茶树应答茶小绿叶蝉为害的蛋白质组分析

王芬; 喻佩; 翟媛; 李传刚; 罗思妤; 裴会敏

茶树应答茶小绿叶蝉为害的蛋白质组分析

黔南民族师范学院生物科学与农学院，贵州　都匀　558000

基金项目: 国家自然基金项目（32260080）；贵州省教育厅项目（QNYSKYTD2018006）；黔南民族师范学院项目(2019xjg0303、2018xjg0520、QNSY2018ZJ006、QNYSXXK2018005)。

详细信息

作者简介:
王芬（1984 —），女，博士，教授，主要从事茶学研究，E-mail：fenmin521@163.com

通讯作者:
裴会敏（1977 —），男，硕士，副教授，主要从事茶学研究，E-mail: huiminpei@163.com

中图分类号: S435
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出版历程
- 收稿日期: 2024-03-25
- 修回日期: 2024-05-09
- 网络出版日期: 2024-10-31

Proteomics analysis on response of Duyun Maojian native tea plant to the invasion of Empoasca vitis Gothe

School of Biological Science and Agriculture, Qiannan Normal University for Nationalities, Duyun, 55800

摘要

摘要: 目的探明都匀毛尖本地种茶树响应茶小绿叶蝉为害相关的蛋白质。方法以被茶小绿叶蝉侵染0、12、24、36、48 h的都匀毛尖本地种茶树叶片为材料，利用串联质谱标签（tandem mass tag, TMT）结合液相色谱-串联质谱（LC-MS/MS）技术对茶小绿叶蝉侵染茶树叶片后的蛋白质进行定性定量分析。结果共鉴定出2 893个蛋白质，0 h与12 h、24 h、36 h、48 h之间分别有0、1、848、849个差异蛋白质，共有2 622个蛋白质被注释，其中2 360个蛋白注释到GO数据库，1 232个注释到KEGG数据库。GO和KEGG分析表明茶小绿叶蝉侵染茶树时可能通过差异蛋白质4-二磷酸胞基-2-c-甲基-d-赤藓糖醇激酶、法尼基二磷酸合成酶、香叶基二磷酸合成酶、香叶基香叶基二磷酸合成酶、过氧化物酶、果胶酯酶、丙二烯氧化环化酶、倍半萜类、三萜类、单萜类、二萜类化合物以及倍半萜和三萜生物合成途径、萜类骨架生物合成途径和单萜生物合成途径来抵御害虫的侵染。结论筛选出4个差异蛋白质（4-二磷酸胞基-2-c-甲基-d-赤藓糖醇激酶、法尼基二磷酸合成酶、香叶基二磷酸合成酶和香叶基香叶基二磷酸合成酶）和萜类化合物可能在应答防御小绿叶蝉侵害时具有重要的作用。研究结果可能为揭示茶树应答茶小绿叶蝉为害的分子机制提供理论依据。
- 茶树 /
- 茶小绿叶蝉 /
- TMT /
- 差异蛋白 /
- 抵御
Abstract: Objective In order to study the proteins related to the response of Duyun Maojian native tea plant infected by Empoasca vitis Gothe. Method The leaves of Duyun Maojian native tea plant were infected by E. vitis Göthe for 0 h, 12 h, 24 h, 36 h and 48 h, were used as research materials. Tandem mass tag (TMT) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) technique was used to qualitatively and quantitatively analyze the proteins of tea leaves infected with E. vitis Göthe. Results We identified a total of 2,893 proteins. There were 0, 1, 848, and 849 differential expression proteins (DEPs) between 0 h and 12 h, 0 h and 24 h, 0 h and 36 h, 0 h and 48 h, respectively. Furthermore, 2,622 proteins were annotated, of which 2,360 proteins were annotated to the GO database and 1,232 proteins were annotated to KEGG database. GO and KEGG analyses showed that tea plants may be in resistance to E. vitis Göthe infestation through CMK, FPPS, GPPS, GGPPS, peroxidase, pectinesterase, and allene-oxide cyclase, sesquiterpenoid, triterpeoid, monoterpenoid, diterpenoid, sesquiterpenoid and triterpenoid biosynthesis, terpenoid backbone biosynthesis, and monoterpenoid biosynthesis. Conclusion Four DEPs (CMK, FPPS, GPPS, and GGPPS) and terpenoids may play important roles in the response and defense against E. vitis Göthe. The research results may provide theoretical basis for revealing the molecular mechanism of tea plant response to the harm of E. vitis Göthe.
- tea plant /
- Empoasca vitis Gothe /
- proteomics /
- tandem mass tag /
- differential expression protein

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图 1 TMT蛋白质组学鉴定的多肽长度、数量、分子质量和覆盖率分布

Figure 1. The distribution of peptide length, peptide number, proteins molecular weight, and coverage

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图 2 鉴定出的蛋白质（A. 主成分分析 B. GO功能分类 C. KEGG分类 D. COG分类）

Figure 2. Analyses of identified proteins(A. Principal component analysis; B. GO annotation; C. KEGG annotation; D. COG annotation)

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图 3 差异表达蛋白质火山图

Figure 3. Volcano plot of DEPs

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图 4 差异蛋白质的GO富集网络

A. 0 h与36 h差异蛋白质的GO富集网络；B. 0 h与48 h差异蛋白质的GO富集网络；C.差异蛋白质GO富集网络；D. 0 h与36 h和0 h与48 h共同的差异蛋白质GO富集网络；E. 0 h与36 h和0 h与48 h不同的差异蛋白质GO富集网络。

Figure 4. GO enrichment networks of DEPs

A 0 h vs 36 h; B. 0 h vs 48 h; C. Merged GO enrichment network of DEPs; D. GO enrichment network of shared DEPs between 0-36 h and 0-48 h; E. GO enrichment network of distinct DEPs between 0-36 h and 0-48 h.

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图 5 差异蛋白质KEGG功能分类（A. 0 h vs 36 h；B. 0 h vs 48 h）

注：1：内吞作用；2：过氧化物酶体；3：吞噬体；4：蛋白酶体；5：蛋白质输出；6：内质网蛋白加工；7：RNA降解；8：泛素介导的蛋白水解；9：核苷酸切除修复；10：剪接体；11：氨酰tRNA生物合成；12：RNA转运；13：核糖体；14：真核生物中核糖体生物发生；15：mRNA监测途径；16：丙氨酸、天冬氨酸和谷氨酸代谢；17：精氨酸和脯氨酸代谢；18：半胱氨酸和蛋氨酸代谢；19：甘氨酸、丝氨酸和苏氨酸的代谢；20：苯丙氨酸代谢；21：苯丙氨酸、酪氨酸和色氨酸:生物合成；22：酪氨酸代谢；23：苯丙素:生物合成；24：氨基糖和核苷酸糖代谢；25：柠檬酸循环；26：果糖和甘露糖代谢；27：糖酵解:/糖质新生；28：乙醛酸盐和二羧酸盐代谢；29：戊糖磷酸途径；30：丙酸代谢；31：丙酮酸代谢；32：淀粉和蔗糖代谢；33：光合生物中:碳固定；34：氧化磷酸化；35：光合作用；36：氨基酸生物合成；37：次级代谢生物合成；38：碳代谢；39：脂肪酸代谢；40：代谢途径；41：脂肪酸生物合成；42：脂肪酸降解；43：α-亚麻酸代谢；44：碳池；45：卟啉和叶绿素代谢；46：维生素B6代谢；47：谷胱甘肽代谢；48：嘌呤代谢；49：嘧啶代谢；50：植物-病原互作；51：精氨酸生物合成；52：烟酸和烟酰胺代谢；53：萜类骨架生物合成。

Figure 5. KEGG classifications of DEPs（A. 0 h and 36 h; B. 0 h and 48 h）

Note: 1: Endocytosis; 2: peroxisome; 3: phagosome; 4: proteasome; 5: protein export; 6: protein processing in endoplasmic reticulum; 7: RNA degradation; 8: ubiquitin mediated proteolysis; 9: nucleotide excision repair; 10: Spliceosome; 11: aminoacyl-tRNA biosynthesis; 12: RNA transport; 13: Ribosome; 14: Ribosome biogenesis in eukaryotes; 15: mRNA surveillance pathway; 16: Alanine, aspartate and glutamate metabolism; 17: Arginine and proline metabolism; 18: Cysteine and methionine metabolism; 19: Glycine, serine, and threonine metabolism; 20: Phenylalanine metabolism; 21: Phenylalanine, tyrosine and tryptophan biosynthesis; 22: Tyrosine metabolism; 23: Phenylpropanoid biosynthesis; 24: Amino sugar and nucleotide sugar metabolism; 25: Citrate cycle (TCA cycle); 26: Fructose and mannose metabolism; 27: Glycolysis/Gluconeogenesis; 28: Glyoxylate and dicarboxylate metabolism; 29: Pentose phosphate pathway; 30: Propanoate metabolism; 31: Pyruvate metabolism; 32: Starch and sucrose metabolism; 33: Carbon fixation in photosynthetic organisms; 34: Oxidative phosphorylation; 35: Photosynthesis; 36: Biosynthesis of amino acids; 37: Biosynthesis of secondary metabolites; 38: Carbon metabolism; 39: Fatty acid metabolism; 40: Metabolic pathways; 41: Fatty acid biosynthesis; 42: Fatty acid degradation; 43: alpha-Linolenic acid metabolism; 44: One carbon pool by folate; 45: Porphyrin and chlorophyll metabolism; 46: Vitamin B6 metabolism; 47: Glutathione metabolism; 48: Purine metabolism; 49: Pyrimidine metabolism; 50: Plant-pathogen interaction; 51: Arginine biosynthesis; 52: Nicotinate and nicotinamide metabolism; 53: Terpenoid backbone biosynthesis.

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图 6 都匀毛尖本地种茶树中的萜类化合物生物合成途径响应茶小绿叶蝉的侵染

Figure 6. Responses of the terpenoid biosynthetic pathway to Empoasca vitis Göthe infestation.

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表 1 差异表达蛋白质

Table 1. Differential expression proteins

差异蛋白 Differential expression protein	差异蛋白数 The number of DEPs	上调 Up-regulated	下调 Down-regulated
0 h vs 12 h	0	0	0
0 h vs 24 h	1	1	0
0 h vs 36 h	848	385	463
0 h vs 48 h	849	374	475

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参考文献(34)

[1]	黄瑞, 夏华富, 代洪苇, 等. 茶树开花相关基因家族的克隆及CsMFT基因的可变剪切分析 [J]. 西北植物学报, 2021, 41(12):2002−2013. doi: 10.7606/j.issn.1000-4025.2021.12.2002 HUANG R, XIA H F, DAI H W, et al. Cloning of flowering-related genes and alternative splicing analysis of CsMFT in tea plant [J]. Acta Botanica Boreali-Occidentalia Sinica, 2021, 41(12): 2002−2013. (in Chinese) doi: 10.7606/j.issn.1000-4025.2021.12.2002
[2]	杨家干. 都匀毛尖成分及其部分生理功能研究[D]. 广州: 华南农业大学, 2016. YANG J G. Study on constituent and part of physiological function from DuyunMaojian [D]. Guangzhou: South China Agricultural University, 2016. (in Chinese)
[3]	邬子惠, 王梦馨, 潘铖, 等. 茶小绿叶蝉发生规律与绿色防控研究进展 [J]. 茶叶通讯, 2021, 48(2):200−206,252. doi: 10.3969/j.issn.1009-525X.2021.02.002 WU Z H, WANG M X, PAN C, et al. Research progress and prospect of occurrence regularity and green control of tea green leafhopper [J]. Journal of Tea Communication, 2021, 48(2): 200−206,252. (in Chinese) doi: 10.3969/j.issn.1009-525X.2021.02.002
[4]	李金玉, 王庆森, 李良德等. 茶小绿叶蝉种名变更及其种群发生与生物生态环境关系的研究进展 [J]. 福建农业学报, 2022, 37(1):123−130. LI J Y, WANG Q S, LI L D, et al. Research progress on the dominant species identification of tea green leafhopper and the relationship between its population and the biological and ecological environment [J]. Fujian Journal of Agricultural Sciences, 2022, 37(1): 123−130. (in Chinese)
[5]	潘杰. 茶树抗性相关蛋白(Cs-cystatin, Cs-Rip)对假眼小绿叶蝉消化道组织的影响[D]. 厦门: 厦门大学, 2014. PAN J. Toxic effects of resistance associated proteins (Cs-cystatin, Cs-Rip) of tea on empoasca vitis [D]. Xiamen: Xiamen University, 2014. (in Chinese)
[6]	杨会敏. 茶树抗假眼小绿叶蝉相关基因的克隆及表达研究[D]. 信阳: 信阳师范学院, 2010. YANG H M. Cloning and expression of anti-Empoasca vitis-related genes in tea [D]. Xinyang: Xinyang Normal University, 2010. (in Chinese)
[7]	王梦馨. 叶蝉诱导的茶树重要防御相关基因的功能解析和互利素鉴定应用[D]. 南京: 南京农业大学, 2012. WANG M X. Function analysis of defence-related genes of tea plant (Camellia Sinensis) and identification and application of its synomones induced by tea green leafhopper [D]. Nanjing: Nanjing Agricultural University, 2012. (in Chinese)
[8]	任倩倩. 抗、感茶树品种对茶小绿叶蝉取食诱导的防御反应[D]. 福州: 福建农林大学, 2020. REN Q Q. The defense strategies of resistant and susceptible tea cultivars in response to Empoasca onukii feeding [D]. Fuzhou: Fujian Agriculture and Forestry University, 2020. (in Chinese)
[9]	MEI X, LIU X Y, ZHOU Y, et al. Formation and emission of linalool in tea (Camellia sinensis) leaves infested by tea green leafhopper (Empoasca (Matsumurasca) onukii Matsuda) [J]. Food Chemistry, 2017, 237: 356−363. doi: 10.1016/j.foodchem.2017.05.124
[10]	ZHAO X M, CHEN S, WANG S S, et al. Defensive responses of tea plants (Camellia sinensis) against tea green leafhopper attack: A multi-omics study [J]. Frontiers in Plant Science, 2019, 10: 1705.
[11]	WASINGER V C, CORDWELL S J, CERPA-POLJAK A, et al. Progress with gene-product mapping of the Mollicutes: Mycoplasma genitalium [J]. Electrophoresis, 1995, 16(7): 1090−1094.
[12]	陈玲玲. 敖汉苜蓿小花与种子响应硼胁迫的蛋白质组学与代谢组学分析[D]. 北京: 中国农业大学, 2017. CHEN L L. Proteomic and metabolomics analysis of flowers and seeds of Medicago sativa L. cv. “Aohan” response to boron stress [D]. Beijing: China Agricultural University, 2017. (in Chinese)
[13]	程晓梅, 黄建安, 刘仲华, 等. 茶树蛋白质组学研究进展 [J]. 湖南农业科学, 2014(23):28−31. CHENG X M, HUANG J A, LIU Z H, et al. Research progress in tea proteomics [J]. Hunan Agricultural Sciences, 2014(23): 28−31. (in Chinese)
[14]	庄重光. 不同水分处理下铁观音茶树的生理机制及其差异蛋白质组学研究[D]. 福州: 福建农林大学, 2008. ZHUANG C G. A study on physiological mechanism and differential proteomics of tea (Camelllia sinensis (L. ) O. Kuntze) under different watering regimes [D]. Fuzhou: Fujian Agriculture and Forestry University, 2008. (in Chinese)
[15]	郭春芳. 水分胁迫下茶树的生理响应及其分子基础[D]. 福州: 福建农林大学, 2008. GUO C F. Physiological response and molecular basis of tea plant (Camellia sinensis) exposed to water stress [D]. Fuzhou: Fujian Agriculture and Forestry University, 2008. (in Chinese)
[16]	LI J K, CHEN J, ZHANG Z H, et al. Proteome analysis of tea pollen (Camellia sinensis) under different storage conditions [J]. Journal of Agricultural and Food Chemistry, 2008, 56(16): 7535−7544. doi: 10.1021/jf800885z
[17]	CHIEN H J, YANG M M, WANG W C, et al. Proteomic analysis of “Oriental Beauty” oolong tea leaves with different degrees of leafhopper infestation [J]. Rapid Communications in Mass Spectrometry, 2020, 34(15): e8825. doi: 10.1002/rcm.8825
[18]	邹瑶, 齐桂年, 陈盛相, 等. 蛋白质组学关键技术及其在茶树研究中的应用 [J]. 生物技术通报, 2012, 28(7):60−64. ZOU Y, QI G N, CHEN S X, et al. Key technology of proteomics and its application in tea plant [J]. Biotechnology Bulletin, 2012, 28(7): 60−64. (in Chinese)
[19]	周琼琼, 孙威江. 茶树总蛋白质提取方法及双向电泳体系的建立 [J]. 热带作物学报, 2014, 35(8):1517−1522. doi: 10.3969/j.issn.1000-2561.2014.08.012 ZHOU Q Q, SUN W J. Establishment of extraction methods and two-dimensional electrophoresis conditions for proteomic analysis of Camellia sinensis [J]. Chinese Journal of Tropical Crops, 2014, 35(8): 1517−1522. (in Chinese) doi: 10.3969/j.issn.1000-2561.2014.08.012
[20]	HU Y G, LU Y Z, LU J. Comparative proteomics analysis of tea leaves exposed to subzero temperature: Molecular mechanism of freeze injury [J]. International Journal of Agricultural & Biological Engineering, 2013, 6(4): 27−34.
[21]	XU Q S, WANG Y, DING Z T, et al. Aluminum induced physiological and proteomic responses in tea (Camellia sinensis) roots and leaves [J]. Plant Physiology and Biochemistry, 2017, 115: 141−151. doi: 10.1016/j.plaphy.2017.03.017
[22]	吴致君. 茶树叶发育相关miRNA和转录因子挖掘及采后不同萎凋蛋白质组差异分析[D]. 南京: 南京农业大学, 2017. WU Z J. Discovery of leaf development-related miRNAs and transcription factors and analysis of the proteome differences of postharvest leaves under different treatments in tea plant (Camellia sinensis) [D]. Nanjing: Nanjing Agricultural University, 2017. (in Chinese)
[23]	任燕. 茶树雌蕊自交和杂交的差异蛋白质组学初步研究[D]. 南京: 南京农业大学, 2013. REN Y. Primary differential proteome analysis of self-and cross-pollinated pistils in tea plant (Camellia sinensis) [D]. Nanjing: Nanjing Agricultural University, 2013. (in Chinese)
[24]	BAI B, TAN H, PAGALA V R, et al. Chapter twenty deep profiling of proteome and phosphoproteome by isobaric labeling, extensive liquid chromatography, and mass spectrometry [J]. Methods in Enzymology, 2017, 585: 377−395.
[25]	TYERS M, MANN M. From genomics to proteomics [J]. Nature, 2003, 422(6928): 193−197. doi: 10.1038/nature01510
[26]	LI Z, ADAMS R M, CHOUREY K, et al. Systematic comparison of label-free, metabolic labeling, and isobaric chemical labeling for quantitative proteomics on LTQ Orbitrap Velos [J]. Journal of Proteome Research, 2012, 11(3): 1582−1590. doi: 10.1021/pr200748h
[27]	JIN S, REN Q Q, LIAN L L, et al. Comparative transcriptomic analysis of resistant and susceptible tea cultivars in response to Empoasca onukii (Matsuda) damage [J]. Planta, 2020, 252(1): 10. doi: 10.1007/s00425-020-03407-0
[28]	陈升龙. 诱导茶树抗茶小绿叶蝉挥发物筛选体系的建立及反式: 橙花叔醇诱抗机制初探[D]. 北京: 中国农业科学院, 2020 CHEN S L. Establishment of screening system for volatiles that induces tea plant defense against tea green leafhopper and preliminary study on (E)-nerolidol induction mechanism [D]. Beijing: Chinese Academy of Agricultural Sciences, 2020. (in Chinese)
[29]	梅瑜, 王继华, 蔡时可, 等. 金线莲应答高温胁迫的蛋白质组学分析 [J]. 江苏农业学报, 2020, 36(6):1389−1397. doi: 10.3969/j.issn.1000-4440.2020.06.006 MEI Y, WANG J H, CAI S K, et al. Proteomics analysis on Anoectochilus roxburghii in response to high temperature stress [J]. Jiangsu Journal of Agricultural Sciences, 2020, 36(6): 1389−1397. (in Chinese) doi: 10.3969/j.issn.1000-4440.2020.06.006
[30]	袁清华. 茶树“信阳10号”生态休眠不同阶段的蛋白质组学研究[D]. 信阳: 信阳师范学院, 2020. YUANG Q. Proteomics research of ecodormancy of tea plant variety ‘Xinyang 10’ [D]. Xinyang: Xinyang Normal University, 2020. (in Chinese)
[31]	YANG Y X, AHAMMED G J, WU C J, et al. Crosstalk among jasmonate, salicylate and ethylene signaling pathways in plant disease and immune responses [J]. Current Protein & Peptide Science, 2015, 16(5): 450−461.
[32]	李新岗, 刘惠霞, 黄建. 虫害诱导植物防御的分子机理研究进展 [J]. 应用生态学报, 2008, 19(4):893−900. LI X G, LIU H X, HUANG J. Molecular mechanisms of insect pests-induced plant defense [J]. Chinese Journal of Applied Ecology, 2008, 19(4): 893−900. (in Chinese)
[33]	BLOCK A K, VAUGHAN M M, SCHMELZ E A, et al. Biosynthesis and function of terpenoid defense compounds in maize (Zea mays) [J]. Planta, 2019, 249(1): 21−30. doi: 10.1007/s00425-018-2999-2
[34]	NINKUU V, ZHANG L, YAN J P, et al. Biochemistry of terpenes and recent advances in plant protection [J]. International Journal of Molecular Sciences, 2021, 22(11): 5710. doi: 10.3390/ijms22115710