过表达水稻OsAQP增强转基因拟南芥耐盐性
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摘要
水稻OsAQP是实验室前期从cDNA文库中筛选的功能未知的水通道蛋白质编码基因。本文采用DNA重组技术构建其植物过表达载体,并对拟南芥进行了遗传转化,筛选获得转基因拟南芥。采用50、100、125和150 mmol/L梯度盐胁迫处理,结果显示,转基因拟南芥的发芽率、根长以及鲜重分别比对照至少高17%、40. 8%和14. 29%,且差异达到显著水平(P<0. 05)。在正常条件下,转基因植株叶片中抗坏血酸过氧化物酶(APX)活性显著高于WT;经300 mmol/L NaCl处理,转基因拟南芥叶片中超氧化物歧化酶(SOD)、过氧化物酶(POD)、APX酶活性均升高,与处理前相比分别提高7. 37倍、30. 87倍和1. 77倍,且与WT的酶活性差异达到显著水平(P<0. 05);丙二醛(MDA)含量也在处理后上升,但在转基因植株中的含量低于WT,分别是WT的0. 74倍、0. 68倍和0. 62倍,差异同样达到显著水平(P<0. 05)。本研究提示,OsAQP过表达不仅能够促进拟南芥种子萌发和根系生长,而且在盐胁迫下通过提高拟南芥内源抗氧化酶活性、降低膜脂过氧化程度,增强了转基因植株对一定程度盐胁迫的耐受性。
Aquaporin-encoding gene Os AQP was isolated from rice cDNA library with unidentified functions. In this article, the plant over-expression vector was constructed by DNA recombination technology,and then transformed into Arabidopsis thaliana. After treatment of gradient salt stress( 50,100,125 and 150 mmol/L),the germination rate,root length and fresh weight of transgenic Arabidopsis were at least 17%,40. 8% and 14. 29% significantly higher than those of the control respectively( P <0. 05). Under normal conditions,APX activity in leaves of transgenic plants was significantly higher than that of WT. After treatment of 300 mmol/L Na Cl,the activities of SOD,POD and APX in the leaves of transgenic Arabidopsis increased by at least 7. 37 times,30. 87 times and 1. 77 times,respectively,as compared with those before treatment,and the difference of the activities of SOD,POD and APX with control reached a significant level( P<0. 05). Furthermore,the content of MDA was also increased,but the content of MDA in transgenic plants was significantly lower than that of WT,which was 0. 74,0. 68 and 0. 62 times of WT,respectively( P < 0. 05). This study suggests that ectopic over-expression of Os AQP can not only promote the seed germination and root growth of transgenic Arabidopsis,but also enhance the tolerance of transgenic plant to salt stress by increasing the activities of endogenous antioxidant enzymes and reducing the degree of membrane lipid peroxidation.
Aquaporin-encoding gene Os AQP was isolated from rice cDNA library with unidentified functions. In this article, the plant over-expression vector was constructed by DNA recombination technology,and then transformed into Arabidopsis thaliana. After treatment of gradient salt stress( 50,100,125 and 150 mmol/L),the germination rate,root length and fresh weight of transgenic Arabidopsis were at least 17%,40. 8% and 14. 29% significantly higher than those of the control respectively( P <0. 05). Under normal conditions,APX activity in leaves of transgenic plants was significantly higher than that of WT. After treatment of 300 mmol/L Na Cl,the activities of SOD,POD and APX in the leaves of transgenic Arabidopsis increased by at least 7. 37 times,30. 87 times and 1. 77 times,respectively,as compared with those before treatment,and the difference of the activities of SOD,POD and APX with control reached a significant level( P<0. 05). Furthermore,the content of MDA was also increased,but the content of MDA in transgenic plants was significantly lower than that of WT,which was 0. 74,0. 68 and 0. 62 times of WT,respectively( P < 0. 05). This study suggests that ectopic over-expression of Os AQP can not only promote the seed germination and root growth of transgenic Arabidopsis,but also enhance the tolerance of transgenic plant to salt stress by increasing the activities of endogenous antioxidant enzymes and reducing the degree of membrane lipid peroxidation.
引文
[1]Denker BM,Smith BL,Kuhajda FP,et al.Identification,purification,and partial characterization of a novel Mr 28,000integral membrane protein from erythrocytes and renal tubules[J].J Biol Chem,1988,263(30):15634-15642
[2]Preston GM,Carroll TP,Guggino WB,et al.Appearance of water channels in Xenopus oocytes expressing red cell CHIP28protein[J].Science,1992,256(5055):385-387
[3]Danielson JA,Johanson U.Unexpected complexity of the aquaporin gene family in the moss Physcomitrella patens[J].BMCPlant Biol,2008,8:45
[4]Bienert GP,Bienert MD,Jahn TP,et al.Solanaceae XIPs are plasma membrane aquaporins that facilitate the transport of many uncharged substrates[J].Plant J,2011,66(2):306-317
[5]Soto G,Alleva K,Amodeo G,et al.New insight into the evolution of aquaporins from flowering plants and vertebrates:orthologous identification and functional transfer is possible[J].Gene,2012,503(1):165-176
[6]Wang LL,Chen AP,Zhong NQ,et al.The Thellungiella salsuginea tonoplast aquaporin Ts TIP1;2 functions in protection against multiple abiotic stresses[J].Plant Cell Physiol,2014,55(1):148-161
[7]Biela A,Grote K,Otto B,et al.The Nicotiana tabacum plasma membrane aquaporin Nt AQP1 is mercury-insensitive and permeable for glycerol[J].Plant J,1999,18(5):565-570
[8]Maurel C,Verdoucq L,Luu DT,et al.Plant aquaporins:membrane channels with multiple integrated functions[J].Annu Rev Plant Biol,2008,59:595-624
[9]Liao YD,Lin KH,Chen CC,et al.Oryza sativa protein phosphatase 1a(Os PP1a)involved in salt stress tolerance in transgenic rice[J].Mol Breeding,2016,36:22
[10]Hu W,Yuan Q,Wang Y,et al.Overexpression of a wheat aquaporin gene,TaAQP8,enhances salt stress tolerance in transgenic tobacco[J].Plant Cell Physiol,2012,53(12):2127-2141
[11]Chang W,Liu X,Zhu J,et al.An aquaporin gene from halophyte Sesuvium portulacastrum,SpAQP1,increases salt tolerance in transgenic tobacco[J].Plant Cell Rep,2016,35(2):385-395
[12]Xin S,Yu G,Sun L,et al.Expression of tomato Sl TIP2;2enhances the tolerance to salt stress in the transgenic Arabidopsis and interacts with target proteins[J].J Plant Res,2014,127(6):695-708
[13]Xu Y,Hu W,Liu J,et al.A banana aquaporin gene,MaPIP1;1,is involved in tolerance to drought and salt stresses[J].BMCPlant Biol,2014,14:59
[14]Wang X,Li Y,Ji W,et al.A novel Glycine soja tonoplast intrinsic protein gene responds to abiotic stress and depresses salt and dehydration tolerance in transgenic Arabidopsis thaliana[J].JPlant Physiol,2011,168(11):1241-1248
[15]刘悦霞,梁卫红.水稻Os AQP基因的生物信息学分析[J].河南师范大学学报(自然科学版)(Liu YX,Liang WH.Bioinformatical analysis of Os AQP gene from rice[J].J Henan Normal Univ(Nat Sci)),2008,36(2):117-119
[16]Liang WH,Li L,Zhang F,et al.Effects of abiotic stress,light,phytochromes and phytohormones on the expression of Os AQP,a rice aquaporin gene[J].Plant Growth Regul,2013,69(1):21-27
[17]张静,梁卫红.2种非生物胁迫和7种激素对水稻Os AQP基因表达的影响[J].河南师范大学学报(自然科学版)(Zhang J,Liang WH.Effects of seven phytohormones and two abiotic stresses on the expression of rice Os AQP gene[J].J Henan Normal Univ(Nat Sci)),2016,44(1):105-109
[18]Li ZQ,Zhang JL,Li JX,et al.The functional and regulatory mechanisms of the Thellungiella salsuginea Ascorbate Peroxidase6(Ts APX6)in response to salinity and water deficit stresses[J].PLoS One,2016,11(4):e0154042
[19]Clough SJ,Bent AF.Floral dip:a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana[J].Plant J,1998,16(6):735-743
[20]李合生.植物生理生化实验原理和技术[M].高等教育出版社(Li HS.Principles and Techniques of Plant Physiological Biochemical Experiments[M].Higher Education Press),2000:261-263
[21]任小林,李嘉瑞.杏果实成熟衰老过程中活性氧和几种生理指标的变化[J].植物生理学通讯(Ren XL,Li JR.Changes of active oxygen and some physiological indicators in the processes of maturation and senescence ripening of apricot fruits[J].Plant Physiol Commun),1991,27(1):34-36
[22]沈文飚,徐朗莱,叶茂炳,等.抗坏血酸过氧化物酶活性测定的探讨[J].植物生理学通讯(Sheng WB,Xu LL,Ye MB,et al.Study on determination of ASP activity[J].Plant Physiol Commun),1996,32(3):203-205
[23]Stewart RR,Bewley JD.Lipid peroxidation associated with accelerated aging of soybean axes[J].Plant Physiol,1980,65(2):245-248
[24]Gao Z,He X,Zhao B,et al.Overexpressing a putative aquaporin gene from wheat,Ta NIP,enhances salt tolerance in transgenic Arabidopsis[J].Plant Cell Physiol,2010,51(5):767-775
[25]Sade N,Vinocur BJ,Diber A,et al.Improving plant stress tolerance and yield production:is the tonoplast aquaporin SlTIP2;2 a key to isohydric to anisohydric conversion?[J].New Phytol,2009,181(3):651-661
[26]Peng Y,Lin W,Cai W,et al.Overexpression of a Panax ginseng tonoplast aquaporin alters salt tolerance,drought tolerance and cold acclimation ability in transgenic Arabidopsis plants[J].Planta,2007,226(3):729-740
[27]Jiang M,Zhang J.Water stress-induced abscisic acid accumulation triggers the increased generation of reactive oxygen species and up-regulates the activities of antioxidant enzymes in maize leaves[J].J Exp Bot,2002,53(379):2401-2410
[28]Cakmak I,Horst WJ.Effect of aluminium on lipid peroxidation,superoxide dismutase,catalase and peroxidase activities in root tips of soybean(Glycine max)[J].Physiol Plant,1991,83(3):463-468
[29]Jaleel CA,Riadh K,Gopi R,et al.Antioxidant defense responses:physiological plasticity in higher plants under abiotic constraints[J].Acta Physiol Plant,2009,31(3):427-436
[30]Li GW,Peng YH,Yu X,et al.Transport functions and expression analysis of vacuolar membrane aquaporins in response to various stresses in rice[J].Physiol Plant,2008,165(18):1879-1888
[31]张帆,李玉萍,孙卫宁,等.液泡膜内在蛋白基因Os TIP1;1异位表达对油菜根皮层细胞水分关系参数的影响[J].植物生理学报(Zhang F,Li YP,Sun WN,et al.Effects of Os TIP1;1ecotopic expression on root cortex cell water related parameters of Brassica napus[J].Acta Phytophysiol Sin),2014,50(12):1863-1870
[2]Preston GM,Carroll TP,Guggino WB,et al.Appearance of water channels in Xenopus oocytes expressing red cell CHIP28protein[J].Science,1992,256(5055):385-387
[3]Danielson JA,Johanson U.Unexpected complexity of the aquaporin gene family in the moss Physcomitrella patens[J].BMCPlant Biol,2008,8:45
[4]Bienert GP,Bienert MD,Jahn TP,et al.Solanaceae XIPs are plasma membrane aquaporins that facilitate the transport of many uncharged substrates[J].Plant J,2011,66(2):306-317
[5]Soto G,Alleva K,Amodeo G,et al.New insight into the evolution of aquaporins from flowering plants and vertebrates:orthologous identification and functional transfer is possible[J].Gene,2012,503(1):165-176
[6]Wang LL,Chen AP,Zhong NQ,et al.The Thellungiella salsuginea tonoplast aquaporin Ts TIP1;2 functions in protection against multiple abiotic stresses[J].Plant Cell Physiol,2014,55(1):148-161
[7]Biela A,Grote K,Otto B,et al.The Nicotiana tabacum plasma membrane aquaporin Nt AQP1 is mercury-insensitive and permeable for glycerol[J].Plant J,1999,18(5):565-570
[8]Maurel C,Verdoucq L,Luu DT,et al.Plant aquaporins:membrane channels with multiple integrated functions[J].Annu Rev Plant Biol,2008,59:595-624
[9]Liao YD,Lin KH,Chen CC,et al.Oryza sativa protein phosphatase 1a(Os PP1a)involved in salt stress tolerance in transgenic rice[J].Mol Breeding,2016,36:22
[10]Hu W,Yuan Q,Wang Y,et al.Overexpression of a wheat aquaporin gene,TaAQP8,enhances salt stress tolerance in transgenic tobacco[J].Plant Cell Physiol,2012,53(12):2127-2141
[11]Chang W,Liu X,Zhu J,et al.An aquaporin gene from halophyte Sesuvium portulacastrum,SpAQP1,increases salt tolerance in transgenic tobacco[J].Plant Cell Rep,2016,35(2):385-395
[12]Xin S,Yu G,Sun L,et al.Expression of tomato Sl TIP2;2enhances the tolerance to salt stress in the transgenic Arabidopsis and interacts with target proteins[J].J Plant Res,2014,127(6):695-708
[13]Xu Y,Hu W,Liu J,et al.A banana aquaporin gene,MaPIP1;1,is involved in tolerance to drought and salt stresses[J].BMCPlant Biol,2014,14:59
[14]Wang X,Li Y,Ji W,et al.A novel Glycine soja tonoplast intrinsic protein gene responds to abiotic stress and depresses salt and dehydration tolerance in transgenic Arabidopsis thaliana[J].JPlant Physiol,2011,168(11):1241-1248
[15]刘悦霞,梁卫红.水稻Os AQP基因的生物信息学分析[J].河南师范大学学报(自然科学版)(Liu YX,Liang WH.Bioinformatical analysis of Os AQP gene from rice[J].J Henan Normal Univ(Nat Sci)),2008,36(2):117-119
[16]Liang WH,Li L,Zhang F,et al.Effects of abiotic stress,light,phytochromes and phytohormones on the expression of Os AQP,a rice aquaporin gene[J].Plant Growth Regul,2013,69(1):21-27
[17]张静,梁卫红.2种非生物胁迫和7种激素对水稻Os AQP基因表达的影响[J].河南师范大学学报(自然科学版)(Zhang J,Liang WH.Effects of seven phytohormones and two abiotic stresses on the expression of rice Os AQP gene[J].J Henan Normal Univ(Nat Sci)),2016,44(1):105-109
[18]Li ZQ,Zhang JL,Li JX,et al.The functional and regulatory mechanisms of the Thellungiella salsuginea Ascorbate Peroxidase6(Ts APX6)in response to salinity and water deficit stresses[J].PLoS One,2016,11(4):e0154042
[19]Clough SJ,Bent AF.Floral dip:a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana[J].Plant J,1998,16(6):735-743
[20]李合生.植物生理生化实验原理和技术[M].高等教育出版社(Li HS.Principles and Techniques of Plant Physiological Biochemical Experiments[M].Higher Education Press),2000:261-263
[21]任小林,李嘉瑞.杏果实成熟衰老过程中活性氧和几种生理指标的变化[J].植物生理学通讯(Ren XL,Li JR.Changes of active oxygen and some physiological indicators in the processes of maturation and senescence ripening of apricot fruits[J].Plant Physiol Commun),1991,27(1):34-36
[22]沈文飚,徐朗莱,叶茂炳,等.抗坏血酸过氧化物酶活性测定的探讨[J].植物生理学通讯(Sheng WB,Xu LL,Ye MB,et al.Study on determination of ASP activity[J].Plant Physiol Commun),1996,32(3):203-205
[23]Stewart RR,Bewley JD.Lipid peroxidation associated with accelerated aging of soybean axes[J].Plant Physiol,1980,65(2):245-248
[24]Gao Z,He X,Zhao B,et al.Overexpressing a putative aquaporin gene from wheat,Ta NIP,enhances salt tolerance in transgenic Arabidopsis[J].Plant Cell Physiol,2010,51(5):767-775
[25]Sade N,Vinocur BJ,Diber A,et al.Improving plant stress tolerance and yield production:is the tonoplast aquaporin SlTIP2;2 a key to isohydric to anisohydric conversion?[J].New Phytol,2009,181(3):651-661
[26]Peng Y,Lin W,Cai W,et al.Overexpression of a Panax ginseng tonoplast aquaporin alters salt tolerance,drought tolerance and cold acclimation ability in transgenic Arabidopsis plants[J].Planta,2007,226(3):729-740
[27]Jiang M,Zhang J.Water stress-induced abscisic acid accumulation triggers the increased generation of reactive oxygen species and up-regulates the activities of antioxidant enzymes in maize leaves[J].J Exp Bot,2002,53(379):2401-2410
[28]Cakmak I,Horst WJ.Effect of aluminium on lipid peroxidation,superoxide dismutase,catalase and peroxidase activities in root tips of soybean(Glycine max)[J].Physiol Plant,1991,83(3):463-468
[29]Jaleel CA,Riadh K,Gopi R,et al.Antioxidant defense responses:physiological plasticity in higher plants under abiotic constraints[J].Acta Physiol Plant,2009,31(3):427-436
[30]Li GW,Peng YH,Yu X,et al.Transport functions and expression analysis of vacuolar membrane aquaporins in response to various stresses in rice[J].Physiol Plant,2008,165(18):1879-1888
[31]张帆,李玉萍,孙卫宁,等.液泡膜内在蛋白基因Os TIP1;1异位表达对油菜根皮层细胞水分关系参数的影响[J].植物生理学报(Zhang F,Li YP,Sun WN,et al.Effects of Os TIP1;1ecotopic expression on root cortex cell water related parameters of Brassica napus[J].Acta Phytophysiol Sin),2014,50(12):1863-1870