腐皮镰刀菌侵染甘薯的转录组分析

doi:10.3969/j.issn.1004-1524.2023.05.13

浙江农业学报 ›› 2023, Vol. 35 ›› Issue (5): 1097-1107.DOI: 10.3969/j.issn.1004-1524.2023.05.13

腐皮镰刀菌侵染甘薯的转录组分析

罗勤川(), 唐伟, 马居奎, 陈晶伟, 杨冬静, 高方园, 孙厚俊, 谢逸萍, 张成玲()

中国农业科学院甘薯研究所,江苏徐淮地区徐州农业科学研究所,农业农村部甘薯生物学与遗传育种重点实验室,江苏徐州 221131

收稿日期:2022-10-08 出版日期:2023-05-25 发布日期:2023-06-01
作者简介:罗勤川(1994—),男,四川遂宁人,硕士研究生,主要从事甘薯病虫害研究。E-mail:luoqinchuan123@163.com
通讯作者: *张成玲,E-mail:zhchlig5291@163.com
基金资助:
国家甘薯产业技术体系(CARS-10-GW17);国家重点研发计划(2018YFD1000703);国家重点研发计划(2018YFD1000700)

Transcriptomic analysis of Fusarium solani infecting sweetpotato

LUO Qinchuan(), TANG Wei, MA Jukui, CHEN Jingwei, YANG Dongjing, GAO Fangyuan, SUN Houjun, XIE Yiping, ZHANG Chengling()

Key laboratory of Biology and Genetic Improvement of Sweetpotato, Ministry of Agriculture and Rural Affairs, Xuzhou Institute of Agricultural Sciences in Jiangsu Xuhuai Area, Sweet Potato Research Institute of Chinese Academy of Agricultural Sciences Xuzhou 221131, Jiangsu,China

Received:2022-10-08 Online:2023-05-25 Published:2023-06-01

摘要/Abstract

摘要：

腐皮镰刀菌(Fusarium solani)是甘薯上的重要病原菌,易引起甘薯根腐病、腐烂溃疡病等。为明确F. solani在侵染甘薯时病原基因变化趋势,对腐烂溃疡病病菌侵染甘薯不同时期取样,通过Illumina Hiseq 4000高通量测序平台测定及分析序列。结果表明,共获得1 068 861 644条有效数据,F. solani侵染后6 h、24 h、3 d、5 d分别筛选出了1 056、995、737、935个显著差异表达基因,其中上调基因分别有587、679、430、551个,下调基因分别有469、316、307、384个,GO功能分析差异基因富集在分子功能和细胞组分,KEGG通路富集分析属于代谢和遗传信息过程。其中差异表达倍数2倍以上,且表达量水平前20的差异基因,有15个基因参与碳水化合物、能量、核苷酸以及氨基酸代谢;3个基因参与翻译和蛋白质加工和修饰等遗传信息处理;2个基因参与细胞自噬过程。随机筛选9个差异表达基因进行实时荧光定量PCR验证,结果表明,这9个基因的表达模式与转录组数据分析结果基本一致。本研究结果发现,F. solani在侵染甘薯过程中代谢活跃,这可能是其在侵染甘薯过程中需破除寄主防御并在寄主中定殖,需要自身和寄主提供大量的营养物质并转化为能量以及中间产物等物质所致。

关键词: 腐皮镰刀菌, 甘薯, 转录组

Abstract:

Fusarium solani is an important pathogen that causes root rot and canker disease of sweetpotato. To determine the change trend of F. solani genes during different infection stage, the high-throughput sequencing platform, Illumina Hiseq 4000 was used. The results showed that, a total of 1 068 861 644 clean reads were obtained. Total 1 056, 995, 737 and 935 differentially expressed genes were screened out at 6 h, 24 h, 3 d and 5 d after infection respectively, including 587, 679, 430 and 551 up-regulated genes and 469, 316, 307 and 384 down-regulated genes, respectively. These genes mainly belong to molecular function and cell component by GO enrichment analysis, and metabolic and genetic information processes by KEGG pathway analysis. The top 20 genes of expression level in the genes whose differential expression fold more than two times were selected to carry out further analysis. Among them, 15 genes were related to metabolism, involving carbohydrate metabolism, energy metabolism, nucleotide metabolism and amino acid metabolism, 3 genes were related to translation, protein processing and modification, and 2 genes were related to autophagy. Nine genes were randomly selected to verify the expression trend by quantitative real-time fluorescence PCR. The qRT-PCR results showed that the expression trend was consistent with the results of transcriptome data. In this study we found that the genes related to the metabolism of F. solani expressed differentially with significance during the pathogen infecting sweetpotato. The possible reason is that the pathogen needs a lot of nutrients, and then convert them to energy and intermediate, to break out the host defense and colonize in sweetpotato at last.

Key words: Fusarium solani, sweetpotato, RNA-seq

中图分类号:

S531

罗勤川, 唐伟, 马居奎, 陈晶伟, 杨冬静, 高方园, 孙厚俊, 谢逸萍, 张成玲. 腐皮镰刀菌侵染甘薯的转录组分析[J]. 浙江农业学报, 2023, 35(5): 1097-1107.

LUO Qinchuan, TANG Wei, MA Jukui, CHEN Jingwei, YANG Dongjing, GAO Fangyuan, SUN Houjun, XIE Yiping, ZHANG Chengling. Transcriptomic analysis of Fusarium solani infecting sweetpotato[J]. Acta Agriculturae Zhejiangensis, 2023, 35(5): 1097-1107.

图/表 8

参考文献 21

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样品名称 Sample name	原始数据 Raw data/bp	过滤后序列长 Clean data/bp	过滤后碱基数量 The base number of clean reads/Gb	过滤后Q20 The proportion of Q20/%	过滤后Q30 The proportion of Q30/%	过滤后GC The proportion of GC/%
CK-1	10 119 073 500	10 070 938 374	10.07	98.12	94.22	56.03
CK-2	8 860 847 700	8 820 337 043	8.82	97.60	92.78	56.02
CK-3	10 097 356 200	10 049 359 346	10.05	98.08	94.19	56.12
6 h-1	10 908 435 900	10 850 204 955	10.85	97.92	93.97	54.70
6 h-2	9 606 120 000	9 561 584 577	9.56	98.19	94.33	54.54
6 h-3	11 053 800 900	11 001 198 312	11.00	96.57	91.06	54.60
24 h-1	12 372 993 300	12 306 759 585	12.31	97.78	93.64	54.89
24 h-2	11 375 891 100	11 298 967 073	11.30	98.05	94.22	55.03
24 h-3	10 539 713 700	10 480 876 632	10.48	97.72	93.66	54.67
3 d-1	11 251 023 600	11 178 221 150	11.18	97.64	93.24	47.62
3 d-2	10 795 743 300	10 732 313 682	10.73	97.44	92.76	47.49
3 d-3	10 311 817 800	10 199 345 383	10.20	97.87	93.63	48.28
5 d-1	11 497 078 200	11 437 221 773	11.44	97.59	93.06	47.28
5 d-2	11 820 597 600	11 757 361 749	11.76	98.06	94.19	47.54
5 d-3	9 965 246 700	9 915 128 318	9.92	97.41	92.61	47.11

样品名称 Sample name	原始数据 Raw data/bp	过滤后序列长 Clean data/bp	过滤后碱基数量 The base number of clean reads/Gb	过滤后Q20 The proportion of Q20/%	过滤后Q30 The proportion of Q30/%	过滤后GC The proportion of GC/%
CK-1	10 119 073 500	10 070 938 374	10.07	98.12	94.22	56.03
CK-2	8 860 847 700	8 820 337 043	8.82	97.60	92.78	56.02
CK-3	10 097 356 200	10 049 359 346	10.05	98.08	94.19	56.12
6 h-1	10 908 435 900	10 850 204 955	10.85	97.92	93.97	54.70
6 h-2	9 606 120 000	9 561 584 577	9.56	98.19	94.33	54.54
6 h-3	11 053 800 900	11 001 198 312	11.00	96.57	91.06	54.60
24 h-1	12 372 993 300	12 306 759 585	12.31	97.78	93.64	54.89
24 h-2	11 375 891 100	11 298 967 073	11.30	98.05	94.22	55.03
24 h-3	10 539 713 700	10 480 876 632	10.48	97.72	93.66	54.67
3 d-1	11 251 023 600	11 178 221 150	11.18	97.64	93.24	47.62
3 d-2	10 795 743 300	10 732 313 682	10.73	97.44	92.76	47.49
3 d-3	10 311 817 800	10 199 345 383	10.20	97.87	93.63	48.28
5 d-1	11 497 078 200	11 437 221 773	11.44	97.59	93.06	47.28
5 d-2	11 820 597 600	11 757 361 749	11.76	98.06	94.19	47.54
5 d-3	9 965 246 700	9 915 128 318	9.92	97.41	92.61	47.11

样品 Sample	总序列 Total gene sequence/ bp	比对到参考序列上的序列 Aligne to reference sequence		比对到参考序列唯一位置上的序列 Align to the unique position of the reference sequence		比对到参考序列多个位置上的序列 Align to multiple positions in the reference sequence		比对到参考序列外显子区域 Align to the exon region of the reference sequence		比对到参考序列内含子区域 Align to reference sequence column containing sub regions
样品 Sample	总序列 Total gene sequence/ bp	长度 Length/bp	占比 Proportion /%	长度 Length/bp	占比 Proportion /%	长度 Length/bp	占比 Proportion /%	长度 Length/bp	占比 Proportion /%	长度 Length/bp	占比 Proportion /%
CK-1	61 002 848	43 948 488	72.04	43 898 824	71.96	49 664	0.08	21 506 666	90.31	192 479	0.81
CK-2	52 444 612	37 408 991	71.33	37 364 628	71.25	44 363	0.08	18 090 239	90.39	175 130	0.88
CK-3	63 156 424	455 780 661	72.17	45 529 774	72.09	48 292	0.08	21 565 610	90.44	214 765	0.90
6 h-1	65 060 974	36 057 705	55.42	36 011 225	55.35	46 480	0.07	19 145 455	90.93	129 676	0.62
6 h-2	40 119 272	23 265 840	57.99	23 240 761	57.93	25 079	0.06	12 090 755	90.81	86 066	0.65
6 h-3	60 484 944	34 090 092	56.36	34 051 092	56.30	39 000	0.06	17 475 090	90.67	129 079	0.67
24 h-1	72 035 904	41 211 305	57.21	41 156 718	57.13	54 587	0.08	19 966 747	90.90	156 719	0.71
24 h-2	66 444 752	37 586 165	56.57	37 539 588	56.50	46 577	0.07	18 645 057	90.81	147 741	0.72
24 h-3	62 022 092	35 613 524	57.42	35 567 080	57.35	46 444	0.07	17 247 258	90.23	141 544	0.74
3 d-1	74 243 204	428 662	0.58	428 002	0.58	660	<0.01	212 565	91.18	1 392	0.60
3 d-2	70 796 204	407 700	0.58	407 016	0.57	684	<0.01	209 351	91.78	1 209	0.53
3 d-3	67 674 158	3 097 165	4.58	3 093 673	4.57	3 492	0.01	1 525 732	90.60	12 309	0.73
5 d-1	75 670 110	443 750	0.59	443 096	0.59	654	<0.01	226 227	92.15	1 356	0.55
5 d-2	7 7046 624	1 291 467	1.68	1 289 821	1.67	1 646	<0.01	626 377	90.68	4 417	0.64
5 d-3	65 710 952	292 812	0.45	292 299	0.44	513	<0.01	149 896	91.83	940	0.58

样品 Sample	总序列 Total gene sequence/ bp	比对到参考序列上的序列 Aligne to reference sequence		比对到参考序列唯一位置上的序列 Align to the unique position of the reference sequence		比对到参考序列多个位置上的序列 Align to multiple positions in the reference sequence		比对到参考序列外显子区域 Align to the exon region of the reference sequence		比对到参考序列内含子区域 Align to reference sequence column containing sub regions
样品 Sample	总序列 Total gene sequence/ bp	长度 Length/bp	占比 Proportion /%	长度 Length/bp	占比 Proportion /%	长度 Length/bp	占比 Proportion /%	长度 Length/bp	占比 Proportion /%	长度 Length/bp	占比 Proportion /%
CK-1	61 002 848	43 948 488	72.04	43 898 824	71.96	49 664	0.08	21 506 666	90.31	192 479	0.81
CK-2	52 444 612	37 408 991	71.33	37 364 628	71.25	44 363	0.08	18 090 239	90.39	175 130	0.88
CK-3	63 156 424	455 780 661	72.17	45 529 774	72.09	48 292	0.08	21 565 610	90.44	214 765	0.90
6 h-1	65 060 974	36 057 705	55.42	36 011 225	55.35	46 480	0.07	19 145 455	90.93	129 676	0.62
6 h-2	40 119 272	23 265 840	57.99	23 240 761	57.93	25 079	0.06	12 090 755	90.81	86 066	0.65
6 h-3	60 484 944	34 090 092	56.36	34 051 092	56.30	39 000	0.06	17 475 090	90.67	129 079	0.67
24 h-1	72 035 904	41 211 305	57.21	41 156 718	57.13	54 587	0.08	19 966 747	90.90	156 719	0.71
24 h-2	66 444 752	37 586 165	56.57	37 539 588	56.50	46 577	0.07	18 645 057	90.81	147 741	0.72
24 h-3	62 022 092	35 613 524	57.42	35 567 080	57.35	46 444	0.07	17 247 258	90.23	141 544	0.74
3 d-1	74 243 204	428 662	0.58	428 002	0.58	660	<0.01	212 565	91.18	1 392	0.60
3 d-2	70 796 204	407 700	0.58	407 016	0.57	684	<0.01	209 351	91.78	1 209	0.53
3 d-3	67 674 158	3 097 165	4.58	3 093 673	4.57	3 492	0.01	1 525 732	90.60	12 309	0.73
5 d-1	75 670 110	443 750	0.59	443 096	0.59	654	<0.01	226 227	92.15	1 356	0.55
5 d-2	7 7046 624	1 291 467	1.68	1 289 821	1.67	1 646	<0.01	626 377	90.68	4 417	0.64
5 d-3	65 710 952	292 812	0.45	292 299	0.44	513	<0.01	149 896	91.83	940	0.58

序号 Number	基因号 Gene number	标签 Symbol	上调/下调 Up/down	途径或功能 Pathway or function
1	ncbi_9673094	pdcA	下调Down	糖酵解和糖异生、次生代谢物生物合成 Glycolysis and gluconeogenesis, biosynthesis of secondary metabolites
2	ncbi_9674521	pma-1	下调Down	氧化磷酸化Oxidative phosphorylation
3	ncbi_9671178	DPS1	下调Down	翻译,氨酰基-tRNA的生物合成Translation, biosynthesis of amyl-tRNA
4	ncbi_9677451	SPCC 1620.06c	下调Down	次生代谢产物的生物合成、抗生素合成、氨基酸生物合成、磷酸戊糖途径 Biosynthesis of secondary metabolites, antibiotic synthesis, amino acid biosynthesis,pentose phosphate pathway
5	ncbi_9672983	des	上调Up	氧化磷酸化 Oxidative phosphorylation
6	ncbi_9673795	gdh-1	下调Down	丙氨酸,天冬氨酸和谷氨酸代谢、精氨酸生物合成、氮代谢、牛磺酸代谢 Alanine, aspartic acid and glutamate metabolism, arginine biosynthesis, nitrogen metabolism, taurine metabolism
7	ncbi_9674832	FES1	下调Down	内质网中的蛋白质加工Protein processing in the endoplasmic reticulum
8	ncbi_9664755	CLAH10	上调Up	次生代谢产物的生物合成、抗生素合成、精氨酸和脯氨酸代谢、色氨酸代谢、缬氨酸,亮氨酸和异亮氨酸的降解等 Biosynthesis of secondary metabolites, antibiotic synthesis, metabolism of arginine and proline, metabolism of tryptophan, degradation of valine, leucine and isoleucine, etc
9	ncbi_9671992	pep-4	上调Up	运输和分解代谢,自噬 Transport and catabolism, autophagy
10	ncbi_9671150	imqH	下调Down	过氧化物酶体、甘氨酸、丝氨酸和苏氨酸的代谢、赖氨酸降解 Metabolism of peroxisome, glycine, serine, threonine, lysine degradation
11	ncbi_9671105	glkA	上调Up	次生代谢产物的生物合成、抗生素合成、碳代谢、淀粉和蔗糖代谢、氨基糖和核苷酸糖代谢、果糖和甘露糖代谢 Biosynthesis of secondary metabolites, antibiotic synthesis, carbon metabolism, starch and sucrose metabolism, amino sugars and nucleotide sugars metabolism, fructose and mannose metabolism
12	ncbi_9679169	nuo40	上调Up	氧化磷酸化Oxidative phosphorylation
13	ncbi_9664067	SOD1	上调Up	过氧化物酶体Peroxisome
14	ncbi_9678178	ykt6	上调Up	SNARE在水泡运输中的相互作用、自噬 Interaction and autophagy of SNARE in blister transport
15	ncbi_9674908	HYR1	上调Up	谷胱甘肽代谢、花生四烯酸代谢Glutathione metabolism, arachidonic acid metabolism
16	ncbi_9664589	Cda	上调Up	嘧啶代谢Pyrimidine metabolism
17	ncbi_9668438	mcsA	上调Up	碳水化合物代谢;柠檬酸甲基合酶前体、氨基酸生物合成 Carbohydrate metabolism; Citrate methyl synthase precursor, amino acid biosynthesis
18	ncbi_9673059	Acaa1b	上调Up	α-亚麻酸代谢、缬氨酸,亮氨酸和异亮氨酸的降解、脂肪酸降解等 α-linolenic acid metabolism, degradation of valine, leucine and isoleucine, fatty acid degradation, etc
19	ncbi_9665574	ACAD10	上调Up	抗生素生物合成、β-丙氨酸代谢、缬氨酸,亮氨酸和异亮氨酸的降解 Antibiotic biosynthesis, β-alanine metabolism, degradation of valine, leucine and isoleucine
20	ncbi_9674141	KATG2	上调Up	次生代谢物生物合成、色氨酸代谢、丙氨酸代谢 Secondary metabolite biosynthesis, tryptophan metabolism, alanine metabolism

腐皮镰刀菌侵染甘薯的转录组分析

Transcriptomic analysis of Fusarium solani infecting sweetpotato

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