fv4_2	96101768	95247160	95.13	0.05	95.26
Fv4_3	84949414	83492592	94.76	0.04	93.59
Fv12_1	103162526	102019788	95.00	0.07	95.27
Fv12_2	107524620	106427588	95.46	0.05	95.37
Fv12_3	107586942	106395294	95.25	0.06	95.20
Fv72_1	108575700	107478816	93.31	2.27	95.27
Fv72_2	104267510	103103634	91.63	2.27	95.43
Fv72_3	82027324	81228748	92.25	3.21	95.30
-：未比对 Not blast

侵染时间 Infection time	GO ID	GO功能描述 GO description	功能类别 Term type	基因数量 Gene number	P value
4 h	GO:0042542	对过氧化氢的反应Response to hydrogen peroxide	BP	15	3.90E-11
	GO:0000302	对活性氧的反应Response to reactive oxygen species	BP	16	5.02E-11
	GO:0009628	对非生物刺激的反应Response to abiotic stimulus	BP	52	5.25E-11
	GO:0004497	单加氧酶活性Monooxygenase activity	MF	25	3.31E-08
	GO:0016491	氧化还原酶活性Oxidoreductase activity	MF	73	1.69E-06
	GO:0004568	几丁质酶活性Chitinase activity	MF	6	5.52E-05
	GO:0022857	跨膜转运蛋白活性Transmembrane transporter activity	MF	48	0.00040527
12 h	GO:0042542	对过氧化氢的反应Response to hydrogen peroxide	BP	15	5.02E-07
	GO:0009628	对非生物刺激的反应Response to abiotic stimulus	BP	85	2.20E-06
	GO:0009808	木质素代谢过程Lignin metabolic process	BP	6	0.005347
	GO:0004568	几丁质酶活性Chitinase activity	MF	13	0.002782
72 h	GO:0009699	苯丙素生物合成过程Phenylpropanoid biosynthetic process	BP	20	2.29E-07
	GO:0009809	木质素生物合成过程Lignin biosynthetic process	BP	13	2.60E-07
	GO:0009620	对真菌的反应Response to fungus	BP	22	3.56E-07
	GO:0072593	活性氧代谢过程Reactive oxygen species metabolic process	BP	29	6.85E-07
	GO:0009694	茉莉酸代谢过程Jasmonic acid metabolic process	BP	9	1.55E-05
	GO:0009696	水杨酸代谢过程Salicylic acid metabolic process	BP	5	5.05E-05
	GO:0008610	脂质生物合成过程Lipid biosynthetic process	BP	54	0.000134
	GO:0004568	几丁质酶活性Chitinase activity	MF	11	1.46E-06
	GO:0045548	苯丙氨酸解氨酶活性Phenylalanine ammonia-lyase activity	MF	7	3.87E-06
	GO:0004601	过氧化物酶活性Peroxidase activity	MF	27	1.50E-05