灰霉菌侵染葡萄叶片初期的蛋白质组学研究

doi:10.16420/j.issn.0513-353x.2024-0264

摘要/Abstract

摘要：

利用非标记定量蛋白质组学技术，对易感灰霉病的欧洲葡萄（Vitis vinifera）品种‘黑比诺’和高抗灰霉病的野生山葡萄（V. amurensis）品种‘双优’的幼嫩叶片进行灰霉菌接种，以接种无菌水为对照，分析二者在灰霉菌侵染初期（接种后12 h）的差异表达蛋白。2个品种共鉴定出12 210个蛋白质，在‘黑比诺’中，检测出868个差异表达蛋白，‘双优’中检测出1 946个差异表达蛋白。通过GO和KEGG富集分析，发现差异表达蛋白主要集中于代谢通路、次生代谢物、氨基酸代谢等信号途径中。在2个品种中共筛选出10个关键差异表达蛋白，并且在高抗品种‘双优’中筛选出的过氧化物酶、谷胱甘肽转移酶、酯氧合酶、乙醛脱氢酶和二苯乙烯合酶的表达量发生上调，NADH脱氢酶和碳酸酐酶的表达量受到抑制，说明以上蛋白在‘双优’叶片受到灰霉菌侵染过程中起重要作用，其中过氧化物酶在易感品种‘黑比诺’中也有表达，表明过氧化物酶在葡萄叶片受灰霉菌侵染初期积极响应，是重要差异表达蛋白。

关键词: 葡萄, 灰霉菌, 蛋白质组学, 差异表达蛋白, 实时荧光定量PCR

Abstract:

Inoculating young leaves of susceptible Vitis vinifera‘Pinot Noir’and highly resistant V. amurensis‘Shuangyou’with Botrytis cinerea and treating them with sterile water as the control，label-free quantitative proteomics technology were used to analyze the differentially expressed proteins in the initial stage of B. cinerea infection（Inoculation for 12 h）. A total of 12 210 proteins were identified from the two grapevine cultivars. In‘Pinot Noir’，868 differentially expressed proteins were detected，while 1 946 differentially expressed proteins were detected in‘Shuangyou’. Through Gene Ontology（GO）and Kyoto Encyclopedia of Genes and Genomes（KEGG）enrichment analyses，it was determined that differentially expressed proteins were primarily enriched in metabolic pathways，secondary metabolite biosynthesis，and amino acid metabolism，among other signaling pathways. A total of 10 key differently expressed proteins were screened from the two grapevine varieties，and the expression levels of peroxidase，glutathione transferase，lipoxygenase，aldehyde dehydrogenase and stilbene synthase were up-regulated，while the expression levels of NADH dehydrogenase and carbonic anhydrase were down-regulated in highly resistant V. amurensis‘Shuangyou’. The above proteins play an important role in the infection of ‘Shuangyou’leaves by B. cinerea. Peroxidase was also expressed in the susceptible V. vinifera‘Pinot Noir’，indicating that peroxidase was an important differently expressed protein in the early stage of B. cinerea infection in grapevine leaves.

Key words: grapevine, Botrytis cinerea, proteomics, differentially expressed protein, quantitative Real-time PCR

赵钰磊, 李珊, 李承男, 马金龙, 马红义, 尹晓. 灰霉菌侵染葡萄叶片初期的蛋白质组学研究[J]. 园艺学报, 2025, 52(2): 279-291.

ZHAO Yulei, LI Shan, LI Chengnan, MA Jinlong, MA Hongyi, YIN Xiao. Proteomics Studies on the Early Stages of Botrytis cinerea Infection in Grapevine Leaves[J]. Acta Horticulturae Sinica, 2025, 52(2): 279-291.

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蛋白 Protein	基因ID Gene ID	描述 Description	引物序列（5′-3′） Primer sequence
A0A438C3M1	PNC1_16	过氧化物酶Peroxidase	F：GTTTGTGCCCTGGGGTTGTTTC
A0A438C3M1	PNC1_16		R：ACTCGCCGTGGTTGAATCTCTTC
A0A438C992	GSTU10_1	谷胱甘肽转移酶Glutathione transferase	F：TGAGGTAGTTGGTGTGGTTGTCG
A0A438C992	GSTU10_1		R：GTCTCCTTCATCAGTGGGCAGTC
D7TAQ3	XM_002265469	脂氧合酶Lipoxygenase	F：TGAGGACAAGACTGTGGAAGGTG
D7TAQ3	XM_002265469		R：AGAAGACGACGACGACGACAAC
D7SL57	XM_002285430	乙醛脱氢酶Aldehyde dehydrogenase	F：TTCACACACGAGAAGGCGATCC
D7SL57	XM_002285430		R：TCAAGCCCAAGAGGACAAGCAG
A5AEM3	XM_003634025	二苯乙烯合酶Stilbene synthase 4	F：AACGACGACAGGTGAAGGATTGG
A5AEM3	XM_003634025		R：TGGAGCACAACGGTCTCAATGG
D7TU30	XM_010649825	碳酸酐酶Carbonic anhydrase	F：TGGCCTTCTCATCCTCCTCTCTC
D7TU30	XM_010649825		R：TGGGTCTGCTCAATTACGCTCAG
A5C501	XM_002265668	NADH脱氢酶NADH dehydrogenase [ubiquinone] iron-sulfur protein 4，mitochondrial	F：AGTCACTGCAACGGCTATGGAG R：CGATCTCGCCCGGTTTGATTTC
A0A438JBL2	POX2_1	脯氨酸脱氢酶Proline dehydrogenase	F：GCAGGATGTGGAGGAGGCTTG
A0A438JBL2	POX2_1		R：AGGTGGCGTTATCGGTGGTATG
A0A438CM14	POXN1_5	过氧化物酶Peroxidase	F：GAGGGCTGCGTGGATTGAATTTC
A0A438CM14	POXN1_5		R：TCCCAGTCTTCACCCCAATGTTG
A0A438IMB3	VvCHDp000857_1	葡萄糖-6-磷酸脱氢酶 Glucose-6-phosphate-dehydrogenase	F：TTCGTCTTTCTCGGCACCCTTC R：AATGGCGGCTGATGGTACTGAG

蛋白 Protein	基因ID Gene ID	描述 Description	引物序列（5′-3′） Primer sequence
A0A438C3M1	PNC1_16	过氧化物酶Peroxidase	F：GTTTGTGCCCTGGGGTTGTTTC
A0A438C3M1	PNC1_16		R：ACTCGCCGTGGTTGAATCTCTTC
A0A438C992	GSTU10_1	谷胱甘肽转移酶Glutathione transferase	F：TGAGGTAGTTGGTGTGGTTGTCG
A0A438C992	GSTU10_1		R：GTCTCCTTCATCAGTGGGCAGTC
D7TAQ3	XM_002265469	脂氧合酶Lipoxygenase	F：TGAGGACAAGACTGTGGAAGGTG
D7TAQ3	XM_002265469		R：AGAAGACGACGACGACGACAAC
D7SL57	XM_002285430	乙醛脱氢酶Aldehyde dehydrogenase	F：TTCACACACGAGAAGGCGATCC
D7SL57	XM_002285430		R：TCAAGCCCAAGAGGACAAGCAG
A5AEM3	XM_003634025	二苯乙烯合酶Stilbene synthase 4	F：AACGACGACAGGTGAAGGATTGG
A5AEM3	XM_003634025		R：TGGAGCACAACGGTCTCAATGG
D7TU30	XM_010649825	碳酸酐酶Carbonic anhydrase	F：TGGCCTTCTCATCCTCCTCTCTC
D7TU30	XM_010649825		R：TGGGTCTGCTCAATTACGCTCAG
A5C501	XM_002265668	NADH脱氢酶NADH dehydrogenase [ubiquinone] iron-sulfur protein 4，mitochondrial	F：AGTCACTGCAACGGCTATGGAG R：CGATCTCGCCCGGTTTGATTTC
A0A438JBL2	POX2_1	脯氨酸脱氢酶Proline dehydrogenase	F：GCAGGATGTGGAGGAGGCTTG
A0A438JBL2	POX2_1		R：AGGTGGCGTTATCGGTGGTATG
A0A438CM14	POXN1_5	过氧化物酶Peroxidase	F：GAGGGCTGCGTGGATTGAATTTC
A0A438CM14	POXN1_5		R：TCCCAGTCTTCACCCCAATGTTG
A0A438IMB3	VvCHDp000857_1	葡萄糖-6-磷酸脱氢酶 Glucose-6-phosphate-dehydrogenase	F：TTCGTCTTTCTCGGCACCCTTC R：AATGGCGGCTGATGGTACTGAG

品种 Cultivar	显著差异蛋白 Significant difference protein	上调 Up-regulation	下调 Down-regulation
黑比诺Pinot Noir	868	543	325
双优Shuangyou	1 946	969	977

品种 Cultivar	显著差异蛋白 Significant difference protein	上调 Up-regulation	下调 Down-regulation
黑比诺Pinot Noir	868	543	325
双优Shuangyou	1 946	969	977

品种 Cultivar	表达 Express	蛋白ID Protein ID	描述 Description	log₂FC
黑比诺 Pinot Noir	上调 Up-regulated	A0A438JBL2	脯氨酸脱氢酶Proline dehydrogenase	3.71
		A0A438CM14	过氧化物酶Peroxidase	3.66
		A0A438IMB3	葡萄糖-6-磷酸脱氢酶Glucose-6-phosphate-dehydrogenase	2.15
双优 Shuangyou	上调 Up-regulated	A0A438C3M1	过氧化物酶Peroxidase	3.56
		A0A438C992	谷胱甘肽转移酶Glutathione S-transferase	3.31
		D7TAQ3	脂氧合酶Lipoxygenase	2.11
		D7SL57	乙醛脱氢酶Aldehyde dehydrogenase	2.09
		A5AEM3	二苯乙烯合酶Stilbene synthase 4	1.65
	下调 Down-regulated	D7TU30	碳酸酐酶Carbonic anhydrase	-2.89
	下调 Down-regulated	A5C501	NADH脱氢酶NADH dehydrogenase[ubiquinone] iron-sulfur protein 4，mitochondrial	-2.72