基于iTRAQ技术的不同耐热性水稻花药差异蛋白分析
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摘要
为从蛋白质水平揭示耐热与热敏感水稻花药响应高温胁迫的分子机制,本研究以耐热水稻品系996和热敏感水稻品系4628为材料,采用iTRAQ技术对高温和适温条件下水稻花药进行差异蛋白质组学分析。结果表明,在996高温-996适温、4628高温-4628适温、996高温-4628高温和996适温-4628适温4个比较组中,共筛选到957个差异表达蛋白,其中上调表达蛋白398个,覆盖率达20%以上的蛋白占鉴定总蛋白的50. 96%。GO分析显示,抽穗开花期花药差异蛋白主要集中于代谢过程和细胞过程,在细胞组成方面主要分布于胞内部分、细胞器和细胞,分子功能主要涉及催化活性、绑定等。花药差异蛋白通路分析显示,来源于bZIP和DOF家族的2个转录因子差异表达,DOF家族基因上调,b ZIP家族基因下调; 18个HSPs基因中大部分表达上调;与植物激素和信号传导相关基因大部分表达下调; 10个活性氧(ROS)相关基因中5个表现为上调;与次生代谢相关基因大部分下调。本研究结果为揭示水稻花药高温胁迫的应答分子调控机制提供了一定的理论基础。
To reveal the molecular mechanism of heat tolerant and heat sensitive anther response to high temperature stress from protein level.The heat tolerance line 996 and heat sensitive line 4628 were chosen as research materials and the differential proteomics analysis was carried out on the rice anthers under high temperature and optimum temperature condition by using iTRAQ technique. The results showed that totally 957 differential expressed proteins were screened out in four comparable groups,996 high temperature-996 optimal temperature,4628 high temperature-4628 optimal temperature,996 high temperature-4628 high temperature,996 optimal temperature-4628 optimal temperature,among which 398 differential proteins were up-regulated,the proteins withover 20% coverage was 50. 96% of the total protein number. GO analysis showed that the anther differentially expressed proteins in heading and flowering stage were mainly concentrated on metabolic processes,cell processes and other biological processes. The differentially expressed proteins were mainly distributed in intracellular parts,organelles and cells on cell composition and their molecular functions were majorly catalytic activity and binding. Analysis of anther differential protein pathway showed that 2 transcription factor( TF) genes belonging to bZIP and DOF family were differently expressed,the DOF family gene was up-related,and the bZIP family gene was down-regulated; majority of 18 HSPs genes were up-regulated. The genes related to plant hormone and signal transduction were mainly down-regulated. 5 genes of 10 differentially expressed genes related to reactive oxygen species( ROS) were differently expressed,5 genes were up-regulated. The genes related to secondary metabolism were mainly down-regulated. The study can provide theoretical basis for revealing the mechanism of molecular regulation on rice anther response to heat stress.
To reveal the molecular mechanism of heat tolerant and heat sensitive anther response to high temperature stress from protein level.The heat tolerance line 996 and heat sensitive line 4628 were chosen as research materials and the differential proteomics analysis was carried out on the rice anthers under high temperature and optimum temperature condition by using iTRAQ technique. The results showed that totally 957 differential expressed proteins were screened out in four comparable groups,996 high temperature-996 optimal temperature,4628 high temperature-4628 optimal temperature,996 high temperature-4628 high temperature,996 optimal temperature-4628 optimal temperature,among which 398 differential proteins were up-regulated,the proteins withover 20% coverage was 50. 96% of the total protein number. GO analysis showed that the anther differentially expressed proteins in heading and flowering stage were mainly concentrated on metabolic processes,cell processes and other biological processes. The differentially expressed proteins were mainly distributed in intracellular parts,organelles and cells on cell composition and their molecular functions were majorly catalytic activity and binding. Analysis of anther differential protein pathway showed that 2 transcription factor( TF) genes belonging to bZIP and DOF family were differently expressed,the DOF family gene was up-related,and the bZIP family gene was down-regulated; majority of 18 HSPs genes were up-regulated. The genes related to plant hormone and signal transduction were mainly down-regulated. 5 genes of 10 differentially expressed genes related to reactive oxygen species( ROS) were differently expressed,5 genes were up-regulated. The genes related to secondary metabolism were mainly down-regulated. The study can provide theoretical basis for revealing the mechanism of molecular regulation on rice anther response to heat stress.
引文
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[18]张宪政.作物生理研究法[M].北京:中国农业出版社,1992
[19]陈蕊红,张改生,刘卫,叶景秀,牛娜.小麦花药蛋白质组双向电泳技术体系的优化[J].核农学报,2008,22(4):404-409
[20]王涛,唐颂豪,何梅,谢寅峰,张往祥.热激蛋白与植物耐热性关系的研究进展[J].西北林学院学报,2014,29(6):72-79
[21] Gonzalez-Schain N,Dreni L,Lawas L M,Galbiati M,Colombo L,Heuer S,Jagadish K S,Kater M M. Genome-wide transcrip-tome analysis during anthesis reveals new insights into the molecular basis of heat stress responses in tolerant and sensitive rice varieties[J].Plant Cell Physiology,2016,57(1):57-68
[22] Zou J,Liu C,Liu A,Zou D,Chen X. Overexpression of Os Hsp17. 0and Os Hsp23. 7 enhances drought and salt tolerance inrice[J].Journal of Plant Physiology,2012,169(6):628-635
[23]郭秀林,刘子会,赵会嶶,徐婧,李慧聪.热锻炼和高温胁迫下冬小麦热激蛋白与抗氧化酶基因表达[J].华北农学报,2014,29(4):13-18
[24] Prasad T K. Mechanisms of chilling-induced oxidative stress injury and tolerance:change in antioxidant system,oxidation of proteins and lipids and protease activities[J]. Plant Journal,1996,10:1017-1026
[25] Rhee S G,Woo H A,Kil I S,Bae S H. Peroxiredoxin functions as a peroxidase and a regulator and sensor of local peroxides[J].Journal Biology Chemistry,2012,287(7):4403-4410
[26]汪宝卿,解备涛,张海燕,董顺旭,段文学,王庆美,张立明.基于i TRAQ技术的不同耐旱性甘薯苗期根系差异蛋白分析[J].核农学报,2017,31(10):1904-1912
[27] Xu C, Sibicky T, Huang B. Protein profile analysis of saltresponsive proteins in leaves and roots in two cultivars of creeping bentgrass differing in salinity tolerance[J]. Plant Cell Reports,2010,29(6):595-615
[28] Wang Y,Wang Q Q,Zhao Y,Han G,Zhu S. Systematic analysis of maize classⅢperoxidase gene family reveals a conserved subfamily involved in abiotic stress response[J]. Gene,2015,566(1):95-108
[29] Akter S,Huang J,Waszczak C,Jacques S,Gevaert K,Breusegem F,Messens J. Cysteines under ROS attack in plants:a proteomics view[J]. Journal of Experimental Botany,2015,66(10):2935-2944
[30] Chi W C,Fu S F,Huang X L,Chen Y A,Chen C C,Huang H J.Identification of transcriptome profiles and signaling pathways for the allelochemical juglone in rice roots[J]. Plant Molecular Biology,2011,77(6):591-607
[31] Zou M,Guan Y,Ren H,Zhang F,Chen F. A bZIP transcription factor,Os ABI5 is involved in rice fertility and stress tolerance[J].Plant Molecular Biology,2008,66(6):675-683
[32] Peter J C,Mark R A,Virginia W. Impact of low-temperature stress on general phenylpropanoid and anthocyanin pathways:enhancement of transcript abundance and anthocyanin pigmentation in maize seedlings[J]. Planta,1994,194(4):541-549
[33] Kentaro M,Nami G Y,Masahiko K,Hashizume K. Loss of anthocyanins in red-wine grape under high temperature[J]. Journal of Experimental Botany,2007,58(8):1935
[2]段骅,杨建昌.高温对水稻的影响及其机制的研究进展[J].2012,26(4):393-400
[3]杨军,李迎春,刘丹,周大虎.江西一季稻花期高温热害发生规律及其对产量的影响[J].江苏农业科学,2017,45(19):170-175
[4]杨惠成,黄仲青,蒋之埙,王相文. 2003年安徽早中稻花期热害及防御技术[J].安徽农业科学,2004,32(1):3-4
[5] Lin S K,Chang M C,Tsai Y G,Lur H S. Proteomic analysis of the expression of proteins related to rice quality during caryopsis development and the effect of high temperature on expression[J].Proteomics,2005,5(8):2140-2156
[6]廖江林,宋宇,钟平安,周会汶,张宏玉,黄英金.耐热和热敏感水稻应答灌浆初期高温胁迫过程中的差异表达蛋白质鉴定[J].中国农业科学,2014,47(16):3121-3131
[7]谢秀枝,王欣,刘丽华,董世雷,皮雄娥,刘伟. i TRAQ技术及其在蛋白质组学中的应用[J].中国生物化学与分子生物学报,2011,27(7):616-621
[8]欧阳玥,孙晓红,闫静,褚金芳,闫存玉.稳定同位素标记技术在生物分子相对定量分析研究中的应用与进展[J].科学通报,2013,58(27):2762-2778
[9]陈凌华,程祖锌,许明,郑金贵. i TRAQ技术及其在水稻蛋白质组学中的应用研究进展[J].中国农业科技导报,2017,19(12):14-23
[10] Neilson K A, Mariani M,Haynes P A. Quantitative proteomic analysis of cold-responsive proteins in rice[J]. Proteomics,2011,11(9):1696-1706
[11] Zhang H Y,Lei G,Zhou H W,He C,Liao J L,Huang Y J.Quantitative i TRAQ based proteomic analysis of rice grains to assess high night temperature stress[J]. Proteomics,2017,17(5):365-372
[12]黄小平,张宏玉,雷刚,王志美,章智,贺超,廖江林,黄英金.灌浆期夜间高温胁迫下耐热和热敏感水稻籽粒的比较蛋白质组分析[J].中国水稻科学,2017,13(1):13-22
[13]黄福灯,曹珍珍,李春寿,陆艳婷,潘刚,程方民.花期高温对水稻花器官性状和结实的影响[J].核农学报,2016,30(3):565-570
[14]汤日圣,郑建初,张大栋,金之庆,陈留根,黄益洪,石春林,葛道阔.高温对不同水稻品种花粉活力及籽粒结实的影响[J].江苏农业学报,2006,22(4):369-373
[15]张桂莲,张顺堂,肖浪涛,武小金,肖应辉,陈立云.花期高温胁迫对水稻花药生理特性及花粉性状的影响[J].作物学报,2013,39(1):177-183
[16]张桂莲.两个早稻品种耐热性生理及分子遗传基础研究[D].长沙:湖南农业大学,2006
[17]李合生,孙群,赵世杰,章文华.植物生理生化试验原理和技术[M].北京:高等教育出版社,2000:167-169
[18]张宪政.作物生理研究法[M].北京:中国农业出版社,1992
[19]陈蕊红,张改生,刘卫,叶景秀,牛娜.小麦花药蛋白质组双向电泳技术体系的优化[J].核农学报,2008,22(4):404-409
[20]王涛,唐颂豪,何梅,谢寅峰,张往祥.热激蛋白与植物耐热性关系的研究进展[J].西北林学院学报,2014,29(6):72-79
[21] Gonzalez-Schain N,Dreni L,Lawas L M,Galbiati M,Colombo L,Heuer S,Jagadish K S,Kater M M. Genome-wide transcrip-tome analysis during anthesis reveals new insights into the molecular basis of heat stress responses in tolerant and sensitive rice varieties[J].Plant Cell Physiology,2016,57(1):57-68
[22] Zou J,Liu C,Liu A,Zou D,Chen X. Overexpression of Os Hsp17. 0and Os Hsp23. 7 enhances drought and salt tolerance inrice[J].Journal of Plant Physiology,2012,169(6):628-635
[23]郭秀林,刘子会,赵会嶶,徐婧,李慧聪.热锻炼和高温胁迫下冬小麦热激蛋白与抗氧化酶基因表达[J].华北农学报,2014,29(4):13-18
[24] Prasad T K. Mechanisms of chilling-induced oxidative stress injury and tolerance:change in antioxidant system,oxidation of proteins and lipids and protease activities[J]. Plant Journal,1996,10:1017-1026
[25] Rhee S G,Woo H A,Kil I S,Bae S H. Peroxiredoxin functions as a peroxidase and a regulator and sensor of local peroxides[J].Journal Biology Chemistry,2012,287(7):4403-4410
[26]汪宝卿,解备涛,张海燕,董顺旭,段文学,王庆美,张立明.基于i TRAQ技术的不同耐旱性甘薯苗期根系差异蛋白分析[J].核农学报,2017,31(10):1904-1912
[27] Xu C, Sibicky T, Huang B. Protein profile analysis of saltresponsive proteins in leaves and roots in two cultivars of creeping bentgrass differing in salinity tolerance[J]. Plant Cell Reports,2010,29(6):595-615
[28] Wang Y,Wang Q Q,Zhao Y,Han G,Zhu S. Systematic analysis of maize classⅢperoxidase gene family reveals a conserved subfamily involved in abiotic stress response[J]. Gene,2015,566(1):95-108
[29] Akter S,Huang J,Waszczak C,Jacques S,Gevaert K,Breusegem F,Messens J. Cysteines under ROS attack in plants:a proteomics view[J]. Journal of Experimental Botany,2015,66(10):2935-2944
[30] Chi W C,Fu S F,Huang X L,Chen Y A,Chen C C,Huang H J.Identification of transcriptome profiles and signaling pathways for the allelochemical juglone in rice roots[J]. Plant Molecular Biology,2011,77(6):591-607
[31] Zou M,Guan Y,Ren H,Zhang F,Chen F. A bZIP transcription factor,Os ABI5 is involved in rice fertility and stress tolerance[J].Plant Molecular Biology,2008,66(6):675-683
[32] Peter J C,Mark R A,Virginia W. Impact of low-temperature stress on general phenylpropanoid and anthocyanin pathways:enhancement of transcript abundance and anthocyanin pigmentation in maize seedlings[J]. Planta,1994,194(4):541-549
[33] Kentaro M,Nami G Y,Masahiko K,Hashizume K. Loss of anthocyanins in red-wine grape under high temperature[J]. Journal of Experimental Botany,2007,58(8):1935