嫁接对网纹甜瓜幼苗叶片氮代谢及转录因子CmHY5表达的影响
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摘要
嫁接技术在甜瓜生产上的应用在一定程度上缓解了施肥和产量之间的矛盾,并可有效地防治土传病害,缓解土壤连作障碍。以白籽南瓜圣砧1号嫁接的网纹甜瓜幼苗为处理,以自根网纹甜瓜幼苗为对照,测定接穗生物量、氮素含量、氮代谢关键酶活性、氮代谢关键基因及其转运蛋白相关基因表达变化,并对氮代谢和转运蛋白相关基因上游的调控因子CmHY5转录因子时空表达特性进行分析,以期进一步探析嫁接影响网纹甜瓜幼苗叶片氮素代谢的生理和分子机制。试验结果表明:嫁接能够显著提高接穗生物量和叶片中NO3--N和NH4+-N含量。嫁接网纹甜瓜幼苗叶片氮代谢关键酶NR与GS的酶活性及其相关基因CmNR和CmGS相对表达量显著增加,硝酸转运蛋白基因NRT(CmNRT2.4、CmNRT2.5)和铵盐转运蛋白基因AMT(CmAMT2、CmAMT3)相对表达量显著上调。自然光照条件下,嫁接处理显著促进CmHY5表达,其相对表达量为对照的2.68倍;但在弱光和黑暗条件下,嫁接处理与对照间无显著差异。嫁接处理能够显著提高CmHY5在网纹甜瓜幼苗根、茎、叶中表达,嫁接处理叶片中的CmHY5相对表达量是对照的5.98倍。可见,转录因子CmHY5在嫁接促进网纹甜瓜幼苗叶片氮素代谢及其转运过程中具有重要调控作用。
Grafting technology applied in muskmelon production could not only alleviate contradictions between fertilization and yield as well as continuous cropping obstacle, but also prevent soil diseases. In this experiment, we used the treatment of muskmelon seedlings grafted with white-seed pumpkin 'Sheng-zhen No.1' and self-rooted muskmelon seedlings as control to measure the plants' scion biomass, nitrogen contents, key enzyme activities, relative genes expression of nitrogen metabolism and nitrogen translocator. We also analyzed the spatio-temporal expression of CmHY5, an upstream regulator of the relative genes involved in nitrogen metabolism and nitrogen translocator in order to further explore physiological and molecular mechanism of muskmelon seedling leaves by grafting. The results showed that grafting significantly increased the scions' biomass and contents of NO3--N and NH4+-N in seedling leaves. NR and GS activities, relative expression quantities of CmNR and CmGS in leaves of grafted muskmelon seedling significantly not only increased, but also CmNRT2.4, CmNRT2.5 of nitrate translocator and CmAMT2,CmAMT3 of ammonium translocator were up-regulated by grafting. Meanwhile, we found that grafting remarkably induced up-regulated expression of CmHY5 in muskmelon seedling leaves. Under natural lighting condition, the grafting treatment promoted CmHY5 expression which were 2.68 folds of that of control. Under weak light treatment and dark condition, there was no significant difference between grafting treatment and control. CmHY5 expression was increased in root, stem and leaf of muskmelon seedling under grafting treatment, for example, CmHY5 expression in leaf was 5.98 folds of that of the control.Therefore, CmHY5, a transcription factor, played a pivotal, regulatory role in the process of grafting to accelerate the nitrogen metabolism and transport.
Grafting technology applied in muskmelon production could not only alleviate contradictions between fertilization and yield as well as continuous cropping obstacle, but also prevent soil diseases. In this experiment, we used the treatment of muskmelon seedlings grafted with white-seed pumpkin 'Sheng-zhen No.1' and self-rooted muskmelon seedlings as control to measure the plants' scion biomass, nitrogen contents, key enzyme activities, relative genes expression of nitrogen metabolism and nitrogen translocator. We also analyzed the spatio-temporal expression of CmHY5, an upstream regulator of the relative genes involved in nitrogen metabolism and nitrogen translocator in order to further explore physiological and molecular mechanism of muskmelon seedling leaves by grafting. The results showed that grafting significantly increased the scions' biomass and contents of NO3--N and NH4+-N in seedling leaves. NR and GS activities, relative expression quantities of CmNR and CmGS in leaves of grafted muskmelon seedling significantly not only increased, but also CmNRT2.4, CmNRT2.5 of nitrate translocator and CmAMT2,CmAMT3 of ammonium translocator were up-regulated by grafting. Meanwhile, we found that grafting remarkably induced up-regulated expression of CmHY5 in muskmelon seedling leaves. Under natural lighting condition, the grafting treatment promoted CmHY5 expression which were 2.68 folds of that of control. Under weak light treatment and dark condition, there was no significant difference between grafting treatment and control. CmHY5 expression was increased in root, stem and leaf of muskmelon seedling under grafting treatment, for example, CmHY5 expression in leaf was 5.98 folds of that of the control.Therefore, CmHY5, a transcription factor, played a pivotal, regulatory role in the process of grafting to accelerate the nitrogen metabolism and transport.
引文
[1]张新英.嫁接对甜瓜(Cucumis melo L.)苗期耐盐性和果实发育期品质的影响[D].北京:中国农业科学院,2014.
[2]吴凤芝,安美君.西瓜枯萎病抗性及其嫁接对根际土壤微生物数量及群落结构的影响[J].中国农业科学,2011,44(22):4636-4644.
[3]郑长英.抗性砧木嫁接番茄控制土传病害的研究[D].北京:中国农业大学,2004.
[4]徐敬华,黄丹枫,支月娥.PAL活性与嫁接西瓜枯萎病抗性传递的相关性[J].上海交通大学学报(农业科学版),2004(1):12-16.
[5] MA Q X,CAO X C,TAN X L,et al.Effects of cadmium stress on pakchoi(Brassica chinensis L)growth and uptake of inorganic and organic nitrogenous compounds[J].Environmental and Experimental Botany,2017,137:49-57.
[6] LILLO C.Signalling cascades integrating light-enhanced nitrate metabolism[J].Biochemical Journal,2008,415(1):11-19.
[7]郑冬超.调节转运蛋白At NRT1.1、At NRT2.1及PdAMT1.1基因表达对拟南芥和杨树氮素吸收的影响研究[D].北京:北京林业大学,2013.
[8]刘玉梅.不同施氮水平对嫁接黄瓜不同部位硝酸盐含量的影响[J].西北农业学报,2008(2):225-228.
[9]张合琼,张汉马,梁永书,等.植物硝酸盐转运蛋白研究进展[J].植物生理学报,2016,52(2):141-149.
[10]贾宏昉,张洪映,刘维智,等.高等植物硝酸盐转运蛋白的功能及其调控机制[J].生物技术通报,2014(6):14-21.
[11]谢丹.水稻硝酸盐转运蛋白基因OsNPF7.9表达模式分析及过表达对水稻生长和氮素积累的影响[D].南京:南京农业大学,2014.
[12] HUANG L,ZHANG H,ZHANG H,et al.HY5 regulates nitrite reductase 1(NIR1)and ammonium translocator1;2(AMT1;2)in Arabidopsis seedlings[J].Plant Science,2015,238:330-339.
[13] CHEN X,YAO Q,GAO X,et al.Shoot-to-root mobile transcription factor HY5 coordinates plant carbon and nitrogen acquisition[J].Current Biology,2016,26(5):640-646.
[14] SHI Q M,YANG X,SONG L,et al.Arabidopsis MSBP1 is activated by HY5 and HYH and is involved in photomorphogenesis and brassinosteroid sensitivity regulation[J].Molecular plant,2011,4(6):1092-1104.
[15] JONASSEN E M,SANDSMARK B A A,LILLO C.Unique status of NIA2 in nitrate assimilation:NIA2 expression is Promoted by HY5/HYH and inhibited by PIF4[J].Plant signaling&behavior,2009,4(11):1084-1086.
[16] JONASSEN E M,SéVIN D C,LILLO C.The bZIP transcription factors HY5 and HYH are positive regulators of the main nitrate reductase gene in Arabidopsis leaves,NIA2,but negative regulators of the nitrate uptake gene NRT1.1[J].Journal of Plant Physiology,2009,166(18):2071-2076.
[17]王丽萍,孙锦,郭世荣,等.白籽南瓜嫁接对不同盐胁迫下黄瓜幼苗氮代谢和蛋白表达的影响[J].植物营养与肥料学报,2012,18(3):689-698.
[18]刘燕.黄瓜嫁接苗对NO3-胁迫的生理响应[D].泰安:山东农业大学,2010.
[19]薛亮,马忠明,杜少平.嫁接与施氮对甜瓜产量和氮素吸收、利用的影响[J].应用生态学报,2017,28(6):1909-1916.
[20]付新星.嫁接对西瓜苗期硝酸盐吸收和利用的影响[D].杭州:浙江大学,2014.
[21]孙培琪.甜樱桃(Prunus avium L.)果实发育期氮素代谢特性研究[D].泰安:山东农业大学,2011.
[22]王学奎,李合生,刘武定.光对小麦叶片谷氨酰胺合成酶调节机理初探[J].华中农业大学学报,2000(2):102-105.
[23]陈胜勇,李彩凤,侯静,等.蛋白核酸合成抑制剂对甜菜谷氨酰胺合成酶活性调控分析[J].东北农业大学学报,2009,40(1):18-22.
[24]杨邵,姚小华,龙伟,等.普通油茶不同嫁接组合对苗木根系生长发育的影响[J].安徽农业大学学报,2016,43(1):83-87.
[25]李跃建,刘小俊,刘独臣,等.黄瓜自根苗、嫁接苗根系活力、净光合速率和蛋白质表达差异研究[J].西南农业学报,2007(5):979-981.
[26]马凌之.基质湿度影响下嫁接黄瓜(Cucumis sativus L.)根系生长冗余的初步研究[D].兰州:甘肃农业大学,2007.
[27]司亚平,程孙亮,陈殿奎.西瓜断根嫁接育苗根际营养体积对根系活力和秧苗质量的影响[J].华北农学报,2005(3):67-69.
[28]齐红岩,李天来,刘轶飞,等.嫁接对薄皮甜瓜光合特性、产量与含糖量的影响[J].沈阳农业大学学报,2006,37(2):155-158.
[29]刘轶飞.嫁接砧木对厚皮甜瓜叶片光合物质生产的影响[J].园艺学报,2010,37(S):2176.
[30]魏述英,吴震,黄俊.砧木对网纹甜瓜嫁接植株生长和光合特性的影响[J].上海农业学报,2006(4):114-117.
[31]许传强,李天来,齐红岩.嫁接对网纹甜瓜光合特性、生长状况及产量的影响[J].中国西瓜甜瓜,2005(2):5-7.
[32]周宝利,姜荷,赵鑫.不同砧木嫁接茄子抗黄萎病特性及其与根系分泌物关系[J].沈阳农业大学学报,2001,32(6):414-417.
[33] GANGAPPA S N,BOTTO J F.The multifaceted roles of HY5 in plant growth and development[J].Molecular Plant,2016,9(10):1353-1365.
[34] ABBAS N,MAURYA J P,SENAPATI D,et al.Arabidopsis CAM7 and HY5 physically interact and directly bind to the HY5promoter to regulate its expression and thereby promote photomorphogenesis[J].The Plant Cell,2014,26(3):1036-1052.
[35] ANG L H,CHATTOPADHYAY S,WEI N,et al.Molecular interaction between COP1 and HY5 defines a regulatory switch for light control of Arabidopsis development[J].Molecular cell,1998,1(2):213-222.
[36] HUANG X,OUYANG X,YANG P,et al.Arabidopsis FHY3 and HY5 positively mediate induction of COP1 transcription in response to photomorphogenic UV-B light[J].The Plant Cell,2012,24(11):4590-4606.
[37] YU Y,WANG J,ZHANG Z,et al.Ethylene promotes hypocotyl growth and HY5 degradation by enhancing the movement of COP1 to the nucleus in the light[J].PLoS Genet,2013,9(12):e1004025.
[38]黄远,别之龙,孔秋生,等.嫁接对西瓜和甜瓜果实品质影响的研究进展[J].中国蔬菜,2012(4):10-18.
[39] JONASSEN E M,LEA U S,LILLO C.HY5 and HYH are positive regulators of nitrate reductase in seedlings and rosette stage plants[J].Planta,2008,227(3):559-564.
[2]吴凤芝,安美君.西瓜枯萎病抗性及其嫁接对根际土壤微生物数量及群落结构的影响[J].中国农业科学,2011,44(22):4636-4644.
[3]郑长英.抗性砧木嫁接番茄控制土传病害的研究[D].北京:中国农业大学,2004.
[4]徐敬华,黄丹枫,支月娥.PAL活性与嫁接西瓜枯萎病抗性传递的相关性[J].上海交通大学学报(农业科学版),2004(1):12-16.
[5] MA Q X,CAO X C,TAN X L,et al.Effects of cadmium stress on pakchoi(Brassica chinensis L)growth and uptake of inorganic and organic nitrogenous compounds[J].Environmental and Experimental Botany,2017,137:49-57.
[6] LILLO C.Signalling cascades integrating light-enhanced nitrate metabolism[J].Biochemical Journal,2008,415(1):11-19.
[7]郑冬超.调节转运蛋白At NRT1.1、At NRT2.1及PdAMT1.1基因表达对拟南芥和杨树氮素吸收的影响研究[D].北京:北京林业大学,2013.
[8]刘玉梅.不同施氮水平对嫁接黄瓜不同部位硝酸盐含量的影响[J].西北农业学报,2008(2):225-228.
[9]张合琼,张汉马,梁永书,等.植物硝酸盐转运蛋白研究进展[J].植物生理学报,2016,52(2):141-149.
[10]贾宏昉,张洪映,刘维智,等.高等植物硝酸盐转运蛋白的功能及其调控机制[J].生物技术通报,2014(6):14-21.
[11]谢丹.水稻硝酸盐转运蛋白基因OsNPF7.9表达模式分析及过表达对水稻生长和氮素积累的影响[D].南京:南京农业大学,2014.
[12] HUANG L,ZHANG H,ZHANG H,et al.HY5 regulates nitrite reductase 1(NIR1)and ammonium translocator1;2(AMT1;2)in Arabidopsis seedlings[J].Plant Science,2015,238:330-339.
[13] CHEN X,YAO Q,GAO X,et al.Shoot-to-root mobile transcription factor HY5 coordinates plant carbon and nitrogen acquisition[J].Current Biology,2016,26(5):640-646.
[14] SHI Q M,YANG X,SONG L,et al.Arabidopsis MSBP1 is activated by HY5 and HYH and is involved in photomorphogenesis and brassinosteroid sensitivity regulation[J].Molecular plant,2011,4(6):1092-1104.
[15] JONASSEN E M,SANDSMARK B A A,LILLO C.Unique status of NIA2 in nitrate assimilation:NIA2 expression is Promoted by HY5/HYH and inhibited by PIF4[J].Plant signaling&behavior,2009,4(11):1084-1086.
[16] JONASSEN E M,SéVIN D C,LILLO C.The bZIP transcription factors HY5 and HYH are positive regulators of the main nitrate reductase gene in Arabidopsis leaves,NIA2,but negative regulators of the nitrate uptake gene NRT1.1[J].Journal of Plant Physiology,2009,166(18):2071-2076.
[17]王丽萍,孙锦,郭世荣,等.白籽南瓜嫁接对不同盐胁迫下黄瓜幼苗氮代谢和蛋白表达的影响[J].植物营养与肥料学报,2012,18(3):689-698.
[18]刘燕.黄瓜嫁接苗对NO3-胁迫的生理响应[D].泰安:山东农业大学,2010.
[19]薛亮,马忠明,杜少平.嫁接与施氮对甜瓜产量和氮素吸收、利用的影响[J].应用生态学报,2017,28(6):1909-1916.
[20]付新星.嫁接对西瓜苗期硝酸盐吸收和利用的影响[D].杭州:浙江大学,2014.
[21]孙培琪.甜樱桃(Prunus avium L.)果实发育期氮素代谢特性研究[D].泰安:山东农业大学,2011.
[22]王学奎,李合生,刘武定.光对小麦叶片谷氨酰胺合成酶调节机理初探[J].华中农业大学学报,2000(2):102-105.
[23]陈胜勇,李彩凤,侯静,等.蛋白核酸合成抑制剂对甜菜谷氨酰胺合成酶活性调控分析[J].东北农业大学学报,2009,40(1):18-22.
[24]杨邵,姚小华,龙伟,等.普通油茶不同嫁接组合对苗木根系生长发育的影响[J].安徽农业大学学报,2016,43(1):83-87.
[25]李跃建,刘小俊,刘独臣,等.黄瓜自根苗、嫁接苗根系活力、净光合速率和蛋白质表达差异研究[J].西南农业学报,2007(5):979-981.
[26]马凌之.基质湿度影响下嫁接黄瓜(Cucumis sativus L.)根系生长冗余的初步研究[D].兰州:甘肃农业大学,2007.
[27]司亚平,程孙亮,陈殿奎.西瓜断根嫁接育苗根际营养体积对根系活力和秧苗质量的影响[J].华北农学报,2005(3):67-69.
[28]齐红岩,李天来,刘轶飞,等.嫁接对薄皮甜瓜光合特性、产量与含糖量的影响[J].沈阳农业大学学报,2006,37(2):155-158.
[29]刘轶飞.嫁接砧木对厚皮甜瓜叶片光合物质生产的影响[J].园艺学报,2010,37(S):2176.
[30]魏述英,吴震,黄俊.砧木对网纹甜瓜嫁接植株生长和光合特性的影响[J].上海农业学报,2006(4):114-117.
[31]许传强,李天来,齐红岩.嫁接对网纹甜瓜光合特性、生长状况及产量的影响[J].中国西瓜甜瓜,2005(2):5-7.
[32]周宝利,姜荷,赵鑫.不同砧木嫁接茄子抗黄萎病特性及其与根系分泌物关系[J].沈阳农业大学学报,2001,32(6):414-417.
[33] GANGAPPA S N,BOTTO J F.The multifaceted roles of HY5 in plant growth and development[J].Molecular Plant,2016,9(10):1353-1365.
[34] ABBAS N,MAURYA J P,SENAPATI D,et al.Arabidopsis CAM7 and HY5 physically interact and directly bind to the HY5promoter to regulate its expression and thereby promote photomorphogenesis[J].The Plant Cell,2014,26(3):1036-1052.
[35] ANG L H,CHATTOPADHYAY S,WEI N,et al.Molecular interaction between COP1 and HY5 defines a regulatory switch for light control of Arabidopsis development[J].Molecular cell,1998,1(2):213-222.
[36] HUANG X,OUYANG X,YANG P,et al.Arabidopsis FHY3 and HY5 positively mediate induction of COP1 transcription in response to photomorphogenic UV-B light[J].The Plant Cell,2012,24(11):4590-4606.
[37] YU Y,WANG J,ZHANG Z,et al.Ethylene promotes hypocotyl growth and HY5 degradation by enhancing the movement of COP1 to the nucleus in the light[J].PLoS Genet,2013,9(12):e1004025.
[38]黄远,别之龙,孔秋生,等.嫁接对西瓜和甜瓜果实品质影响的研究进展[J].中国蔬菜,2012(4):10-18.
[39] JONASSEN E M,LEA U S,LILLO C.HY5 and HYH are positive regulators of nitrate reductase in seedlings and rosette stage plants[J].Planta,2008,227(3):559-564.
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