不同抗性苹果品种应答轮纹病菌胁迫的差异蛋白质组分析

doi:10.11983/CBB19204

摘要/Abstract

摘要： 为鉴定不同抗性苹果(Malus domestica)品种响应轮纹病菌胁迫的抗性相关蛋白表达差异, 以抗病品种华月及易感品种金冠为试材, 采用高通量同位素标记定量(IBT)技术结合液相色谱-串联质谱(LC-MS)鉴定技术, 对病原菌处理前后抗、感病品种叶片的蛋白质组差异表达进行分析, 共鉴定出171个差异表达蛋白(DEPs)。GO富集及KEGG通路分析表明, 在细胞组分、分子功能和生物过程3类中共注释到686个GO条目, 其中52个DEPs注释于KEGG通路的18个显著差异途径(P<0.05)。亚细胞定位预测分析表明, 171个DEPs中有170个分别定位于8类细胞器。蛋白功能注释分析表明, 46个DEPs注释于7类抗性相关蛋白, 包括类甜蛋白、过氧化物酶、多酚氧化酶、过敏原蛋白、几丁质酶、内切葡聚糖酶以及主乳胶蛋白。此外, 还对抗性相关蛋白的表达特点及基因定量结果进行了分析。该研究结果可为进一步解析抗、感病苹果品种应答轮纹病菌胁迫的抗性机制提供参考。

关键词: 苹果, 抗、感病, IBT定量蛋白质组学, 轮纹病, 抗性相关蛋白

Abstract: The aim of this study was to investigate the differential expression of resistance related proteins in resistant and susceptible apple leaves in response to ring rot disease. The resistant and susceptible apple cultivars Huayue and Golden Delicious were analyzed by high-throughput isobaric tag (IBT) marker quantitative analysis combined with liquid chromatography-tandem mass spectrometry (LC-MS), proteome differential expression of resistant and susceptible apple leaves before and after pathogen treatment was analyzed, and 171 differential expression proteins (DEPs) were identified. GO enrichment and KEGG pathway analysis showed that 686 GO entries were annotated in cell components, molecular functions, and biological processes, of which 52 DEPs were annotated in 18 distinct pathways of KEGG pathway (P<0.05). Subcellular localization prediction analysis showed that 170 DEPs in 171 DEPs were located in 8 organelles. Functional annotation analysis showed that 46 DEPs were annotated in 7 classes of resistance-related proteins, including the thaumatin-like proteins, peroxidases, polyphenol oxidases, Mal d1 proteins, chitinases, endo-1,3-beta-glucosidases and MLP-like proteins. In addition, the expression characteristics and gene quantitative results of resistance-related proteins were analyzed. The results of this study provided a technical reference for further understanding the resistance mechanism of resistant and susceptible apples to the stress of ring rot disease.

Key words: apple, resistant and susceptible, IBT proteomics, ring rot disease, resistance-related proteins

张彩霞,袁高鹏,韩晓蕾,李武兴,丛佩华. 不同抗性苹果品种应答轮纹病菌胁迫的差异蛋白质组分析. 植物学报, 2020, 55(4): 430-441.

Caixia Zhang,Gaopeng Yuan,Xiaolei Han,Wuxing Li,Peihua Cong. Proteome Analysis of Different Resistant Apple Cultivars in Response to the Stress of Ring Rot Disease. Chinese Bulletin of Botany, 2020, 55(4): 430-441.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB19204

https://www.chinbullbotany.com/CN/Y2020/V55/I4/430

图/表 7

表1 荧光定量PCR引物序列

Table 1 Primers used in fluorescent quantitative PCR

Accession No.	Forward primer (5'-3')	Reverse primer (5'-3')
gi\|657977120	TCACCTTAGCCATCTTCTTCGC	TGCTAACTCGAACCCTGTGG
gi\|658027651	CTCCACTGTGCCTATTGCGA	GAGCCGGGTTAGCGAACAA
gi\|658061109	TTGATGCCAGCCCTGCAAAT	GGGCTTGAAGCTCGTTGTTG
gi\|661567324	GATTGCACCCCAGGCAATCA	TTGTGCTTCACGTAGCCGTA
gi\|658009573	AGGCATTCCCTCAGGACTAC	TTCCGACTTCATCCACTGC
MdActin	TGACCGAATGAGCAAGGAAATTACT	TACTCAGCTTTGGCAATCCACATC

图1 抗、感病苹果品种华月和金冠应答轮纹病菌侵染的发病特征比较 R: 抗病品种华月; S: 感病品种金冠。Bars=1 cm

Figure 1 Comparison of morphological characteristics of resistant and susceptible apple cultivars Huayue and Golden Delicious in response to ring rot disease R: Resistant cultivar Huayue; S: Susceptible cultivar Golden Delicious. Bars=1 cm

图2 抗、感病苹果品种叶片应答轮纹病菌胁迫的差异表达蛋白(DEPs)分布 R: 抗病品种华月; S: 感病品种金冠

Figure 2 Distribution of differentially expressed proteins (DEPs) of apple leaves susceptible and resistant to ring rot disease, respectively R: Resistant cultivar Huayue; S: Susceptible cultivar Golden Delicious

图3 抗、感病品种应答轮纹病菌胁迫的差异表达蛋白(DEPs)的Gene Ontology (GO)分析

Figure 3 Enriched Gene Ontology (GO) analysis of differentially expressed proteins (DEPs) in apple leaves resistant and susceptible to ring rot disease, respectively

表2 差异表达蛋白(DEPs)的KEGG Pathway富集分析

Table 2 KEGG pathways analysis of differentially expressed proteins (DEPs)

No.	Pathway	Pathway ID	DEPs with pathway annotation (326 in total)	All proteins with pathway annotation (14916 in total)	P-value
1	Fatty acid biosynthesis	ko00061	2 (0.61%)	19 (0.13%)	0.011
2	Fatty acid elongation	ko00062	3 (0.92%)	9 (0.06%)	0.040
3	Arginine biosynthesis	ko00220	2 (0.61%)	16 (0.11%)	0.050
4	Alanine, aspartate and glutamate metabolism	ko00250	2 (0.61%)	23 (0.15%)	0.037
5	Cyanoamino acid metabolism	ko00460	2 (0.61%)	20 (0.13%)	0.022
6	Other glycan degradation	ko00511	2 (0.61%)	14 (0.09%)	0.011
7	Monoterpenoid biosynthesis	ko00902	5 (1.53%)	12 (0.08%)	0.035
8	Sesquiterpenoid and triterpenoid biosynthesis	ko00909	1 (0.31%)	2 (0.01%)	0.027
9	Nitrogen metabolism	ko00910	4 (1.23%)	28 (0.19%)	0.014
10	Sulfur metabolism	ko00920	1 (0.31%)	12 (0.08%)	0.036
11	Phenylpropanoid biosynthesis	ko00940	3 (0.92%)	47 (0.32%)	0.041
12	Isoquinoline alkaloid biosynthesis	ko00950	2 (0.61%)	16 (0.11%)	0.028
13	Biosynthesis of unsaturated fatty acids	ko01040	2 (0.61%)	9 (0.06%)	0.016
14	Fatty acid metabolism	ko01212	2 (0.61%)	32 (0.21%)	0.030
15	Ribosome	ko03010	13 (3.99%)	200 (1.34%)	0.005
16	RNA transport	ko03013	2 (0.61%)	33 (0.22%)	0.031
17	Homologous recombination	ko03440	1 (0.31%)	5 (0.03%)	0.039
18	Circadian rhythm-plant	ko04712	3 (0.92%)	9 (0.06%)	0.026

图4 抗、感病苹果叶片应答轮纹病菌胁迫的差异表达蛋白(DEPs)的亚细胞定位 Chlo: 叶绿体; Cysk: 细胞骨架; Cyto: 胞液; Extr: 胞外; Mito: 线粒体; Nucl: 细胞核; Plas: 质膜; Vacu: 液泡膜

Figure 4 Subcellular localization of differentially expressed proteins (DEPs) in apple leaves resistant and susceptible to ring rot disease, respectively Chlo: Chloroplast; Cysk: Cytoskeleton; Cyto: Cytosol; Extr: Extracellular; Mito: Mitochondria; Nucl: Nucleus; Plas: Plasma membrane; Vacu: Vacuolar membrane

图5 抗、感病苹果叶片轮纹病关键抗病相关蛋白编码基因的实时荧光定量PCR分析 (A) 类甜蛋白编码基因(MD04G1018400); (B) Mal d1编码基因(MD16G1160700); (C) β-1,3-葡聚糖酶编码基因(MD11G1189000); (D) 多酚氧化酶编码基因(MD10G1299100); (E) MLP423编码基因(MD16G1088600)。R: 抗病品种华月; S: 感病品种金冠; 不同小写字母表示抗病与感病品种间差异显著(P<0.05)。

Figure 5 Quantitative real-time PCR analysis of genes encoding key resistance-related proteins in apple leaves resistance and susceptible to ring rot disease (A) A gene encoding a thaumatin (MD04G1018400); (B) The gene encoding Mal d1 (MD16G1160700); (C) A gene encoding a beta-1,3-glucosidase (MD11G1189000); (D) A gene encoding a polyphenol oxidase (MD10G1299100); (E) The gene encoding MLP423 (MD16G1088600). R: Resistant cultivars Huayue; S: Susceptible cultivars Golden Delicious; Different lowercase letters indicate significant differences between resistant and susceptible cultivars (P<0.05).

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