新疆小拟南芥烯醛双键还原酶基因对转基因烟草耐盐性的影响
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摘要
为了研究前期从新疆小拟南芥中克隆的编码烯醛双键还原酶(alkenal reductase)Ap AER基因的功能,本研究构建了带有花椰菜花叶病毒(Ca MV)的35S启动子的植物过表达载体35S:Ap AER,并转化至农杆菌GV3101中,采用叶盘法转化烟草获得了转35S:Ap AER基因的烟草。用250 mmol/L的Na Cl溶液分别胁迫转基因和野生型烟草2和4 d,并测定了脯氨酸(Proline)和丙二醛(MDA)含量、过氧化氢酶(CAT)及过量化物酶(POD)酶活力等生理指标。结果表明:高盐胁迫下14 d,转基因烟草的长势明显优于野生型烟草,转基因烟草普遍植株较高、叶片大、叶色深;转基因烟草的Proline含量、CAT及POD酶活力均高于对照,而反映质膜受损情况的MDA含量显著低于对照。表明过表达Ap AER基因,能提高转基因植株清除醛自由基的能力、提高脯氨酸等渗透调节物质的含量及相关还原酶活性,从而显著提高转基因烟草的耐盐性。
We previously identified a Ap AER gene encoding alkenal reductase from Arabidopsis thaliana. To study the function of the gene, we constructed a plant overexpression vector 35 S:Ap AER under control of the cauliflower mosaic virus(Ca MV) 35 S promoter in the present study, and then it was transformed into Agrobacterium bacteria GV3101.Transgenic tobacco plants were generated by Agrobacturium-mediated leaf disk transformation method. The transgenic and wild-type tobacco plants were treated with 250 mmol/L Na Cl for 2 days and 4 days, respectively. Malondialdehyde(MDA)and proline contents, superoxide dismutase(SOD) and peroxidase(POD) activities were determined respectively. The results showed that under high salt stress, the growth of transgenic tobacco plants were obviously better than that of wild-type controls. The transgenic plants were higher; their leaves were larger and darker. Under high salt stress, proline content, CAT and POD enzyme activity of transgenic tobacco were higher than those of control, whereas the content of MDA which reflects the damage of plasma membrane was obviously lower than that of the control lines. These results indicate that the overexpression of Ap AER can improve the ability of scavenging aldehyde free radicals, increase the content of related osmotic adjustment like proline and reductase activities, thereby obviously improving the salt tolerance of transgenic tobacco.
We previously identified a Ap AER gene encoding alkenal reductase from Arabidopsis thaliana. To study the function of the gene, we constructed a plant overexpression vector 35 S:Ap AER under control of the cauliflower mosaic virus(Ca MV) 35 S promoter in the present study, and then it was transformed into Agrobacterium bacteria GV3101.Transgenic tobacco plants were generated by Agrobacturium-mediated leaf disk transformation method. The transgenic and wild-type tobacco plants were treated with 250 mmol/L Na Cl for 2 days and 4 days, respectively. Malondialdehyde(MDA)and proline contents, superoxide dismutase(SOD) and peroxidase(POD) activities were determined respectively. The results showed that under high salt stress, the growth of transgenic tobacco plants were obviously better than that of wild-type controls. The transgenic plants were higher; their leaves were larger and darker. Under high salt stress, proline content, CAT and POD enzyme activity of transgenic tobacco were higher than those of control, whereas the content of MDA which reflects the damage of plasma membrane was obviously lower than that of the control lines. These results indicate that the overexpression of Ap AER can improve the ability of scavenging aldehyde free radicals, increase the content of related osmotic adjustment like proline and reductase activities, thereby obviously improving the salt tolerance of transgenic tobacco.
引文
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[14]Babiychuk E,Kushnir S,Bellesboix E,et al.Arabidopsis thaliana NADPH oxidoreductase homologs confer tolerance of yeasts toward the thiol-oxidizing drug diamide[J].Journal of Biological Chemistry,1995,270(44):26224-26231.
[15]张婷婷,周亚平,曾幼玲.新疆短命植物小拟南芥与模式植物拟南芥的抗逆表型比较[J].生物技术通报,2013(8):43-50.Zhang T T,Zhou Y P,Zeng Y L.Comparison on stressresistent phenotypes between Arabidopsis pumila and Arabidopsis thaliana and expression analysis[J].Biotechnology Bulletin,2013(8):43-50.
[16]刘慧,郭丹丽,蔡大润,等.小拟南芥Ap ZFP基因异源超表达促进拟南芥开花并提高耐逆性[J].植物学报,2016,51(3):296-305.Liu H,Guo D L,Cai D R,et al.Heterologous overexpression of Ap ZFP promotes flowering and improves abiotic tolerance in Arabidopsis thaliana[J].Bulletin of Botany,2016,51(3):296-305.
[17]院海英,顾超,徐芳,等.小拟南芥Na+/H+逆向转运蛋白基因的克隆及生物信息学分析[J].石河子大学学报(自然科学版),2011,29(4):401-407.Yuan H Y,Gu C,Xu F,et a1.Identification and bioinformatics analysis of a vacuolar Na+/H+antiporter gene in Arabidopsis pumila[J].Journal of Shihezi University:Natural Science,2011,29(4):401-407.
[18]院海英,徐芳,吕新华,等.小拟南芥高盐胁迫诱导c DNA文库的构建与测序分析[J].石河子大学学报(自然科学版),2012,30(1):1-4.Yuan H Y,Xu F,Lv X H,et a1.Construction and sequencing analysis of c DNA hbrary induced by high salt stress in Olimarabidops pumila[J].Joumal of Shihezi University:Natural Science,2012,30(1):1-4.
[19]郑丽洁,林军,黄先忠.小拟南芥双键还原酶基因Op AER的克隆及表达分析[J].石河子大学学报(自然科学版),2016,34(2):251-258.Zhen L J,Lin J,Huang X Z.Molecular cloning and expression analysis of Op AER from Olimarabidopsis pumila[J].Journal of Shihezi University:Natural Science,2016,34(2):251-258.
[20]Shi H,Quintero F J,Pardo J M,et al.The putative plasma membrane Na+/H+antiporter SOS1 controls long-distance Na+transport in plants[J].Plant Cell,2002,14(2):465-477.
[21]Guo Y,Halfter U,Ishitani M,et al.Molecular characterization of functional domains in the protein kinase SOS2 that is required for plant salt tolerance[J].Plant Cell,2001,13(6):1383-1400.
[22]Ishitani M,Liu J,Halfter U,et al.SOS3 Function in plant salt tolerance requires N-myristoylation and calcium binding[J].Plant Cell,2000,12(9):1667-1678.
[23]陈鑫,马超,杨永娟,等.转碱蓬Ss NHX1基因烟草的耐盐抗旱性研究[J].中国生态农业学报,2017,25(10):1518-1526.Chen X,Ma C,Yang Y J,et al.Overexpression of Suaeda salsa Ss NHX1 gene enhanced salt and drought tolerance of transgenic tobacco[J].Chinese Journal of Eco-Agriculture,2017,25(10):1518-1526.
[24]唐绯绯,赵玉琳,王培龙,等.刚毛柽柳Th P5CR基因的克隆及抗逆功能分析[J].林业科学,2017,53(7):1-9.Tang F F,Zhao Y L,Wang P L,et al.Cloning and ctress tolerance analysis of Th P5CR from Tamarix hispida[J].Scientia Silvae Sinicae,2017,53(7):1-9.
[25]王伟英,李海明,戴艺民,等.中国水仙Nt PLATZ1正、反义植物表达载体构建及转化烟草的研究[J].园艺学报,2017,44(12):2399-2407Wang W Y,Li H M,Dai Y M,et al.Research on construction of sense and antisense plant expression vectors of Nt PLATZ1 gene in Narcissus tazetta var.chinensis and genetic transformation to tobacco plants[J].Acta Horticulturae Sinica,2017,44(12):2399-2407
[26]李小芳,张志良.植物生理学实验指导[M].北京:高等教育出版社,2016:154-155.
[27]张秋萍,吴霞红,郑剑恒,等.生物样本中丙二醛测定方法的研究进展[J].理化检验(化学分册),2016,52(8):979-985.Zhang Q P,Wu X H,Zheng J H,et al.Progress of researches on methods for determination of malondialdehyde in biological samples[J].Physical Testing and Chemical Analysis Part B(Chemical Analysis),2016,52(8):979-985.
[28]李忠光,龚明.愈创木酚法测定植物过氧化物酶活性的改进[J].植物生理学通讯,2008(2):323-324.
[29]Foolad M R,Jones R A.Mapping salt-tolerance genes in tomato Lycopersicon esculentum using trait-based marker analysis[J].Tag.theoretical&Applied Genetics.theoretische Und Angewandte Genetik,1993,87(1-2):184-192.
[30]王艳,杜蒙蒙,王小冬,等.癸二烯醛对3种常见浮游植物生长和光合作用的影响[J].植物学报,2015,50(3):346-353.Wang Y,Du M M,Wang X D,et a1.The effects of 2E,4Edecadienal on the growth and photosynthesis of three species of phytoplankton[J].Bulletin of Botany,2015,50(3):346-353.
[31]魏宇昆.青蒿α,β-双键还原酶基因的克隆及功能鉴定[D].上海:中国科学院上海生命科学研究院植物生理生态研究所,2009.
[32]袁琳,克热木·伊力,张利权.Na Cl胁迫对阿月浑子实生苗活性氧代谢与细胞膜稳定性的影响[J].植物生态学报,2005,29(6):985-991.Yuan L,Krem Elia,Zhang L Q.Effects of Na Cl stress on active oxygen metabolism and memberane stability in Pistacia Vera seedings[J].Chinese Journal of Plant Ecologyl,2005,29(6):985-991.
[33]邓凤飞,杨双龙,龚明.细胞信号分子对非生物胁迫下植物脯氨酸代谢的调控[J].植物生理学报,2015,51(10):1573-1582.Deng F F,Yang S L,Gong M.Regulation of cell signaling molecules on proline metabolism in plants under abiotic stress[J].Plant Physiology Communications,2015,51(10):1573-1582.
[34]Willekens H,Langebartels C,TiréC,et al.Differential expression of catalase genes in Nicotiana plumbaginifolia L.[J].Proc Natl Acad Sci U S A,1994,91(22):10450-10454.
[2]董起广,何振嘉,高红贝,等.沿黄地区盐碱地种植水稻土壤理化性质的比较[J].植物资源与环境学报,2017,(2):110-112.Dong Q G,He Z J,Gao H B,et al.Comparison on soil physicochemical properties of saline and alkaline land planted with Oryza sativa in the area along the Yellow River[J].Journal of Plant Resources and Environment,2017,(2):110-112.
[3]Guo S,Yin H,Zhang X,et al.Molecular cloning and characterization of a vacuolar H+-pyrophosphatase gene,Ss VP,from the halophyte Suaeda salsa and its overexpression increases salt and drought tolerance of Arabidopsis[J].Plant Molecular Biology,2006,60(1):41-50.
[4]闫慧芳,毛培胜,夏方山.植物抗氧化剂谷胱甘肽研究进展[J].草地学报,2013,21(3):428-434.Yan F H,Mao P S,Xia F S.Research progress in plant antioxidant glutathione[J].Acta Agrectir Sinica,2013,21(3):428-434.
[5]Apel K,Hirt H.Reactive oxygen species:metabolism,oxidative stress,and signal transduction[J].Annual Review of Plant Biology,2004,55(5):373-399.
[6]Moller I M,Jensen P E,Hansson A.Oxidative modifications to cellular components in plants[J].Annual Review of Plant Biology,2007,58(1):459-481.
[7]Elstner E F.Oxygen activation and oxygen toxicity[J].Annual Review of Plant Physiology,1982,33(1):73-96.
[8]Gardner H W,Hamberg M.Oxygenation of(3Z)-nonenal to(2E)-4-hydroxyl-2-nonenal in the broad bean(Vicia faba L.)[J].Journal of Biological Chemistry,1993,268(10):6971-6977.
[9]Noordermeer M A,Feussner I,Kolbe A,et a1.Oxygenation of(3Z)-alkenals to 4-hydroxy-(2E)-alkenals in plant extracts:a nonenzymatic process[J].Biochemical&Biophysical Research Communications,2000,277(1):112-116.
[10]Mano J,Bellesboix E,Babiychuk E,et al.Protection against photooxidative injury of tobacco leaves by 2-alkenal reductase.detoxication of lipid peroxide-derived reactive carbonyls[J].Plant Physiology,2005,139(4):1773-83.
[11]Kasahara H,Jiao Y,Bedgar D L,et al.Pinus taeda,phenylpropenal double-bond reductase:Purification,c DNA cloning,heterologous expression in Escherichia coli,and subcellular localization in P.taeda[J].Phytochemistry,2006,67(16):1765-1780.
[12]Vance C P,And T K K,Sherwood R T.Lignification as a mechanism of disease resistance[J].Ann Rev Phytopathol,1980,18(1):259-288.
[13]Kraus C,Spiteller G.Comparison of phenolic compounds from galls and shoots of Picea glauca[J].Phytochemistry,1997,44(1):59-67.
[14]Babiychuk E,Kushnir S,Bellesboix E,et al.Arabidopsis thaliana NADPH oxidoreductase homologs confer tolerance of yeasts toward the thiol-oxidizing drug diamide[J].Journal of Biological Chemistry,1995,270(44):26224-26231.
[15]张婷婷,周亚平,曾幼玲.新疆短命植物小拟南芥与模式植物拟南芥的抗逆表型比较[J].生物技术通报,2013(8):43-50.Zhang T T,Zhou Y P,Zeng Y L.Comparison on stressresistent phenotypes between Arabidopsis pumila and Arabidopsis thaliana and expression analysis[J].Biotechnology Bulletin,2013(8):43-50.
[16]刘慧,郭丹丽,蔡大润,等.小拟南芥Ap ZFP基因异源超表达促进拟南芥开花并提高耐逆性[J].植物学报,2016,51(3):296-305.Liu H,Guo D L,Cai D R,et al.Heterologous overexpression of Ap ZFP promotes flowering and improves abiotic tolerance in Arabidopsis thaliana[J].Bulletin of Botany,2016,51(3):296-305.
[17]院海英,顾超,徐芳,等.小拟南芥Na+/H+逆向转运蛋白基因的克隆及生物信息学分析[J].石河子大学学报(自然科学版),2011,29(4):401-407.Yuan H Y,Gu C,Xu F,et a1.Identification and bioinformatics analysis of a vacuolar Na+/H+antiporter gene in Arabidopsis pumila[J].Journal of Shihezi University:Natural Science,2011,29(4):401-407.
[18]院海英,徐芳,吕新华,等.小拟南芥高盐胁迫诱导c DNA文库的构建与测序分析[J].石河子大学学报(自然科学版),2012,30(1):1-4.Yuan H Y,Xu F,Lv X H,et a1.Construction and sequencing analysis of c DNA hbrary induced by high salt stress in Olimarabidops pumila[J].Joumal of Shihezi University:Natural Science,2012,30(1):1-4.
[19]郑丽洁,林军,黄先忠.小拟南芥双键还原酶基因Op AER的克隆及表达分析[J].石河子大学学报(自然科学版),2016,34(2):251-258.Zhen L J,Lin J,Huang X Z.Molecular cloning and expression analysis of Op AER from Olimarabidopsis pumila[J].Journal of Shihezi University:Natural Science,2016,34(2):251-258.
[20]Shi H,Quintero F J,Pardo J M,et al.The putative plasma membrane Na+/H+antiporter SOS1 controls long-distance Na+transport in plants[J].Plant Cell,2002,14(2):465-477.
[21]Guo Y,Halfter U,Ishitani M,et al.Molecular characterization of functional domains in the protein kinase SOS2 that is required for plant salt tolerance[J].Plant Cell,2001,13(6):1383-1400.
[22]Ishitani M,Liu J,Halfter U,et al.SOS3 Function in plant salt tolerance requires N-myristoylation and calcium binding[J].Plant Cell,2000,12(9):1667-1678.
[23]陈鑫,马超,杨永娟,等.转碱蓬Ss NHX1基因烟草的耐盐抗旱性研究[J].中国生态农业学报,2017,25(10):1518-1526.Chen X,Ma C,Yang Y J,et al.Overexpression of Suaeda salsa Ss NHX1 gene enhanced salt and drought tolerance of transgenic tobacco[J].Chinese Journal of Eco-Agriculture,2017,25(10):1518-1526.
[24]唐绯绯,赵玉琳,王培龙,等.刚毛柽柳Th P5CR基因的克隆及抗逆功能分析[J].林业科学,2017,53(7):1-9.Tang F F,Zhao Y L,Wang P L,et al.Cloning and ctress tolerance analysis of Th P5CR from Tamarix hispida[J].Scientia Silvae Sinicae,2017,53(7):1-9.
[25]王伟英,李海明,戴艺民,等.中国水仙Nt PLATZ1正、反义植物表达载体构建及转化烟草的研究[J].园艺学报,2017,44(12):2399-2407Wang W Y,Li H M,Dai Y M,et al.Research on construction of sense and antisense plant expression vectors of Nt PLATZ1 gene in Narcissus tazetta var.chinensis and genetic transformation to tobacco plants[J].Acta Horticulturae Sinica,2017,44(12):2399-2407
[26]李小芳,张志良.植物生理学实验指导[M].北京:高等教育出版社,2016:154-155.
[27]张秋萍,吴霞红,郑剑恒,等.生物样本中丙二醛测定方法的研究进展[J].理化检验(化学分册),2016,52(8):979-985.Zhang Q P,Wu X H,Zheng J H,et al.Progress of researches on methods for determination of malondialdehyde in biological samples[J].Physical Testing and Chemical Analysis Part B(Chemical Analysis),2016,52(8):979-985.
[28]李忠光,龚明.愈创木酚法测定植物过氧化物酶活性的改进[J].植物生理学通讯,2008(2):323-324.
[29]Foolad M R,Jones R A.Mapping salt-tolerance genes in tomato Lycopersicon esculentum using trait-based marker analysis[J].Tag.theoretical&Applied Genetics.theoretische Und Angewandte Genetik,1993,87(1-2):184-192.
[30]王艳,杜蒙蒙,王小冬,等.癸二烯醛对3种常见浮游植物生长和光合作用的影响[J].植物学报,2015,50(3):346-353.Wang Y,Du M M,Wang X D,et a1.The effects of 2E,4Edecadienal on the growth and photosynthesis of three species of phytoplankton[J].Bulletin of Botany,2015,50(3):346-353.
[31]魏宇昆.青蒿α,β-双键还原酶基因的克隆及功能鉴定[D].上海:中国科学院上海生命科学研究院植物生理生态研究所,2009.
[32]袁琳,克热木·伊力,张利权.Na Cl胁迫对阿月浑子实生苗活性氧代谢与细胞膜稳定性的影响[J].植物生态学报,2005,29(6):985-991.Yuan L,Krem Elia,Zhang L Q.Effects of Na Cl stress on active oxygen metabolism and memberane stability in Pistacia Vera seedings[J].Chinese Journal of Plant Ecologyl,2005,29(6):985-991.
[33]邓凤飞,杨双龙,龚明.细胞信号分子对非生物胁迫下植物脯氨酸代谢的调控[J].植物生理学报,2015,51(10):1573-1582.Deng F F,Yang S L,Gong M.Regulation of cell signaling molecules on proline metabolism in plants under abiotic stress[J].Plant Physiology Communications,2015,51(10):1573-1582.
[34]Willekens H,Langebartels C,TiréC,et al.Differential expression of catalase genes in Nicotiana plumbaginifolia L.[J].Proc Natl Acad Sci U S A,1994,91(22):10450-10454.