玉米种胚内质网胁迫相关基因对人工老化处理的响应

doi:10.3864/j.issn.0578-1752.2016.03.003

摘要/Abstract

摘要： 【目的】在植物中，内质网胁迫（endoplasmic reticulum stress，ERS）和未折叠蛋白应答（unfolded protein response，UPR）参与环境胁迫响应过程，然而，玉米种子老化过程中内质网胁迫相关基因表达情况尚未见报道。文章利用基因数字表达谱技术探究玉米种子老化过程中内质网胁迫相关基因表达规律，以期为揭示种子衰老的分子机制提供理论依据。【方法】以玉米杂交种郑单958种子为材料，采用高温（45℃）高湿（相对湿度100%）的方法进行人工老化处理。分别提取未老化处理（对照）和老化处理3 d的玉米种胚总RNA，利用Illumina HiSeqTM 2000平台进行高通量测序。去除原始数据中的接头序列、包含模糊碱基的序列以及低质量序列，获得Clean reads，利用短序列比对软件SOAPaligner/ SOAP2将Clean Reads分别比对到玉米参考基因组和参考基因序列，采用RPKM（reads per kb per million reads）方法计算基因的表达量，根据FDR（false discovery rate）＜0.001和|log2 ratio(T/CK)|≥1的标准筛选差异表达的基因，对获得的差异表达基因（differentially expressed genes，DEGs）进行KEGG（kyoto encyclopedia of genes and genomes）数据库功能注释分析，筛选出响应人工老化的内质网胁迫相关差异表达基因。利用qRT-PCR技术定量分析内质网胁迫相关基因在不同人工老化时间内的表达特性。【结果】基因数字表达谱鉴定结果表明，有104个差异表达基因在人工老化过程中参与内质网蛋白质加工（protein processing in endoplasmic reticulum）通路，其中内质网胁迫相关基因有97个（81个上调表达，16个下调表达）。对差异表达基因功能注释分析表明，内质网胁迫的标志性蛋白基因BiP以及分子伴侣蛋白基因CRT、CNT和GRP94等显著上调表达。参与内质网相关性降解（endoplasmic reticulum-associated degradation，ERAD）途径的有83个差异表达基因（70个上调，13个下调），其中启动ERAD途径的关键酶基因EDEM （ER degradation enhancing mannosidase I-like protein）下调，参与蛋白泛素化的E2泛素结合酶基因UbcH5、E3泛素连接酶基因Hrd1和Doa10等也发生显著的表达变化。qRT-PCR结果表明，内质网胁迫相关基因在不同人工老化时间内表现表达多样性和复杂性。【结论】人工老化处理能造成玉米种胚细胞发生内质网胁迫。细胞通过上调分子伴侣基因表达和诱导ERAD途径响应内质网胁迫，但ERAD途径受阻可能引起错误折叠蛋白聚集，从而进一步加剧细胞损伤，最终导致种子活力降低甚至丧失。

关键词: 玉米, 种子老化, 差异表达基因, 内质网胁迫, 内质网相关性降解

Abstract: 【Objective】 Endoplasmic reticulum (ER) stress and unfolded protein response (UPR) are involved in plant responses to environmental stresses. However, the expression of ER stress-related genes during maize seed aging has not been reported. In this study, the expression of ER stress-related genes during maize seed aging was investigated by Digital Gene Expression Profile (DGE) to provide theoretical support for clarifying the molecular mechanism of seed deterioration.【Method】 Hybrid maize (Zea mays L.) cultivar Zhengdan 958 seeds were used as experimental material and treated by artificial aging treatment (45℃, 100% relative humidity). DGE analysis was carried out on the Illumina Hiseq 2000 platform using total RNA extracted from 3 d artificial aging treatment and the untreated embryos (CK) of maize seeds. The reads with adaptor and ambiguous sequences, and the low-quality reads were filtered out to obtain the high quality clean reads. Clean reads were mapped to the maize reference genome and genes database using SOAPaligner/SOAP2. The gene expression level was calculated by the RPKM (Reads Per kb per million reads) method. A combination of FDR ＜0.001 and the absolute value of |log₂ ratio (T/CK)|≥1 was used as the threshold to determine the significance of gene expression difference. All differentially expressed genes (DEGs) were assigned to the pathways in KEGG (Kyoto Encyclopedia of Genes and Genomes) database and searched for the differentially expressed genes related to ER stress. Quantitative real-time PCR was performed to analyze the expression patterns of ER stress-related genes in the different artificial aging times.【Result】 Analysis of the DGE revealed that 104 DEGs were relevant to the protein processing in ER during the process of artificial aging treatment. A total of 97 DEGs related to ER stress including 81 and 16 genes respectively up- and down-regulated were screened out. The expression levels of ER stress marker BiP gene, as well as ER chaperones, such as CRT, CNT, GRP94 genes were considered to be significantly up-regulated. In particular, 83 DEGs were involved in ER-associated degradation (ERAD) pathway, including 70 up-regulated and 13 down-regulated DEGs. Among them, gene encoding EDEM (ER degradation enhancing mannosidase I-like protein) which is a rate-limiting enzyme of ERAD pathway was down-regulated. Genes involved in protein ubiquitination were altered in expression, including E2 ubiquitin-conjugating enzyme UbcH5, E3 ubiquitin-ligases Hrd1 and Doa10. The results of qRT-PCR analysis validated the diversity and complexity of ER stress-related genes expression in different artificial aging times.【Conclusion】 Artificial aging treatment can cause endoplasmic reticulum stress in maize embryo. The cell can respond to ER stress by inducing up-regulation of ER chaperones genes and activation of the ERAD pathway. Inhibition of ERAD pathway resulted in the accumulation of misfolded proteins in the ER with these leading to further cell damage and subsequently accelerating the loss of seed vigor.

Key words: maize, seed aging, differential expressed genes, ER stress, ERAD

曹广灿，林一欣，薛梅真，邢芦蔓，吕伟增，杨伟飞，陈军营. 玉米种胚内质网胁迫相关基因对人工老化处理的响应[J]. 中国农业科学, 2016, 49(3): 429-442.

CAO Guang-can, LIN Yi-xin, XUE Mei-zhen, XING Lu-man, Lü Wei-zeng, YANG Wei-fei, CHEN Jun-ying . Responses of Endoplasmic Reticulum Stress-Related Genes in Maize Embryo to Artificial Aging Treatment[J]. Scientia Agricultura Sinica, 2016, 49(3): 429-442.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2016.03.003

https://www.chinaagrisci.com/CN/Y2016/V49/I3/429

参考文献

[1] Schröder M, Kaufman R J. ER stress and the unfolded protein response. Mutation Research, 2005, 569: 29-63.

[2] Zhao L H, Ackerman S L. Endoplasmic reticulum stress in health and disease. Current Opinion in Cell Biology, 2006, 18: 444-452.

[3] Xu C Y, Bailly-Maitre B, Reed J C. Endoplasmic reticulum stress: Cell life and death decisions. The Journal of Clinical Investigation, 2005, 115: 2656-2664.

[4] Davies M J. The oxidative environment and protein damage. Biochimica et Biophysica Acta, 2005, 1703: 93-109.

[5] Berlett B S, Stadtman E R. Protein oxidation in ageing, disease, and oxidative stress. The Journal of Biological Chemistry,1997, 272(33): 20313-20316.

[6] Rajjou L, Lovigny Y, Groot S P, Belghazi M, Job C, Job D. Proteome-wide characterization of seed aging in Arabidopsis: A comparison between artificial and natural aging protocols. Plant Physiology, 2008, 148: 620-641.

[7] Rabek J P, Boylston W H, Papaconstantinou J. Carbonylation of ER chaperone proteins in aged mouse liver. Biochemical and Biophysical Research Communications, 2003, 305: 566-572.

[8] Chen H, Osuna D, Colville L, Lorenzo O, Graeber K, Küster H, Leubner-Metzger G, Kranner I. Transcriptome-wide mapping of pea seed ageing reveals a pivotal role for genes related to oxidative stress and programmed cell death. PLoS ONE, 2013, 8(10): e78471.

[9] Schröder M, Kaufman R J. The mammalian unfolded protein response. Annual Review of Biochemistry, 2005, 74: 739-789.

[10] Yoshida H. ER stress and diseases. FEBS Journal, 2007, 274(3): 630-658.

[11] Rao R V, Ellerby H M, Bredesen D E. Coupling endoplasmic reticulum stress to the cell death program. Cell Death and Differentiation, 2004, 11: 372-380.

[12] Fujita M, Mizukado S, Fujita Y, Ichikawa T, Nakazawa M, Seki M, Matsui M, Yamaguchi-Shinozaki K, Shinozaki K. Identification of stress-tolerance-related transcription-factor genes via mini-scale full-length cDNA Over-expressor (FOX) gene hunting system.Biochemical and Biophysical Research Communication, 2007, 364: 250-257.

[13] Jia X Y, Xu C Y, Jing R L, Li R Z, Mao X G, Wang J P, Chang X P. Molecular cloning and characterization of wheat calreticulin (CRT) gene involved in drought-stressed responses. Journal of Experimental Botany, 2008, 59(4): 739-751.

[14] Ruggiano A, Foresti O, Carvalho P. ER-associated degradation: Protein quality control and beyond. The Journal of Cell Biology, 2014, 204(6): 869-879.

[15] Meusser B, Hirsch C, Jarosch E, Sommer T. ERAD: The long road to destruction. Nature Cell Biology, 2005, 7: 766-772.

[16] Hiraishi H, Mochizuki M, Takagi H. Enhancement of stress tolerance in Saccharomyces cerevisiae by overexpression of ubiquitin ligase Rsp5 and ubiquitin-conjugating enzymes. Bioscience Biotechnology and Biochemistry, 2006, 70(11): 2762-2765.

[17] Hong Z, Jin H, Fitchette A C, Xia Y, Monk A M, Faye L, Li J M. Mutations of an α1,6 mannosyl transferase inhibit endoplasmic reticulum-associated degradation of defective brassinosteroid receptors in Arabidopsis. The Plant Cell, 2009, 21: 3792-3802.

[18] Yamamoto M, Kawanabe M, Hayashi Y, Endo T, Nishikawa S. A vacuolar carboxypeptidase mutant of Arabidopsis thaliana is degraded by the ERAD pathway independently of its N-glycan. Biochemical and Biophysical Research Communications, 2010, 393: 384-389.

[19] Liu L, Cui F, Li Q, Yin B, Zhang H, Lin B, Wu Y, Xia R, Tang S, Xie Q. The endoplasmic reticulum-associated degradation is necessary for plant salt tolerance. Cell Research, 2011, 21: 957-969.

[20] Cui F, Liu L, Zhao Q, Zhang Z, Li Q, Lin B, Wu Y, Tang S, Xie Q. Arabidopsis ubiquitin conjugase UBC32 is an ERAD component that functions in Brassinosteroid-mediated salt stress tolerance. The Plant Cell, 2012, 24: 233-244.

[21] Delouche J C, Baskin C C. Accelerated ageing techniques for predicting the relative storability of seed lots. Seed Science and Technology, 1973, 1: 427-452.

[22] 董国军, 胡兴明, 曾大力, 藤本宽, 滕胜, 国广泰史, 郭龙彪, 钱前. 水稻种子人工老化和自然老化的比较研究. 浙江农业科学, 2004(1): 29-31.

Dong G J, Hu X M, Zeng D L, Fujimoto K, Teng S, Kunihiro Y, Guo L B, Qian Q. Comparison between artificial ageing and natural ageing in rice. Journal of Zhejiang Agricultural Sciences, 2004(1): 29-31. (in Chinese)

[23] 吕伟增, 曹广灿, 林一欣, 薛梅真, 陈军营. 玉米种子老化相关MAPK途径基因表达分析. 植物生理学报, 2015, 51(6): 869-881.

Lü W Z, Cao G C, Lin Y X, Xue M Z, Chen J Y. Analysis of MAPK pathway genes expression related to maize seed ageing. Plant Physiology Journal, 2015, 51(6): 869-881. (in Chinese)

[24] 张景龙, 杨伟飞, 吕伟增, 曹广灿, 陈军营. 基于cDNA-AFLP 技术的玉米种子劣变相关基因分析. 植物生理学报, 2014, 50(7): 973-982.

Zhang J L, Yang W F, Lü W Z, Cao G C, Chen J Y. Analysis of genes related to seed deterioration in maize (Zea mays) based on cDNA-AFLP approach. Plant Physiology Journal, 2014, 50(7): 973-982. (in Chinese)

[25] Basak O, Demir I, Mavi K, Matthews S. Controlled deterioration test for predicting seedling emergence and longevity of pepper (Capsicum annuum L.) seed lots.Seed Science and Technology, 2006, 34: 723-734.

[26] Jana S, Choudhur M A. Glycolate metabolism of three submersed aquatic angiosperms during ageing. Aquatic Botany, 1982, 12: 345-354.

[27] 李合生. 植物生理生化实验原理和技术. 北京: 高等教育出版社, 2000: 164-169, 260-261.

Li H S. Experimental Principles and Techniques of Plant Physiological Biochemical. Beijing: Higher Education Press, 2000: 164-169, 260-261. (in Chinese)

[28] 杨伟飞, 张景龙, 吕伟增, 曹广灿, 陈军营. 人工劣变处理对玉米种胚差异基因表达的影响. 中国农业科学, 2014, 47(10): 1878-1893.

Yang W F, Zhang J L, Lü W Z, Cao G C, Chen J Y. Study on the differential genes expression in maize embryo treated by a controlled deterioration treatment. Scientia Agricultura Sinica, 2014, 47(10): 1878-1893. (in Chinese)

[29] Bennett S. Solexa Ltd. Pharmacogenomics, 2004, 5(4): 433-438.

[30] Kanehisa M, Araki M, Goto S, Hattori M, Hirakawa M, Itoh M, Katayama T, Kawashima S, Okuda S, Tokimatsu T, Yamanishi Y. KEGG and the environment for linking genomes to life. Nucleic Acids Research, 2008, 36: 480-484.

[31] Lee A S. The ER chaperone and signaling regulator GRP78/BiP as a monitor of endoplasmic reticulum stress. Methods,2005, 35: 373-381.

[32] Kaneko M, Nomura Y. ER signaling in unfolded protein response. Life Sciences, 2003, 74: 199-205.

[33] Wang B, Heath-Engel H, Zhang D, Nguyen N, Thomas D Y, Hanrahan J W, Shore G C. BAP31 interacts with Sec61 translocons and promotes retrotranslocation of CFTR△F508 via the Derlin-1 Complex. Cell,2008, 133: 1080-1092.

[34] Medicherla B, Kostova Z, Schaefer A, Wolf D H. A genomic screen identifies Dsk2p and Rad23p as essential components of ER- associated degradation. EMBO Reports, 2004, 5: 692-697.

[35] Hendry G A F. Oxygen, free radical processes and seed longevity. Seed Science Research, 1993, 3: 141-153.

[36] Bailly C. Active oxygen species and antioxidants in seed biology. Seed Science Research, 2004, 14: 93-107.

[37] Bailly C, Kranner I. Analyses of reactive oxygen species and antioxidants in relation to seed longevity and germination. Methods in Molecular Biology, 2011, 773: 343-367.

[38] Halliwell B, Gutteridge J M. Oxygen free radicals and iron in relation to biology and medicine: Some problems and concepts. Archives of Biochemistry and Biophysics, 1986, 246: 501-514.

[39] Henzler T, Steudle E. Transport and metabolic degradation of hydrogen peroxide in Chara coralline: Model calculations and measurements with the pressure probe suggest transport of H₂O₂ across water channels. Journal of Experimental Botany, 2000, 51(353): 2053-2066.

[40] Goel A, Goel A K, Sheoran I S. Changes in oxidative stress enzymes during artificial ageing in cotton (Gossypium hirsutum L.) seeds. Journal of Plant Physiology, 2003, 160: 1093-1100.

[41] Kibinza S, Vinel D, Côme D, Bailly C, Corbinea F. Sunflower seed deterioration as related to moisture content during ageing, energy metabolism and active oxygen species scavenging. Physiologia Plantarum, 2006, 128: 496-506.

[42] Yao Z, Liu L, Gao F, Rampitsch C, Reinecke D M, Ozga J A, Ayele B T. Developmental and seed aging mediated regulation of antioxidative genes and differential expression of proteins during pre and post-germinative phases in pea. Journal of Plant Physiology, 2012, 169: 1477-1488.

[43] Lisa S, Domingo B, Martínez J, Gilch S, Llopis J F, Schätzl H M, Gasset M. Failure of prion protein oxidative folding guides the formation of toxic transmembrane forms.Journal of Biological Chemistry, 2012, 287: 36693-36701.

[44] Tu B P, Weissman J S. Oxidative protein folding in eukaryotes: Mechanisms and consequences. The Journal of Cell Biology, 2004, 164(3): 341-346.

[45] Tu B P, Weissman J S. The FAD- and O(2)-dependent reaction cycle of Ero1-mediated oxidative protein folding in the endoplasmic reticulum. Molecular Cell, 2002, 10: 983-994.

[46] Higa A, Chevet E. Redox signaling loops in the unfolded protein response.Cellular Signalling, 2012, 24: 1548-1555.

[47] Malhotra J D, Kaufman R J. Endoplasmic reticulum stress and oxidative stress: A vicious cycle or a double-edged sword? Antioxidants and Redox Signaling, 2007, 9: 2277-2293.

[48] Bravo R, Vicencio J M, Parra V, Troncoso R, Munoz J P, Bui M, Quiroga C, Rodriguez A E, Verdejo H E, Ferreira J, Iglewski M, Chiong M, Simmen T, Zorzano A, Hill J A, Rothermel B A, Szabadkai G, Lavandero S. Increased ER-mitochondrial coupling promotes mitochondrial respiration and bioenergetics during early phases of ER stress. Journal of Cell Science, 2011, 124: 2143-2152.

[49] Yoon H, Kim D S, Lee G H, Kim K W, Kim H R, Chae H J. Apoptosis induced by manganese on neuronal SK-N-MC cell line: Endoplasmic reticulum (ER) stress and mitochondria dysfunction. Environmental Health and Toxicology, 2011, 26: e2011017.

[50] Bays N W, Gardner R G, Seelig L P, Joazeiro C A, Hampton R Y. Hrd1p/Der3p is a membrane-anchored ubiquitin ligase required for ER-associated degradation. Nature Cell Biology, 2001, 3: 24-29.

[51] Loertscher J, Larson L L, Matson C K, Parrish M L, Felthauser A, Sturm A, Tachibana C, Bard M, Wright R. Endoplasmic reticulum- associated degradation is required for cold adaptation and regulation of sterol biosynthesis in the yeast. Eukaryotic Cell, 2006, 5: 712-722.

[52] Kamauchi S, Nakatani H, Nakano C, Urade R. Gene expression in response to endoplasmic reticulum stress in Arabidopsis thaliana. The FEBS Journal, 2005, 272: 3461-3476.

[53] Martínez I M, Chrispeels M J. Genomic analysis of the unfolded protein response in Arabidopsis shows its connection to important cellular processes. The Plant Cell, 2003, 15: 561-576.

[54] Deng Y, Srivastava R, Howell S H. Endoplasmic reticulum (ER) stress response and its physiological roles in plants. International Journal of Molecular Sciences, 2013, 14: 8188-8212.

[55] Avezov E, Frenkel Z, Ehrlich M, Herscovics A, Lederkremer G Z. Endoplasmic reticulum (ER) mannosidase I is compartmentalized and required for N-glycan trimming to Man5-6GlcNAc2 in glycoprotein ER-associated degradation. Molecular Biology of the Cell, 2008, 19: 216-225.

[56] Oda Y, Hosokawa N, Wada I, Nagata K. EDEM as an acceptor of terminally misfolded glycoproteins released from calnexin. Science, 2003, 299(5611): 1394-1397.

[57] Eriksson K K, Vago R, Calanca V, Galli C, Paganetti P, Molinari M. EDEM contributes to maintenance of protein folding efficiency and secretory capacity. The Journal of Cell Biology, 2004, 279(43): 44600-44605.

[58] Molinari M, Calanca V, Galli C, Lucca P, Paganetti P. Role of EDEM in the release of misfolded glycoproteins from the calnexin cycle. Science, 2003, 299(5611): 1397-1400.

[59] Su W, Liu Y D, Xia Y, Hong Z, Li J M. Conserved endoplasmic reticulum-associated degradation system to eliminate mutated receptor-like kinases in Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America, 2011, 108(2): 870-875.

[60] Ye Y, Meyer H H, Rapoport T A. The AAA ATPase Cdc48/p97 and its partners transport proteins from the ER into the cytosol. Nature, 2001, 414(6): 652-656.

[61] Hirsch C, Gauss R, Horn S C, Neuber O, Sommer T. The ubiquitylation machinery of the endoplasmic reticulum. Nature, 2009, 458(7237): 453-460.