‘春甜橘’及其突变体果皮差异相关蛋白质组分析

doi:10.3864/j.issn.0578-1752.2015.24.011

摘要/Abstract

摘要： 【目的】柑橘自然芽变材料是柑橘育种的重要来源。‘明柳甜橘’（Citrus reticuiata Blanco cv.Mingliutianju，MP）是从‘春甜橘’（C. reticuiata Blanco cv. Chuntianju，CP）中选育出的自然芽变品种。分析‘春甜橘’与其自然芽变‘明柳甜橘’果皮差异蛋白质，探讨导致两品种形态差异的原因。【方法】以花后12周和23周的‘明柳甜橘’和‘春甜橘’果皮为材料，采用双向电泳技术（Two-dimensional gel electrophoresis，2-DE）分离获得野生型与突变型差异表达蛋白点，利用MALDI-TOF-MS质谱鉴定技术分析相关差异蛋白质，并进行功能注释和生物信息学分析。【结果】将提取的果皮总蛋白质经双向电泳后，分别采用硝酸银和考马斯亮蓝染色法进行蛋白质凝胶染色，均获得了清晰的2-DE电泳图。2-DE图谱经ImageMaster 2D软件分析表明，硝酸银染色法能检测到约1 200个蛋白点，考马斯亮蓝染色法能检测到约500个蛋白质点，从中各选取20个变化丰度2倍以上且重复性好的蛋白点进行MALDI-TOF-MS质谱鉴定和数据库检索分析，共有33个蛋白质有功能注释，其中17个蛋白质在‘明柳甜橘’果皮中表达上调，16个表达下调。按照33个差异蛋白质的功能，可将其分为11类，它们分别参与糖类/能量代谢、胁迫/防御反应、核酸代谢、氨基酸代谢、转录、氮代谢、脂肪酸代谢、蛋白修饰与降解以及未知类。利用KOBAS（KEGG Orthology-Based Annotation System）在线功能分析平台对33个差异表达蛋白质进行信号通路分析，其中18个差异蛋白质有KO注释，共涉及31条信号通路，按照P值大小排列，类黄酮生物合成过程位居第一，说明该信号通路最有可能参与春甜橘芽变形成过程。【结论】综合分析‘春甜橘’和‘明柳甜橘’基因水平及蛋白质水平的差异，推测可能是由参与类黄酮生物合成过程的差异表达基因尿苷二磷酸葡萄糖基转移酶基因和差异表达蛋白咖啡酰辅酶A-O-甲基转移酶的差异表达导致了二者的表型差异。

关键词: 柑橘, 芽变, 双向电泳, 差异表达蛋白, MALDI-TOF/TOF-MS质谱鉴定技术

Abstract: 【Objective】 Bud mutation of citrus is one of the most important breeding technique for new cultivars. However, little is known about the mechanisms of citrus bud mutation, particularly at the molecular level. Mingliutianju (C. reticuiata Blanco cv. Mingliutianju, MP) is a new cultivar selected from Chuntianju (C. reticuiata Blancocv. Chuntianju, CP) bud mutation. To identify the differentially expressed proteins in peels of Chuntianju (C. reticuiata Blanco cv. Chuntianju, CP) and its bud mutant Mingliutianju (C. reticuiata Blanco cv. Mingliutianju, MP) and to clarify the possible mechanism of this phenotype bud mutation. 【Method】Two-dimensional gel electrophoresis (2-DE) combined with the mass spectrometry (MS) technology were applied to separate the differentially expressed proteins (DEPs) between the MP and CP using peels at 12 weeks and 23 weeks after flowering. Meanwhile, the identified differentially expressed proteins (DEPs) with fold-change of more than 2 were analyzed using MALDI-TOF-MS and Bioinformatics. 【Result】 Clear 2-DE gel maps of peels of both CP and MP were successfully obtained. In total, more than 1200 protein spots and 500 protein spots were detected by silver stained (SS) 2-DE gels and by Coomassie brilliant blue stained (CS) 2-DE gels respectively. Forty DEP spots (20 identified from SS 2-DE gels and 20 from CS 2-DE gels) were further analyzed through MALDI-TOF-MS. Totally, 33 annotated DEPs, including 17 up-regulated and 16 down-regulated DEPs, were identified in MP. Gene ontology analysis revealed that these 33 DEPs could be divided into 11 categories including carbohydrate/energy metabolism, stress/defense response, nucleotide metabolism, amino acid metabolism, transcription, nitrogen metabolism, lipid metabolism, unclassified, protein modification and degradation and unknown pathways. Moreover, by using the web-based platform KOBAS (KEGG Ontology-Based Annotation System), 18 of the 33 DEPs were successfully mapped to 31 KEGG pathways, among which the flavonol biosynthetic process pathway was ranked the first. 【Conclusion】 Intensive studies were conducted to identify the mechanism that lead to the morphological differences between CP and MP from both the gene expression and the protein expression levels. Results generated in this study suggested that the flavonol biosynthetic process play an essential role in CP’s spontaneous bud mutation process. Notably, the differential expression of 2 UDP-glucosyltransferase genes and 1 caffeoyl-CoA O-methyltransferase protein may contribute a lot to the morphological differences.

Key words: citrus, bud mutant, two-dimensional electrophoresis, differentially expressed proteins, MALDI-TOF/TOF-MS

曾继吾，邓贵明，高长玉，姜波，钟云，钟广炎，易干军. ‘春甜橘’及其突变体果皮差异相关蛋白质组分析[J]. 中国农业科学, 2015, 48(24): 4965-4978.

ZENG Ji-wu, DENG Gui-ming, GAO Chang-yu, JIANG Bo, ZHONG Yun, ZHONG Guang-yan, YI Gan-jun. Comparative Proteomic Analysis in Peels of Chuntianju (Citrus reticuiata Blanco) and Its Mutant[J]. Scientia Agricultura Sinica, 2015, 48(24): 4965-4978.

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参考文献

[1] Mendel K. Bud mutations in citrus and their potential commercial value. Proc Int Soc Citricult, 1981, 1: 86-89.

[2] 曾继吾, 彭成绩, 易干军, 杜观兴, 张绍平, 冯春添, 霍合强, 钟云, 周碧容, 黄永红. 晚熟柑橘新品种明柳甜橘. 园艺学报, 2006, 33(5): 1164.

Zeng J W, Peng C J, Yi G J, Du G X, Zhang S P, Feng C T, Huo H Q, Zhong Y, Zhou B R, Huang Y H. A new late ripening citrus variety Mingliu Tianju. Acta Horticulturae Sinica, 2006, 33(5): 1164. (in Chinese).

[3] O’Farrell P H. High resolution two-dimensional electrophoresis of proteins. The Journal of Biological Chemistry, 1975, 250(10): 4007-4021.

[4] Moore G. Oranges and lemons: clues to the taxonomy of citrus from molecular markers. Trends in Genetics, 2001, 17: 536-540.

[5] Fanizza G, Chaabane R, Ricciardi L, Resta P. Analysis of a spontaneous mutant and selected clones of cv. Italia (Vitis vinifera) by AFLP markers. Vitis, 2003, 42: 27-30.

[6] Tao N G, Wei J, Liu Y Z, Cheng Y J, Deng X X. Copia-like retrotransposons in a precocious mutant of trifoliate orange [Poncirus trifoliata (L.) Raf]. Journal of Horticultural Science & Biotechnology, 2006, 81: 1038-1042.

[7] Mase N, Iketani H &, Sato Y. Analysis of bud sport cultivars of peach (Prunus persica (L.) Batsch) by simple sequence repeats (SSR) and restriction landmark genomic scanning (RLGS). Journal of the Japanese Society for Horticultural Science, 2007, 76: 20-27.

[8] Zhang J Z, Ai X Y, Sun L M, Zhang D L, Guo W W, Deng X X, Hu C G. Molecular cloning and functional characterization of genes associated with flowering in citrus using an early-flowering trifoliate orange (Poncirus trifoliata L. Raf.) mutant. Plant Molecular Biology,2011, 76: 187-204.

[9] Albrecht U, Bowman K. Gene expression in Citrus sinensis (L.) Osbeck after infection with the bacterial pathogen Candidatus Liberibacter asiaticus causing Huanglongbing in Florida. Plant Science, 2008, 175: 291-306.

[10] Alos E, Roca M, Iglesias D J, Minguez-Mosquera M I, Damasceno C M B, Thannhauser T W. An evaluation of the basis and consequences of a stay-green mutation in the navel negra citrus mutant using transcriptomic and proteomic profiling and metabolite analysis. Plant Physiology, 2008, 147: 1300-1315.

[11] Liu Q, Zhu A D, Chai L J, Zhou W J, Yu K Q, Ding J, Xu J, Deng X X. Transcriptome analysis of a spontaneous mutant in sweet orange [Citrus sinensis (L.) Osbeck] during fruit development. Journal of Experimental Botany, 2009, 60: 801-813.

[12] Zhang J Z, Li Z M, Yao J L, Hu C G. Identification of flowering-related genes between early flowering trifoliate orange mutant and wild-type trifoliate orange (Poncirus trifoliata L. Raf.) by suppression subtraction hybridization (SSH) and macroarray. Gene, 2009, 430(1/2): 95-104.

[13] Qiu W M, Zhu A D, Wang Y, Chai L J, Ge X X, Deng X X, Guo W W. Comparative transcript profiling of gene expression between seedless Ponkan mandarin and its seedy wild type during floral organ development by suppression subtractive hybridization and cDNA microarray. BMC Genomics, 2012, 13: 397. doi:10.1186/1471-2164- 13-397.

[14] Zeng J W, Gao C Y, Deng G M, Jiang B, Yi G J. Transcriptome analysis of fruit development of a citrus late-ripening mutant by microarray. Scientia Horticulturae, 2012, 134: 32-39.

[15] 曾继吾, 曹水良, 姜波, 钟云, 邓贵明, 周碧容, 彭成绩, 易干军. 春甜橘及其芽变品种明柳甜橘特性的比较研究. 热带作物学报, 2012, 33(9): 1568-1573.

Zeng J W, Cao S L, Jiang B, Zhong Y, Deng G M, Zhou B R, Peng C J, Yi G J. Differences of biological characters between Mingliutianju (C. reticuiata Blanco cv. Mingliutianju) and Chuntianju (C. reticuiata Blancocv. Chuntianju). Chinese Journal of Tropical Crops, 2012, 33(9): 1568-1573. (in chinese)

[16] Carpentier S C, Witters E, Laukens K, Deckers P, Swennen R, Panis B. Preparation of protein extracts from recalcitrant plant tissues: an evaluation of different methods for two-dimensional gel electrophoresis analysis. Proteomics, 2005, 5: 2497-2507.

[17] 邓贵明, 曾继吾, 姜波, 钟云, 周碧容, 易干军, 彭新湘. 柑桔叶片和果皮总蛋白质提取及双向电流体系的建立. 热带亚热带植物学报, 2012, 20(1): 19-25.

Deng G M, Zeng J W, Jing B, Zhong Y, Zhou B R, Yi G J, Peng X X. Extraction of total proteins from leaves and peels of Citrus reticulata and establishment of two-dimensional electrophoresis system. Journal of Tropical and Subtropical Botany, 2012, 20(1): 19-25. (in Chinese)

[18] Bradford M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry ,1976, 7(72): 248-254.

[19] Li Wen, Liu G, Li S Q, Wan C X, Tao J, Xu K Y, Zhang Z J, Zhu Y G. Proteomic analysis of anthers from Honglian cytoplasmic male sterility line rice and its corresponding maintainer and hybrid. Botanical Studies, 2007, 48(3): 293-309.

[20] Canovas F M, Dumas-Gaudot E, Recorbet G, Jorrin J, Mock H P, Rossignol M. Plant proteome analysis. Proteomics, 2004, 4: 285-298.

[21] 李肖芳, 韩和平, 王旭初, 范鹏祥, 李银心. 适用于盐生植物的双向电泳样品制备方法. 生态学报, 2006, 6: 1849-1853.

Li X F, Han H P, Wang X C, Fan P X, Li Y X. A protein extraction method suitable for two_dimensional electrophoresis analysis of halophytes. Acta Ecologica Sinica, 2006, 26(6): 1848-1853. (in Chinese)

[22] Isaacson T, Damasceno C M, Saravanan R S, He Y, Catalá C, Saladié M, Rose J K. Sample extraction techniques for enhanced proteomic analysis of plant tissues. Nature Protocols, 2006, 1(2): 769-774.

[23] Lee J, Katari G, Sachidanandam R. GO bar: a gene ontology based analysis and visualization tool for gene sets. BMC Bioinformatics,2005, 6: 189. http://www.biomedcentral.com/1471-2105/6/189.

[24] Smith B, Williams J, Steffen S. The ontology of the gene ontology. In American Medical Informatics Association, 2003: 609-613.

[25] Fang D Q, Roose M L. Identification of closely related citrus cultivars with inter-simple sequence repeat markers. Theoretical and Applied Genetics, 1997, 95: 408-417.

[26] Breto M P, Ruiz C, Pina J A, Asins M J. The diversification of Citrus clementina Hort, a vegetatively propagated crop species. Molecular Phylogenetics and Evolution, 2001, 21: 285-293.

[27] Mukesh J, Challa G, Annapurna B. Comprehensive expression analysis suggests overlapping and specific roles of rice glutathione S-transferase genes during development and stress responses. BMC Genomics. http://www.biomedcentral.com/1471-2164/11/73.

[28] Liu Q, Xu J, Liu Y Z. Zhao X L, Deng X X, Guo L L, Gu J Q. A novel bud mutation that confers abnormal patterns of 5 lycopene accumulation in sweet orange fruit (Citrus sinensis L. Osbeck). Journal of Experimental Botany, 2007, 58: 4161-4171.

[29] Xu Q, Liu Y L, Zhu A D, Wu X M, Ye J L, Yu K Q, Guo W W, Deng X X. Discovery and comparative profiling of micro RNAs in a sweet orange red-flesh mutant and its wild type. BMC Genomics, 2010, 11: 246.

[30] Pan Z Y, Liu Q, Yun Z, Guan R, Zeng W F, Xu Q, Deng X X. Comparative proteomics of a lycopene-accumulating mutant reveals the important role of oxidative stress on carotenogenesis in sweet orange (Citrus sinensis [L.] osbeck). Proteomics, 2009, 9: 5455-5470.

[31] Xu Q, Yu K Q, Zhu A D, Ye J L, Liu Q, Zhang J C, Deng X X. Comparative transcripts profiling reveals new insight into molecular processes regulating lycopene accumulation in a sweet orange (Citrus sinensis) red-flesh mutant. BMC Genomicsdoi: 10.1186/ 1471-2164-10-540., 2009, 10: 540.

[32] Zhang J Z, Ai X Y, Sun L M, Zhang D L, Guo W W, Deng X X, Hu C G. Transcriptome profile analysis of flowering molecular processes of early flowering trifoliate orange mutant and the wild-type [Poncirus trifoliata (L.) Raf.] by massively parallel signature sequencing. BMC Genomics, 2011, 12: 63. doi: 10.1186/1471-2164-12-63.

[33] Wu J X, Xu Z L, Zhang Y J, Chai L J, Yi H L, Deng X X. An integrative analysis of the transcriptome and proteome of the pulp of a spontaneous late-ripening sweet orange mutant and its wild type improves our understanding of fruit ripening in citrus. Journal of Experimental Botany, 2014, 65(6): 1651-1671.

[34] Yu K Q, Xu Q, Da X L, Guo F, Ding Y D, Deng X X. Transcriptome changes during fruit development and ripening of sweet orange (Citrus sinensis). BMC Genomics, 2012, 13: 10. http://www. biomedcentral.com/1471-2164/12/63

[35] Taylor L P, Grotewold E . Flavonoids as developmental regulators. Curr. Opin. Plant Biol, 2005, 8: 317-323.

[36] Treutter D. Significance of flavonoids in plant resistance. menhancement of their biosynthesis. Plant Biology, 2005, 7: 581-591.

[37] Ringli C, Bigler L, Kuhn B, Leiber R, Diet A, Santelia D, Frey B, Pollmann S, Klein M. The modified flavonol glycosylation profile in the Arabidopsis rol1 mutants results in alterations in plant growth and cell shape formation. The Plant Cell, 2008, 20: 1470-1481.

[38] Ylstra B, Moreno R M, Stöger E, van Tunen A J, Vicente O, Mol J N, Heberle-Bors E. Flavonols stimulate development, germination, and tube growth of tobacco pollen. Plant physiology, 1992, 100: 902-907.

[39] Napoli C, Fahy D, Wang H, Taylor L P. White anther: a petunia mutant that abolishes pollen flavonol accumulation, induces male sterility, and is complemented by a chalcone synthase transgene. Plant Physiology, 1999, 120: 615-622.

[40] Mo Y, Nagel C, Taylor L. Biochemical complementation of chalcone synthase mutants defines a role for flavonols in functional pollen. Proceedings of the National Academy of Sciences USA, 1992, 89: 7213.

[41] Moriguchi T, Kita M, Ogawa K, Tomono Y, Endo T, Omura M. Flavonol synthase gene expression during citrus fruit development. Physiologia Plantarum, 2002, 114: 251-258.

[42] Buer C, Muday G. The transparent test 4 mutation prevents flavonoid synthesis and alters auxin transport and the response of Arabidopsis roots to gravity and light. The Plant Cell, 2004, 16: 1191-1205.

[43] Peer W, Bandyopadhyay A, Blakeslee J, Makam S, Chen R, Masson P, Murphy A. Variation in expression and protein localization of the PIN family of auxin efflux facilitator proteins in flavonoid mutants with altered auxin transport in Arabidopsis thaliana. The Plant Cell, 2004, 16: 1898-1911.