铜胁迫下葡萄植株的生理响应和铜转运蛋白基因VvCTR1的克隆及分析
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摘要
[目的]本文旨在研究重金属铜对葡萄植物生长发育的影响,为揭示重金属铜对植物的伤害机制提供理论依据。[方法]采用过量的硫酸铜以及硫酸铜与其螯合剂混合液处理葡萄植株幼苗,以Hoagland溶液为对照,分析根和叶片的发育情况,叶绿素含量以及铜在植株中的分布情况;克隆了铜转运蛋白基因VvCTR1,分析其在果实发育过程中的表达量,并对其结构特征和物种间的同源性进行分析,同时分析铜胁迫下葡萄根、茎和叶中的VvCTR1表达情况。[结果]与对照相比,过量的硫酸铜、硫酸铜与柠檬酸或EDTA混合处理可以抑制葡萄植株新根的发育数量和长度,促进叶片叶绿素的降解和落叶速率,增加叶柄基部乙烯的产生以及多聚半乳糖醛酸酶和纤维素酶活性,尤其以硫酸铜和EDTA混合处理伤害更明显。Cu~(2+)在植株叶、茎、根中的含量从小到大分别依次为上部叶片、中部叶片、下部叶片,上部茎、中部茎、下部茎,老根、幼根;硫酸铜处理植株中的Cu~(2+)含量高于对照,并且随着处理时间的延长含量不断升高,尤其是硫酸铜与柠檬酸或者EDTA混合处理植株中Cu~(2+)含量更高。克隆的铜转运蛋白基因VvCTR1中无内含子,其编码的蛋白含有3个跨膜区TMD1~3,N端含有1个甲硫氨酸富含区。VvCTR1基因的表达量随着果实发育逐渐降低。无论在根中还是在不同部位的茎和叶中,VvCTR1都能被铜诱导。[结论]过量的铜会对葡萄植株造成伤害,尤其与Cu~(2+)的螯合剂柠檬酸或EDTA同时存在时,能使更多的Cu~(2+)进入植物体内,最终引起葡萄植株死亡。
[Objectives]This study aims to analyze the effects of heavy metal copper on the growth and development of grape plants,and provided theoretical basis for revealing the mechanism of damage by heavy metal copper to plants.[Methods]The excess CuSO_4 and the mixture of CuSO_4 and its chelating agent were applied on grape seedlings,the root and leaf development,leaf chlorophyll content,and the distribution of copper in plants were analyzed;copper transport VvCTR1 gene was isolated,and its expression levels were analyzed during fruit development.Its structure and homology among species were analyzed,and its expression levels were analyzed under the copper stress in roots,stems and leaves.[Results]Compared to the control,excess CuSO_4,the mixture of CuSO_4 and citric acid or EDTA inhibited the development of the number and length of new roots in grape,promoted the degradation of chlorophyll and leaf rate drop,and increased the production of ethylene and the polygalacturonase and cellulase activities in petiole base.Especially there were obvious damage in mixture of CuSO_4 and EDTA treatment.The content of Cu~(2+)in leaves,stems and roots from small to large was the morphologically up to down of the stems and leaves and old roots to new roots in turn.And the content of copper ion in CuSO_4 treated plants was higher than control,and showed an increase with the extension of time,especially the mixture of CuSO_4 and citric acid or EDTA treatment had more copper ions; there was no intron in copper transport VvCTR1 gene,and it contained three transmembrane regions of TMD1-3,which N contained a methionine rich region.Its expression decreased gradually with the fruit development,and the expression of VvCTR1 gene was enhanced under copper both in roots and in different positions of stems and leaves.[Conclusions]Excessive copper can cause damage to grape plants,especially when combined with Cu~(2+)chelatecitric acid or EDTA,which can make more Cu~(2+) enter the plant and ultimately cause grape plant death.
[Objectives]This study aims to analyze the effects of heavy metal copper on the growth and development of grape plants,and provided theoretical basis for revealing the mechanism of damage by heavy metal copper to plants.[Methods]The excess CuSO_4 and the mixture of CuSO_4 and its chelating agent were applied on grape seedlings,the root and leaf development,leaf chlorophyll content,and the distribution of copper in plants were analyzed;copper transport VvCTR1 gene was isolated,and its expression levels were analyzed during fruit development.Its structure and homology among species were analyzed,and its expression levels were analyzed under the copper stress in roots,stems and leaves.[Results]Compared to the control,excess CuSO_4,the mixture of CuSO_4 and citric acid or EDTA inhibited the development of the number and length of new roots in grape,promoted the degradation of chlorophyll and leaf rate drop,and increased the production of ethylene and the polygalacturonase and cellulase activities in petiole base.Especially there were obvious damage in mixture of CuSO_4 and EDTA treatment.The content of Cu~(2+)in leaves,stems and roots from small to large was the morphologically up to down of the stems and leaves and old roots to new roots in turn.And the content of copper ion in CuSO_4 treated plants was higher than control,and showed an increase with the extension of time,especially the mixture of CuSO_4 and citric acid or EDTA treatment had more copper ions; there was no intron in copper transport VvCTR1 gene,and it contained three transmembrane regions of TMD1-3,which N contained a methionine rich region.Its expression decreased gradually with the fruit development,and the expression of VvCTR1 gene was enhanced under copper both in roots and in different positions of stems and leaves.[Conclusions]Excessive copper can cause damage to grape plants,especially when combined with Cu~(2+)chelatecitric acid or EDTA,which can make more Cu~(2+) enter the plant and ultimately cause grape plant death.
引文
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[3]卢树昌,高悦.天津市郊区果园土壤重金属铜积累状况调查分析[J].天津农业科学,2009,15(1):41-45.Lu S C,Gao Y.Investigation and analyses on metal copper accumulation of orchard soil in Tianjin suburb[J].Tianjin Agricultural Sciences,2009,15(1):41-45(in Chinese with English abstract).
[4]王正直,邱德峰.果园土壤铜素的含量、形态及剖面特征研究[J].土壤通报,2002,33(5):369-371.Wang Z Z,Qiu D F.Study on the content,form and characteristics of copper in orchard soils[J].Bulletin of Soil Science,2002,33(5):369-371(in Chinese with English abstract).
[5]单正军,王连生,蔡道基,等.果园土壤铜污染状况及其对作物生长的影响[J].农业环境保护,2002,21(2):119-121.Shan Z J,Wang L S,Cai D J,et al.Pollution of copper in orchard soil and it's effects on plant growth[J].Agro-Environmental Protection,2002,21(2):119-121(in Chinese with English abstract).
[6]郑袁明,陈同斌,郑国砥,等.不同土地利用方式对土壤铜积累的影响——以北京市为例[J].自然资源学报,2005,20(5):690-696.Zheng Y M,Chen T B,Zheng G D,et al.Soil copper accumulation under different land use types:the case of Beijing[J].Journal of Natural Resources,2005,20(5):690-696(in Chinese with English abstract).
[7]张连忠,路克国,杨洪强.苹果幼树铜、镉分布特征与累积规律研究[J].园艺学报,2006,33(l):111-114.Zhang L Z,Lu K G,Yang H Q.The distribution and accumulation of cuprum and cadmium in young apple tree[J].Acta Horticulturae Sinica,2006,33(1):111-114(in Chinese with English abstract).
[8]陈玉胜,陈亚华,王桂萍,等.硫对水稻种子萌发过程中铜毒害的缓解效应[J].南京农业大学学报,2007,30(2):44-48.DOI:10.7685/j.issn.1000-2030.2007.02.009.Chen Y S,Chen Y H,Wang G P,et al.The alleviation effect of exogenous sulfate on copper toxicity in seed germination of rice(Oryza sativa L.)[J].Journal of Nanjing Agricultural University,2007,30(2):44-48(in Chinese with English abstract).
[9]王友保,刘登义.Cu、As及其复合污染对小麦生理生态指标的影响[J].应用生态学报,2001,12(5):773-776.Wang Y B,Liu D Y.Effect of Cu,As and their combination pollution on eco-physiological index of wheat[J].Chinese Journal of Applied Ecology,2001,12(5):773-776(in Chinese with English abstract).
[10]Alva A K,Chen E Q.Effects of external copper concentrations on uptake of trace elements by citrus seedlings[J].Soil Science,1995,159:59-64.
[11]阚世红,孙百晔,刘春生.棕壤中长期低剂量铜胁迫对苹果幼树的毒性效应[J].农业环境科学学报,2010,29(1):38-42.Kan S H,Sun B Y,Liu C S.Toxic effects of long-term low-dose copper(Cu)stress on apple trees in brown soils[J].Journal of Agro-Environment Science,2010,29(1):38-42(in Chinese with English abstract).
[12]孙百晔,阚世红,刘春生,等.农艺措施对苹果幼树长期低剂量铜胁迫的影响及缓解效果分类[J].农业环境科学学报,2010,29(2):251-257.Sun B Y,Kan S H,Liu C S,et al.Effects of agronomic measures on long-term toxicity of low-dose copper for apple trees and classification of amelioration[J].Journal of Agro-Enviroment Science,2010,29(2):251-257.
[13]Lyanguzova I V.Effect of nickel and copper on bilberry seed germination and seedling development[J].Russian J Plant Physiol,1999,46:431-432.
[14]潘逸,周立祥.施用有机物料对土壤中Cu、Cd形态及小麦吸收的影响:田间微区试验[J].南京农业大学学报,2007,30(2):142-146.DOI:10.7685/j.issn.1000-2030.2007.02.030.Pan Y,Zhou L X.Influence of applying organic manures on the chemical form of Cu and Cd in the contaminated soil and on wheat uptake:field micro-plot trials[J].Journal of Nanjing Agricultural University,2007,30(2):142-146(in Chinese with English abstract).
[15]李得孝,郭月霞,员海燕,等.玉米叶绿素含量测定方法研究[J].中国农学通报,2005,21(6):153-155.Li D X,Guo Y X,Yuan H Y,et al.Determined methods of chlorophyll from maize[J].Chinese Agricultural Science Bulletin,2005,21(6):153-155(in Chinese with English abstract).
[16]Sun L,Sun Y,Zhang M,et al.Suppression of 9-cis-epoxycarotenoid dioxygenase,which encodes a key enzyme in abscisic acid biosynthesis,alters fruit texture in transgenic tomato[J].Plant Physiology,2012,158(1):283-298.
[17]贾海锋,刘众杰,赵鹏程,等.转录因子ABI4调控草莓果实成熟的分子机制[J].南京农业大学学报,2015,38(6):908-914.DOI:10.7685/j.issn.1000-2030.2015.06.006.Jia H F,Liu Z J,Zhao P C,et al.The molecular mechanism of transcription factors ABI4 in regulation of strawberry fruit ripening[J].Journal of Nanjing Agricultural University,2015,38(6):908-914(in Chinese with English abstract).
[18]Contreras L,Mella D,Moenne A,et al.Differential responses to copper-induced oxidative stress in the marine macroalgae Lessonia nigrescens and Scytosiphon lomentaria(Phaeophyceae)[J].Aquatic Toxicology,2009,94:94-102.
[19]Marschner.The role of the rhizosphere in nutrient use efficiency in crops[C]//Hawkesford M J,Barraclough P.The Molecular and Physiological Basis of Uutrient Use Efficiency in Crops.Wilipedia:Wiley,2011:47-63.
[20]Nevitt T,Ohrvik H,Thiele D J.Charting the travels of copper in eukaryotes from yeast to mammals[J].Biochimica Biophysica Acta,2012,1823:1580-1593.
[21]Sancenon V,Puig S,Mateu-Andres I,et al.The Arabidopsis copper transporter COPT1 functions in root elongation and pollen development[J].The Journal of Biological Chemistry,2004,279:15348-15355.
[22]Garcia A,Andres N,Perea A,et al.The Arabidopsis COPT6 transport protein functions in copper distribution under copper-deficient conditions[J].Plant Cell Physiol,2013,54(8):1378-1390.
[23]Andres-Colas N,Perea-Garcia A,Puig S,et al.Deregulated copper transport affects Arabidopsis development specially in the absence of environmental cycles[J].Plant Physiology,2010,153:170-184.
[24]Perea-Garcia A,Garcia-Molina A,Andres-Colas N,et al.Arabidopsis copper transport protein COPT2 participates in the cross talk between iron deficiency responses and low-phosphate signaling[J].Plant Physiology,2013,162:180-194.
[25]de Feo C J,Aller S G,Siluvai G S,et al.Three-dimensional structure of the human copper transporter h CTR1[J].Proc Natl Acad Sci USA,2009,106:4237-4242.
[26]Aller S G,Eng E T,de Feo C J,et al.Eukaryotic CTR copper uptake transporters require two faces of the third transmembrane domain for helix packing,oligomerization,and function[J].The Journal of Biological Chemistry,2004,279:53435-53441.
[2]常红岩,孙百晔,刘春生.植物铜素毒害研究进展[J].山东农业大学学报,2000,31(2):227-230.Chang H Y,Sun B Y,Liu C S.Advances in the study of plants copper toxicity[J].Journal of Shandong Agricultural University,2000,31(2):227-230(in Chinese with English abstract).
[3]卢树昌,高悦.天津市郊区果园土壤重金属铜积累状况调查分析[J].天津农业科学,2009,15(1):41-45.Lu S C,Gao Y.Investigation and analyses on metal copper accumulation of orchard soil in Tianjin suburb[J].Tianjin Agricultural Sciences,2009,15(1):41-45(in Chinese with English abstract).
[4]王正直,邱德峰.果园土壤铜素的含量、形态及剖面特征研究[J].土壤通报,2002,33(5):369-371.Wang Z Z,Qiu D F.Study on the content,form and characteristics of copper in orchard soils[J].Bulletin of Soil Science,2002,33(5):369-371(in Chinese with English abstract).
[5]单正军,王连生,蔡道基,等.果园土壤铜污染状况及其对作物生长的影响[J].农业环境保护,2002,21(2):119-121.Shan Z J,Wang L S,Cai D J,et al.Pollution of copper in orchard soil and it's effects on plant growth[J].Agro-Environmental Protection,2002,21(2):119-121(in Chinese with English abstract).
[6]郑袁明,陈同斌,郑国砥,等.不同土地利用方式对土壤铜积累的影响——以北京市为例[J].自然资源学报,2005,20(5):690-696.Zheng Y M,Chen T B,Zheng G D,et al.Soil copper accumulation under different land use types:the case of Beijing[J].Journal of Natural Resources,2005,20(5):690-696(in Chinese with English abstract).
[7]张连忠,路克国,杨洪强.苹果幼树铜、镉分布特征与累积规律研究[J].园艺学报,2006,33(l):111-114.Zhang L Z,Lu K G,Yang H Q.The distribution and accumulation of cuprum and cadmium in young apple tree[J].Acta Horticulturae Sinica,2006,33(1):111-114(in Chinese with English abstract).
[8]陈玉胜,陈亚华,王桂萍,等.硫对水稻种子萌发过程中铜毒害的缓解效应[J].南京农业大学学报,2007,30(2):44-48.DOI:10.7685/j.issn.1000-2030.2007.02.009.Chen Y S,Chen Y H,Wang G P,et al.The alleviation effect of exogenous sulfate on copper toxicity in seed germination of rice(Oryza sativa L.)[J].Journal of Nanjing Agricultural University,2007,30(2):44-48(in Chinese with English abstract).
[9]王友保,刘登义.Cu、As及其复合污染对小麦生理生态指标的影响[J].应用生态学报,2001,12(5):773-776.Wang Y B,Liu D Y.Effect of Cu,As and their combination pollution on eco-physiological index of wheat[J].Chinese Journal of Applied Ecology,2001,12(5):773-776(in Chinese with English abstract).
[10]Alva A K,Chen E Q.Effects of external copper concentrations on uptake of trace elements by citrus seedlings[J].Soil Science,1995,159:59-64.
[11]阚世红,孙百晔,刘春生.棕壤中长期低剂量铜胁迫对苹果幼树的毒性效应[J].农业环境科学学报,2010,29(1):38-42.Kan S H,Sun B Y,Liu C S.Toxic effects of long-term low-dose copper(Cu)stress on apple trees in brown soils[J].Journal of Agro-Environment Science,2010,29(1):38-42(in Chinese with English abstract).
[12]孙百晔,阚世红,刘春生,等.农艺措施对苹果幼树长期低剂量铜胁迫的影响及缓解效果分类[J].农业环境科学学报,2010,29(2):251-257.Sun B Y,Kan S H,Liu C S,et al.Effects of agronomic measures on long-term toxicity of low-dose copper for apple trees and classification of amelioration[J].Journal of Agro-Enviroment Science,2010,29(2):251-257.
[13]Lyanguzova I V.Effect of nickel and copper on bilberry seed germination and seedling development[J].Russian J Plant Physiol,1999,46:431-432.
[14]潘逸,周立祥.施用有机物料对土壤中Cu、Cd形态及小麦吸收的影响:田间微区试验[J].南京农业大学学报,2007,30(2):142-146.DOI:10.7685/j.issn.1000-2030.2007.02.030.Pan Y,Zhou L X.Influence of applying organic manures on the chemical form of Cu and Cd in the contaminated soil and on wheat uptake:field micro-plot trials[J].Journal of Nanjing Agricultural University,2007,30(2):142-146(in Chinese with English abstract).
[15]李得孝,郭月霞,员海燕,等.玉米叶绿素含量测定方法研究[J].中国农学通报,2005,21(6):153-155.Li D X,Guo Y X,Yuan H Y,et al.Determined methods of chlorophyll from maize[J].Chinese Agricultural Science Bulletin,2005,21(6):153-155(in Chinese with English abstract).
[16]Sun L,Sun Y,Zhang M,et al.Suppression of 9-cis-epoxycarotenoid dioxygenase,which encodes a key enzyme in abscisic acid biosynthesis,alters fruit texture in transgenic tomato[J].Plant Physiology,2012,158(1):283-298.
[17]贾海锋,刘众杰,赵鹏程,等.转录因子ABI4调控草莓果实成熟的分子机制[J].南京农业大学学报,2015,38(6):908-914.DOI:10.7685/j.issn.1000-2030.2015.06.006.Jia H F,Liu Z J,Zhao P C,et al.The molecular mechanism of transcription factors ABI4 in regulation of strawberry fruit ripening[J].Journal of Nanjing Agricultural University,2015,38(6):908-914(in Chinese with English abstract).
[18]Contreras L,Mella D,Moenne A,et al.Differential responses to copper-induced oxidative stress in the marine macroalgae Lessonia nigrescens and Scytosiphon lomentaria(Phaeophyceae)[J].Aquatic Toxicology,2009,94:94-102.
[19]Marschner.The role of the rhizosphere in nutrient use efficiency in crops[C]//Hawkesford M J,Barraclough P.The Molecular and Physiological Basis of Uutrient Use Efficiency in Crops.Wilipedia:Wiley,2011:47-63.
[20]Nevitt T,Ohrvik H,Thiele D J.Charting the travels of copper in eukaryotes from yeast to mammals[J].Biochimica Biophysica Acta,2012,1823:1580-1593.
[21]Sancenon V,Puig S,Mateu-Andres I,et al.The Arabidopsis copper transporter COPT1 functions in root elongation and pollen development[J].The Journal of Biological Chemistry,2004,279:15348-15355.
[22]Garcia A,Andres N,Perea A,et al.The Arabidopsis COPT6 transport protein functions in copper distribution under copper-deficient conditions[J].Plant Cell Physiol,2013,54(8):1378-1390.
[23]Andres-Colas N,Perea-Garcia A,Puig S,et al.Deregulated copper transport affects Arabidopsis development specially in the absence of environmental cycles[J].Plant Physiology,2010,153:170-184.
[24]Perea-Garcia A,Garcia-Molina A,Andres-Colas N,et al.Arabidopsis copper transport protein COPT2 participates in the cross talk between iron deficiency responses and low-phosphate signaling[J].Plant Physiology,2013,162:180-194.
[25]de Feo C J,Aller S G,Siluvai G S,et al.Three-dimensional structure of the human copper transporter h CTR1[J].Proc Natl Acad Sci USA,2009,106:4237-4242.
[26]Aller S G,Eng E T,de Feo C J,et al.Eukaryotic CTR copper uptake transporters require two faces of the third transmembrane domain for helix packing,oligomerization,and function[J].The Journal of Biological Chemistry,2004,279:53435-53441.