本生烟响应蛋白激发子PevD1的差异表达基因鉴定与分析

doi:10.3864/j.issn.0578-1752.2019.21.008

摘要/Abstract

摘要：

目的通过RNA-Seq筛选本生烟（Nicotiana benthamiana）响应大丽轮枝菌（Verticillium dahliae）蛋白激发子PevD1的差异表达基因（differentially expressed gene,DEG）,分析PevD1诱导植物产生抗病性的潜在分子机制。方法用10 μmol·L ^-1的PevD1蛋白液渗入4周龄的本生烟叶片,分别在处理后6、12和24 h取样提取RNA,构建mRNA文库后采用BGISEQ-500平台进行测序。筛选各时间点的差异表达基因进行GO和KEGG分析;重点分析与诱导抗病相关的富含亮氨酸重复序列类受体蛋白激酶（leucine-rich repeats RLKs,LRR-RLKs）、转录因子（transcription factor,TF）以及病程相关蛋白（pathogenesis related protein,PR蛋白）家族差异表达基因;采用qRT-PCR对差异表达基因进行定量验证。结果 GO功能富集以及KEGG通路富集分析表明,PevD1诱导6 h后的差异表达基因主要与细胞识别、光合作用、光收割等功能相关,显著富集在光合作用-天线蛋白通路、萜类化合物合成通路、黄酮和黄酮醇等次生代谢产物合成相关通路中;12 h和24 h的差异表达基因主要与细胞识别和胞内激酶等生物学功能相关,显著富集在植物-病原互作通路、倍半萜和三萜生物合成通路、黄酮和黄酮醇生物合成通路、亚麻酸代谢等次生代谢产物合成相关通路中。与光合作用相关的差异表达基因主要呈下调趋势,与萜类、黄酮类等抗病相关次生代谢产物合成通路相关的差异表达基因主要呈上调趋势。PevD1诱导后大量的LRR-RLKs、TF以及PR蛋白家族基因显著上调表达,这些基因与激发子识别、基因转录调控和抗病性相关。经qRT-PCR验证后,所检测基因的表达趋势与转录组测序结果一致。结论 PevD1诱导本生烟中大量基因转录重排,大量LRR-RLKs、TF和PR蛋白家族基因上调表达,激活了植物免疫系统,使植物产生抗病性。研究结果可为今后深入探讨PevD1诱导植物免疫的机理提供依据。

关键词: 本生烟, 大丽轮枝菌, 蛋白激发子PevD1, RNA-Seq, LRR-RLKs, 转录因子, PR蛋白

Abstract:

【Objective】 The objective of this study is to screen the differentially expressed genes (DEGs) induced by protein elicitor PevD1 of Verticillium dahliae in Nicotiana benthamiana by RNA-Seq, and to analyze the potential mechanism of PevD1-induced disease resistance. 【Method】 Leaves of 4-week-old N. benthamiana were infiltrated with 10 μmol·L ^-1 PevD1 solution, and samples were taken at 6, 12 and 24 h after PevD1 treatment, then RNA was extracted. The mRNA libraries were constructed and sequenced by BGISEQ-500 platform. The DEGs at each time point were screened for GO and KEGG analysis. The leucine-rich repeat receptor-like kinases (LRR-RLKs), transcription factors (TFs) and pathogenesis-related (PR) proteins family in DEGs were analyzed, and qRT-PCR was used to verify the expression levels of relevant DEGs.【Result】 GO functional enrichment and KEGG pathway enrichment analysis indicated that the DEGs at 6 h post infiltration (hpi) were mainly related to cell recognition, photosynthesis, light-harvesting, and were significantly enriched in photosynthesis-antenna protein pathways, terpenoid synthesis pathway, flavonoid and flavonol, and other secondary metabolite synthesis pathways. The DEGs at 12 hpi and 24 hpi were mainly associated with cell recognition and biological functions such as intracellular kinase activity, and were significantly enriched in plant-pathogen interaction pathway, sesquiterpene and triterpenoid biosynthetic pathway, flavonoid and flavonol biosynthetic pathway, linolenic acid metabolism. The unigenes in photosynthesis-antenna proteins, photosynthesis and porphyrin, chlorophyll metabolism pathway were mainly down-regulated, and the unigenes in sesquiterpenoid and triterpenoid biosynthesis, flavone and flavonol biosynthesis, plant-pathogen interaction, phenylpropanoid biosynthesis and terpenoid backbone biosynthesis pathway were mainly up-regulated. After PevD1 induction, a large number of LRR-RLKs, TFs and PR proteins family genes were significantly up-regulated, which were related to elicitor recognition, gene transcriptional regulation and disease resistance. qRT-PCR analysis showed that the expression pattern of the detected DEGs was consistent with the RNA-Seq results.【Conclusion】 PevD1 induced a large number of gene transcriptional rearrangements in N. benthamiana. Lots of LRR-RLKs, TFs and PR proteins family genes were up-regulated, which activated the plant immune system and conferred to plants disease resistant. These results can provide a basis for further study on the mechanism of PevD1-induced immunity in the future.

Key words: Nicotiana benthamiana, Verticillium dahliae, protein elicitor PevD1, RNA-Seq, LRR-RLKs, transcription factor, PR protein

梁颖博,李泽,邱德文,曾洪梅,李广悦,杨秀芬. 本生烟响应蛋白激发子PevD1的差异表达基因鉴定与分析[J]. 中国农业科学, 2019, 52(21): 3794-3805.

LIANG YingBo,LI Ze,QIU DeWen,ZENG HongMei,LI GuangYue,YANG XiuFen. Identification and Analysis of Differentially Expressed Genes Induced by Protein Elicitor PevD1 in Nicotiana benthamiana[J]. Scientia Agricultura Sinica, 2019, 52(21): 3794-3805.

图/表 8

表1

图1

图2

图3

表2

差异表达基因中LRR-RLKs的筛选"

Unigene ID	log₂FC			蛋白特性 Protein property
Unigene ID	6 hpi	12 hpi	24 hpi	蛋白特性 Protein property
Niben101Scf00160g06027	2.80	3.42	2.12	Putative receptor-like protein kinase At3g47111
Niben101Scf00182g06001	—	5.47	—	Pollen receptor-like kinase 2
Niben101Scf00245g00006	10.00	6.88	4.19	Probable LRR receptor-like serine/threonine-protein kinase At1g74361
Niben101Scf00742g01037	—	2.35	—	Protein STRUBBELIG-RECEPTOR FAMILY 3-like isoform X4
Niben101Scf00744g08015	2.10	1.82	—	Receptor-like protein kinase HAIKU3
Niben101Scf00887g04011	1.80	2.26	—	Putative receptor-like protein kinase At3g47111
Niben101Scf00953g00004	2.65	3.06	1.66	Receptor-like protein kinase HAIKU3
Niben101Scf01203g01002	2.12	4.39	2.65	Probable LRR receptor-like serine/threonine-protein kinase At3g47570 isoform X2
Niben101Scf01205g02008	-2.00	—	-2.10	Probable LRR receptor-like serine/threonine-protein kinase At4g36181
Niben101Scf01225g04031	2.51	2.85	2.08	Probably inactive leucine-rich repeat receptor-like protein kinase At5g48381
Niben101Scf01278g09008	—	2.04	1.54	Probable LRR receptor-like serine/threonine-protein kinase At1g56130 isoform X3
Niben101Scf01673g02002	—	6.26	—	Probable LRR receptor-like serine/threonine-protein kinase RLK
Niben101Scf01819g01001	1.31	2.21	—	Phytosulfokine receptor 2
Niben101Scf02217g05010	1.65	2.29	—	Receptor-like protein kinase HSL2
Niben101Scf02531g00007	—	2.88	—	LRR receptor-like serine/threonine-protein kinase EFR
Niben101Scf02862g00007	-1.90	-2.80	—	Probable LRR receptor-like serine/threonine-protein kinase At4g36181
Niben101Scf03374g08019	3.80	3.71	1.10	Probable LRR receptor-like serine/threonine-protein kinase At3g47571
Niben101Scf03816g01001	2.03	2.07	1.39	AHW85126.2//leucine-rich repeat receptor-like kinase
Niben101Scf04053g03008	2.06	2.50	1.79	Receptor-like protein kinase HAIKU3
Niben101Scf04099g05004	2.38	2.35	1.72	Leucine-rich repeat receptor-like serine/threonine/tyrosine-protein kinase SOBIR2
Niben101Scf05349g01040	1.30	2.32	2.20	Protein STRUBBELIG-RECEPTOR FAMILY 5 isoform X2
Niben101Scf05928g03007	5.32	6.75	3.32	Probable LRR receptor-like serine/threonine-protein kinase At1g74361
Niben101Scf06509g02006	5.34	5.87	2.71	Receptor-like protein kinase 6
Niben101Scf08137g01034	2.82	3.29	—	Putative receptor-like protein kinase
Niben101Scf08564g00001	1.15	3.10	—	Probable LRR receptor-like serine/threonine-protein kinase At1g56141
Niben101Scf10381g03006	—	3.10	2.42	Probable LRR receptor-like serine/threonine-protein kinase At4g08851
Niben101Scf20124g00014	2.27	2.20	1.73	Leucine-rich repeat receptor-like tyrosine-protein kinase PXC4

表2

图4

表3

PevD1诱导病程相关蛋白家族差异表达基因的筛选"

PR 家族基因 PR family gene	Unigene ID	log₂ Ratio (WT_PevD1/WT_CK)			蛋白特性 Protein property
PR 家族基因 PR family gene	Unigene ID	6 hpi	12 hpi	24 hpi	蛋白特性 Protein property
PR1	Niben101Scf00107g03008	-2.10	2.38	4.32	Pathogenesis-related protein 1A
	Niben101Scf00953g03008	—	5.98	3.86	Basic form of pathogenesis-related protein 1
	Niben101Scf00953g03009	4.60	7.73	5.58	Basic form of pathogenesis-related protein 1-like
	Niben101Scf01999g07002	—	3.17	2.58	Pathogenesis-related protein 1C-like
	Niben101Scf03376g03004	-6.50	2.87	6.24	Pathogenesis-related protein 1A
	Niben101Scf04053g02006	1.64	5.47	3.81	Basic form of pathogenesis-related protein 1-like
	Niben101Scf04053g02007	5.23	8.30	6.87	Basic form of pathogenesis-related protein 1
PR2	Niben101Ctg13736g00004	—	3.09	4.15	Glucan endo-1,3-beta-glucosidase
	Niben101Scf01001g00003	—	2.86	3.91	Glucan endo-1,3-beta-glucosidase
	Niben101Scf01001g00004	—	2.86	3.91	Glucan endo-1,3-beta-glucosidase
	Niben101Scf01001g00005	—	2.86	3.91	Glucan endo-1,3-beta-glucosidase
	Niben101Scf01001g00006	—	—	3.70	Glucan endo-1,3-beta-glucosidase
	Niben101Scf01934g02004	—	5.40	3.21	Glucan endo-1,3-beta-glucosidase
	Niben101Scf03905g02017	1.32	5.13	2.91	Glucan endo-1,3-beta-glucosidase
	Niben101Scf04869g03002	—	3.25	3.39	Glucan endo-1,3-beta-glucosidase
PR3	Niben101Scf02041g00002	—	1.57	3.85	Acidic endochitinase Q
	Niben101Scf03200g01014	—	2.22	2.15	Endochitinase A
	Niben101Scf03949g00023	—	4.49	1.38	Endochitinase 3
	Niben101Scf07491g00003	—	4.87	3.48	Endochitinase A
PR4	Niben101Scf01015g01002	1.84	4.45	—	Wound-induced protein WIN1-like
	Niben101Scf02171g00007	2.01	5.84	3.06	Endochitinase PR4-like
	Niben101Scf12045g06025	1.41	4.73	6.29	Pathogenesis-related protein PR-4B
PR5	Niben101Scf03937g02019	—	—	1.69	Pathogenesis-related protein 5-like
PR5	Niben101Scf05732g04031	—	—	4.88	Pathogenesis-related protein 5-like
PR6	Niben101Scf04053g01004	—	—	6.66	Pathogenesis-related leaf protein 6-like
PR10	Niben101Scf10735g00018	1.36	2.10	2.50	Pathogenesis-related protein PR-10
PR11	Niben101Scf01789g04010	2.80	3.62	2.77	Chitotriosidase-1-like
PR11	Niben101Scf06295g04023	1.91	2.85	2.19	Chitotriosidase-1-like
PR17	Niben101Scf03385g02011	—	2.02	3.33	Uncharacterized protein LOC104232799

表3

图5

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基因ID Gene ID	基因名称Gene name	正向引物Forward primer	反向引物Reverse primer
Niben101Scf00245g03004	LHCA4	CTCCTACTTATCTTGATGGAA	CACTGGTGAGAACCTCAG
Niben101Scf01094g03014	PTI5	TTCCCAGAAATGTCCTTC	GCTTCTTCTGCTGTTTCA
Niben101Scf01297g04006	WRKY7	GGTCTCCTATCAGTAAGTC	GCTTGTGGTTTGAACTAC
Niben101Scf01426g00001	ERF3	CTTCGAGCTGGATCTCAC	GTCGCCACAAAGAATCAA
Niben101Scf02430g03006	WRKY8	CCTACTGAACTCTTGGAC	CTGCTCTTCCAGTTAAAAG
Niben101Scf03160g01004	NAC90	TGTCGAGTTTACGTCATA	TCAGCTGCTAATTCTTGA
Niben101Scf03816g01001	SOBIR1	CACCAGAATACCATCAGA	GATCCTCAGAAGTCATTACA
Niben101Scf04504g03008	LHCB3	CCAAGTAGTGCTAATGGG	AGTGGGTCAAAGTACTGG
Niben101Scf06017g02002	MYB4	CACCCACTTGAAGAAGAG	CTTGGATGTTGTTAAAATTGTG
Niben101Scf06509g02006	RLK5	GCTGTTAAGAGGATTTGG	GCAACAAAGCAGTTTTAC
Niben101Scf10735g00018	PR10	CTGGAAATGGAGGATGTA	AACGGTCTTGAAAAGTTC
Niben101Scf18667g01002	MKS1	GCTTCTCTTCCACCTATA	GGGCTAGATAAATAACTGTG