SeNHX1耐盐分子机制与耐盐洋桔梗培育
|
摘要
盐碱胁迫是造成世界农作物减产的主要原因。通过种植耐盐碱植物既能改良土壤，又有一定的效益，是一种相对耗资少，见效快的盐碱地农业发展方式。随着植物耐盐分子生物学的发展，植物耐盐基因工程在培育耐盐新品种中成为研究热点。本文在研究SeNHX1于酵母中的功能、定位和SeNHX1转基因烟草的耐盐性及耐盐机制的基础上，将SeNHX1转化洋桔梗，以期培育耐盐洋桔梗新品种。
SeNHX1基因是从盐角草克隆得到。在研究中，构建了pYES2-SeNHX1，pYES2-GFP和pYES2-SeNHX1-GFP三种酵母表达载体和pBin438-SeNHX1植物表达载体。带有SeNHX1目的基因的酵母表达载体转化酵母nhx1缺陷型菌株296H，可部分恢复Na~+的解毒功能，并提高野生型菌株W303的耐盐性；荧光观察表明，SeNHX1定位于酵母液泡膜。
植物表达载体pBin438-SeNHX1转化烟草，过量表达SeNHX1的转基因烟草在离体和盆栽盐胁迫下，均可提高其耐盐性。盆栽盐胁迫下，经过相同盐量的处理，盆栽土Na~+含量大于9.7mg/g时，SeNHX1转基因烟草下部老叶Na~+含量比野生型相应部位的Na~+含量高2.3mg/g FW，嫩叶中的脯氨酸含量比野生型高0.36mg/g FW，两者均达到显著水平；而丙二醛（MDA）含量显著更低。这些结果可以初步推断SeNHX1转基因烟草提高其耐盐性的机制：首先，转基因烟草可吸收盐土中更多的Na~+，促进水份的吸收与利用。其次启动离子分配机制，将吸收的Na~+更多地积累于老叶，防止幼嫩组织受到Na~+的毒害；在细胞水平上，SeNHX1转基因烟草将更多Na~+的区隔化于液泡，防止膜脂和酶活性受到伤害。
植物表达载体pBin438-SeNHX1转化洋桔梗，通过检测表明SeNHX1基因已经整合到洋桔梗基因组中，并实现转录。过量表达SeNHX1的转基因洋桔梗组培苗可在含有150mmol/L NaCl培养基中正常生长，叶片中脯氨酸含量比野生型高0.5mg/g FW，两者间达到显著差异，表现了更强的耐盐性。通过移栽管理，获得10株苗龄2个月的转基因洋桔梗。
Salinity stress is one of the most serious factors limiting the growth of plants.Many research topics focus on breeding salt-resistant varieties by geneticengineering. In this study, In order to breed salt-resistant variety of EustomaGradiflorum, we firstly analyzed the function, localization and salt–tolerancemechanism of SeNHX1in yeast and tobacco, and then transformed it into targetplants.
Yeast expression vector pYES2-SeNHX1，pYES2-GFP, pYES2-SeNHX1-GFP,and binary plant expression vector PBin438-SeNHX1were constructed, and then wasintroduced into S. cerevisiae and plants respectively. SeNHX1was localized on thevacuolar membrane of yeast by fluorescence assay. The function of Na~+-detoxicationin the nhx1mutant strain296H and higher salt tolerance in the strain W303, whichall contained the vectors of pYES2-SeNHX1or pYES2-SeNHX1-GFP, were gained.
Overexpression of SeNHX1in tobacco increased salt tolerance in both “in vitro”and pot culture. when the Na~+content in pot soil was more than9.7mg/g, comparedto wild-type, transgenic plants accumulated2.3mg/g FW greater amount of Na~+inold leaves and0.36mg/g FW higher proline in young leaves, but themalondialdehyde (MDA) content was significantly lower, The research findingsdisplay a strategy to improve tolerance to salt in SeNHX1-transgenic tobacco by (1)absorbing more Na~+for growth in saline soil,(2) accumulating Na~+in old leaves toprevent young tissue from Na~+-toxicity,(3) then, pumping Na~+into vacuole bySeNHX1for detoxification.
In addition, Eustoma Gradiflorum was transformed by C58withpBin438-SeNHX1, and salt tolerance transgenic plants of Eustoma Gradiflorum havegrown normally for two months in greenhouse.
SeNHX1基因是从盐角草克隆得到。在研究中，构建了pYES2-SeNHX1，pYES2-GFP和pYES2-SeNHX1-GFP三种酵母表达载体和pBin438-SeNHX1植物表达载体。带有SeNHX1目的基因的酵母表达载体转化酵母nhx1缺陷型菌株296H，可部分恢复Na~+的解毒功能，并提高野生型菌株W303的耐盐性；荧光观察表明，SeNHX1定位于酵母液泡膜。
植物表达载体pBin438-SeNHX1转化烟草，过量表达SeNHX1的转基因烟草在离体和盆栽盐胁迫下，均可提高其耐盐性。盆栽盐胁迫下，经过相同盐量的处理，盆栽土Na~+含量大于9.7mg/g时，SeNHX1转基因烟草下部老叶Na~+含量比野生型相应部位的Na~+含量高2.3mg/g FW，嫩叶中的脯氨酸含量比野生型高0.36mg/g FW，两者均达到显著水平；而丙二醛（MDA）含量显著更低。这些结果可以初步推断SeNHX1转基因烟草提高其耐盐性的机制：首先，转基因烟草可吸收盐土中更多的Na~+，促进水份的吸收与利用。其次启动离子分配机制，将吸收的Na~+更多地积累于老叶，防止幼嫩组织受到Na~+的毒害；在细胞水平上，SeNHX1转基因烟草将更多Na~+的区隔化于液泡，防止膜脂和酶活性受到伤害。
植物表达载体pBin438-SeNHX1转化洋桔梗，通过检测表明SeNHX1基因已经整合到洋桔梗基因组中，并实现转录。过量表达SeNHX1的转基因洋桔梗组培苗可在含有150mmol/L NaCl培养基中正常生长，叶片中脯氨酸含量比野生型高0.5mg/g FW，两者间达到显著差异，表现了更强的耐盐性。通过移栽管理，获得10株苗龄2个月的转基因洋桔梗。
Salinity stress is one of the most serious factors limiting the growth of plants.Many research topics focus on breeding salt-resistant varieties by geneticengineering. In this study, In order to breed salt-resistant variety of EustomaGradiflorum, we firstly analyzed the function, localization and salt–tolerancemechanism of SeNHX1in yeast and tobacco, and then transformed it into targetplants.
Yeast expression vector pYES2-SeNHX1，pYES2-GFP, pYES2-SeNHX1-GFP,and binary plant expression vector PBin438-SeNHX1were constructed, and then wasintroduced into S. cerevisiae and plants respectively. SeNHX1was localized on thevacuolar membrane of yeast by fluorescence assay. The function of Na~+-detoxicationin the nhx1mutant strain296H and higher salt tolerance in the strain W303, whichall contained the vectors of pYES2-SeNHX1or pYES2-SeNHX1-GFP, were gained.
Overexpression of SeNHX1in tobacco increased salt tolerance in both “in vitro”and pot culture. when the Na~+content in pot soil was more than9.7mg/g, comparedto wild-type, transgenic plants accumulated2.3mg/g FW greater amount of Na~+inold leaves and0.36mg/g FW higher proline in young leaves, but themalondialdehyde (MDA) content was significantly lower, The research findingsdisplay a strategy to improve tolerance to salt in SeNHX1-transgenic tobacco by (1)absorbing more Na~+for growth in saline soil,(2) accumulating Na~+in old leaves toprevent young tissue from Na~+-toxicity,(3) then, pumping Na~+into vacuole bySeNHX1for detoxification.
In addition, Eustoma Gradiflorum was transformed by C58withpBin438-SeNHX1, and salt tolerance transgenic plants of Eustoma Gradiflorum havegrown normally for two months in greenhouse.
引文
[1]李银心，抗盐、耐海水蔬菜的生物技术培育与海水无土栽培应用，[博士论文]，北京；中国科学院植物研究所，2001
[2]Zhu JK, Salt and drought stress signal transduction in plants. Ann Rev Plant Biol，2002，53：247~273．
[3]Zaccai M, Lewinsohn E, Pichersky E, Modifying lisianthus traits by genetic engineering，ActaHorticulturae,2001,552:137~142
[4]陈景明，植物耐盐逆性的研究概况，安徽农业科学，2006，34(14)：3277~3278
[5]Greenway H, Munns R, Mechanisms of salt tolerance in nonhalophytes，Annual Review ofPlant Physiology,1980,31:149~190
[6]Haro R, Banuelos M, Quintero FJ, et a1, Genetic basis of sodium exclution and sodiumtolerance in yeast, Plant Physiol,1993,89:868~874
[7] SANTA-CRUZ A, ACOSTA M, RUS A, et a1, Short-term Salt Tolerance Mechanisms inDifferentially Salt Tolerant Tomar to Species, Plant Physiol Biochem,1999,37(1):65~71
[8]Xiang Dian-jun, Hu Xiang-yang, Zhang Yu, Over-Expression of ICE1Gene in TransgenicRice Improves Cold Tolerance Rice Science,2008,15(3):173~178
[9] Ashraf M, Foolad, MR, Roles of glycine betaine and proline in improving plant abiotic stressresistance, Environmental and Experimental Botany,2007,59(2):206~216
[10]郭北海，甜菜碱醛脱氢酶基因转化小麦及其表达，植物学报，2000，42(3)：279~283
[11]宁顺斌，宋运淳，王玲，等，盐胁迫诱导的植物细胞凋亡—植物抗盐的可能生理机制，实验生物学报，2000，33(3)：245~253
[12]Rubio F, Gassmann W, Schroeder JI, Sodium driven potassium uptake by the plant potassiumtransporter HKT1and mutations conferring salt tolerance, Science,1995,270:1660~1663
[13]Flowers TJ, Troke PF, Yeo AR, The mechanism of salt tolerance in halophytes, Ann RevPlant Physiol,1977,28:89~121
[14]Mansour MF, NaC1alteration of plasma membrane of A lliumcepa epidermal cells,alleviation by calcium, Plant Physiol,1994,145:726~730
[15]Tester M, Davenport R, Na tolerance and Na transport in higher plants, Ann, Bot,2003,91：1~25
[16]Maser P, Eckelman B, Vaidyanathan R, et a1, Altered shoot／root Na distribution andbifurcating salt sensitivity in A rabidopsis by genetic disruption of the Nat transporterAtHKT1. FEBS Letts,2002,531:157~161
[17]张淑红，张恩平，庞金安,植物耐盐性研究进展，北方园艺，2000，3：19~20
[18]Michelet, Boutry M,The plasma membrane H+-ATPase, A highly regulated enzyme withmultiple physiological functions, Plant Physiol,1995,108:1~6
[19]王宝山，赵可夫，邹琦,作物耐盐机理研究进展及提高作物抗盐的对策，植物学通报，1997，14(增刊)：25~30
[20]余叔文，汤章城，植物生理与分子生物学，北京：科学出版社，1998，752~769
[21]许祥明，叶和春，李国凤，植物抗盐机理的研究进展，应用与环境生物学报，2000，6(4)：379~387
[22]康绍忠，新的农业科技革命与21世纪我国节水农业的发展，干旱地区农业研究，l998，l6(1)：ll~l7
[23] Carios GB, Facundo G, Dana EM, et a1, Mitochondrisa are the main target for oxidativedamage in leaves0f wheat (Triticum aestivum L.), pesticide of Experimental Botany,2004,55:403~407
[24] Wu YX, Andreas VT, Physiological Effects0f Azoxystrobin and Epoxiconazole onsenescence and the oxidative Status of Wheat, Pesticide Biochemistry and Physiology,200l,71:l~l0
[25]Salin ML, Toxic oxygen species and protective systems of the ch1oroplast, Physio1.plant,l987,72:681~689
[26]Asada K, Ascorbate peroxidase-a hydrogen pero xide scavenging enzyme in plants, Physio1,Plant,1992,85:235~241
[27]Frid chI, Superoxide dismute, Ann Rev. Biochem, l975,44:l47~l59
[27]陈洁，林栖风，植物耐盐生理及耐盐机研究进展，海南大学学报自然科学版，2003，21(2):177~182
[28]杨素欣，王振镒，盐胁迫下小麦愈伤组织生理生化特性的变化，西北农业大学学报，1999，27(2)：48~51
[29]Ramiro HL, Mariana NM, Celina ML, et a1, Effect of photooxidative stress induced byparaquat in two wheat cultivars with differential tolerance to water, Plant Science,2003, l64：84l~848
[30]蒋明义，荆家海，王韶唐，渗透胁迫对水稻幼苗膜脂过氧化及体内保护酶系统的影响，植物生理学报，l99l，l7(1)：80~84
[31]毛桂莲，许兴，徐兆桢，对盐生理生化研究进展，中国生态农业学报，2004，12(1)：43~46。
[32]李辉，李德芳，陈安国等，植物耐盐研究概况，中国麻业科学，2007，29（4）：227~232
[33]GomezCadenas A, Tadeo FR，PrimoMillo E, Talon M, Involvement of abscic acid andethylene in the responses of citrus seedlings to salt shock，Plant Physio1,1998,103:475~484
[34] Gomez Cadenas A, Arbona V, Jacas J, et al, Abscisic acid reduces leaf abscission andincreases salt tolerance in citrus plants, Plant Growth Regu,2002,21:234~240
[35] Noaman MM, Dvorak J, Dong JM, Genes inducing Salt tolerance in wheat, Lophopyrumelongatum and amphiploid and their responses to ABA under salt stress, Prospects for SalineAgriculture (Series：Tasks for Vegetation Science),2002,37:139~144
[36]Kuiper D, Schuit PJC, Auctal cytokinln concentration in plant tissue as an indicator for saltresistance in cerea1, Plant and Soil,1990:243～250
[37]翟凤林，曹鸣庆，植物耐盐性及其改良，北京:农业出版社，1989:5~7
[38]牛庆杰，李伟，李慧英，等，向日葵耐盐碱材料筛选的新途径，吉林农业科学，1998(3):27~28.
[39]赵连城，刘孟雨，武田和义，等，国外大、小麦种质资源在盐碱地直接筛选可利用材料初报，作物品种资源，1994，4:19~20
[40]翁跃进，马雅琴，耐盐春小麦新品系SW-99-10和SW-99-12，中国种业，2002，2:45
[41]史湘华，殷鸣放，赵辉，等，盐碱地与耐盐碱树种的选育，中国林副特产，2005(2):60~62
[42] Ashraf M. Breeding for salinity Tolerance in Plants, Plant Sci,1994,13:17~42
[43]顾红艳，于福安，魏天权，等，耐盐碱水稻新品种津源85选育及栽培技术，农业科技通讯，2007，1:20~21
[44]张雪艳，郜玉田，抗盐碱优质牧草新品种谷稗，农村科学实验，2008，3:34
[45]张荃，赵彦，张慧，等，海水农业:梦想与现实，山东科学，1993，3(12):1~5
[46]Jones RA, High salt tolerance potential in Lycopersion species during germinateon,Euphytica,1986,35:575~582
[47]King IP，Introgression of salt-tolerance from Thinopyrum bessarabicum into wheat, Newphytol,1997,137:25~83
[48]高玉红，李云，植物离体培养筛选耐盐突变体的研究，核农学报，2004，18(6):448~452
[49]Wang CQ, Song H, Wang XF, et al, Selection of salt-tolerant variant from China pink,Acta-Horticulutrae-Sinica,2001,28(5):469~471
[50]刘凤华，郭岩，陈受宜，等，转甜菜碱脱氢酶基因植物的耐盐性研究，遗传学报，1997，24(1)：54~58
[51]贾庚祥，BADH转化番茄提高耐盐性的研究，[博士学位论文]，北京；中国科学院植物研究所，2002
[52]Kishor, Overexpression of△-pyrroline-5-carboxylate synthetase increases prolineproduction and confers osmotolerance in transgenic plants, Plant Physiol1995,108:1387~1394
[53]刘俊君，彭学贤，王慧中，等，转mtlD／gutD双价转基因水稻的耐盐性，科学通报，2000，45(7)：724~729
[54]Flowers TJ, Garcia M, Koyama M, et a1, Breeding for salt tolerance in crop plants-the roleof molecular biology, Acta Physiologiae Plantarum,1997,19:427~433
[55]Yang SX, Zhao YX, Zhang Q, et a1, HAL1Mediate Salt Adaptation in Arabidopsis Thaliana,Cell Research,2001, ll (2):142~148
[56]Bordas M, Montesinos C, Dabauza M, et a1, Transfer of the Yeast Salt Tolerance Gene HAL1to Cucujmis Melo L, Cultivars and in Vitro Evaluation of Salt Tolerance, TransgenicResearch,1997,6(1):41~50
[57]张荃，王淑芳，赵彦修，等，HALl基因转化番茄及耐盐转基因番茄的鉴定，生物工程学报，2001，17(6)：658~662
[58]Gisbert C, Rus A M, Bolarin MC, et a1, The Yeast HAL1Gene Improves Salt Tolerance ofTransgenic Tomato, Plant Physiol,2000,123(1):393~402
[59]Arrillagada I, Mascarell RG, Gisbert C, et a1, Expression of the Yeast HAL2Gene in TomatoIncreases the in Vitro Salt Tolerance of Transgenic Progenies，Plant Science,1998,136(2):219~226
[60]Gervera M, Ortega C, Navarro A, et a1, Generation of Transgenic Citrus Plants with theTolerance to Salinity Gene HAL2from Yeast, Journal of Horticulture Science&Biotechnology,2000,75(1):26~30
[61]黄绍兴，阎隆飞，高等植物对渗透胁迫的基因表达，农业生物技术学报，1995，3(3)：l~6
[62]刘昀，李冉辉，郑易之，等，大豆PM2蛋白及其结构可提高烟草耐盐性，深圳大学学报理工版，2007，24（1）：95~101
[63]Kazuo T, Kyoko K, A recessive Arobidopsis mutant that grows photoautotrohically undersalt stress shows enhanced active oxygen detoxification, Plant Cell,1999, ll:1195~1206
[64]Mckersie DB, Murnaghan J, Jones SK, et al, Iron-su-peroxide dismutase expression intransgenic in afalfa increases winter survival without a detectable increase in photosyntheticoxidative stress tolerance, Plant Physiol,2000,122:1427~1437
[65]Du J, Zhu Z, Li WC, Over-expression of exotic superoxide dismutase gene MnSOD andincrease in stress resistance in maize, Plant Physiol Mol Biol,2006,32:57~63
[66]Perl AC, Perl-Treves R, Galili S, et a1, Enhanced oxidative stress defense in transgenicpotato expression tomato CulZn superoxide dismutase, Theor Appl Genet,1993,85:568~576
[67]Kasuga M, Liu Q, Miura S, et a1, Improving plant drought, salt, and freezing tolerance bygene transfer of a single stress-inducible transcription factor, Nat Biotech,1999,17:287~291
[68]Yamaguchi-Shinozaki k, Shinozaki K, A novel cis-acting element in all Arabidopsis gene isinvolved in responsiveness to drought, low-temperature or high-salt stress, Plant Cell,1994,6:251~264
[69]Winicov I, Bastola DR, Transgenic overexpression ofthe transcription factor Alfinl enhancesexpression of the endogenous MsPRP2gene in alfalfa an d improves salinity tolerance of theplants, Plant Physiol,1999,120:473~480
[70]Ratner A, Jacoby B, Effect of K+its coumter anion and pH on sodium efflux from barleyroots, J Exp Physiol,1976,148:425~433
[71]Zhu JK, Genetic analysis of plant salt tolerance using arabidopsis，Plant Physiol，2000，124(3):941~948
[72]Shi H, Ishitani M, Kim C, et a1, The Arabidopsis thaliana salt tolerance gene SOS1encodesa putative Na+/H+antiporter, Prec Natl Acad Sci USA,2000,97(12):6896~6901
[73]Watanabe Y, Oshima N, Tamai Y, Co-expression of the Na+/H+antiporter and H-ATPasegenes of the salt tolerant yeast Zygosaccharomyces rouxii in Saccharomyces cerevisiae,FEMS Yeast Res,2005,5(4-5):411~417
[74]Waditee R, Hibino T, Nakamura T, et a1, Overexpression of a Na+/H+antiporer confers salttolerance on a freshwater cyanobacterlum, making it capable of growth in sea water, ProcNatl Acad Sci U S A,2002,99(6):4109~4114
[75]Shi H, Lee B H, Wu S J, et a1, Overexpression of a plasma membrane Na+/H+antiportergene improves salt tolerance in Arabidopsis thaliana, Nat Biotech,2003,21(1):81~85
[76]Gaxiola R A, Rao R, Sherman A, et al, The Arabidopsis thaliana ptoton transporters.AtNHX1and Avpl, can founction in canon detoxify-cation in yest, Proc Natl AcadSci,1999,96:1480~1485
[77]Fukuda A, Nakamura A, Tanska Y, Molecular cloning and expression of the Na+/H+antiporter gene in Oryza sativa.Biochem Bioph Acta,1999,1446:149~155
[78]Hamada A, Shono M, Xia T, et al, Isolation and characterization of a Na+/H+antiporter genefrom the halophyte Atriplex gmelini, Plant Mol Bio1,2001,46:35~42
[79]Apse MP, Aharon GS, Blumwale E, et al, Identification and characterzation of aNaCl-inducible vacuolar Na+/H+antiporterin Beta vulgaris, Physiol Plant,2002,116:206~212
[80]Quintero FJ, Blatt MR, Pardo JM, Functional conservation between yeast and plantendosomal Na+/H+antiporter, FEBS Letter,2000,471:224~228
[81] Barkla BJ, Apse MP, Manolson MF, et al, The plant vacuolar Na+/H+antiport， Symposiaof the Society for Experimental Biology，1994,48:141~153
[82]Garbaino J, Dupont FM，Rapid Induction of Na+/H+Exchanger Activity In Barler RootTonoplast, Plant Physiol,1989,89:1~4
[83]Ballesteros E, Blumwald E, et al, Na+/H+Antiport Activity in Tonoplast Vesicles IsolatedFrom Sunflower Roots Induced By NaCl Stress, Physiologia Plantrum,1997,99:328~334
[84]Blumwald E, Poole, RJ, Na+/H+Antiport In Isolated Tonoplast Vesicles From StorageTissue of Beta Vulgarius, Plant Physiol,1985,78:163~167
[85]Yokoi S, Quintero FJ, Cubero B, et a1, Differential expression and function of Arabidopsisthaliana NHX Na+/H+antiporters in the salt stress response, The Plant Journal,2002,30(5):529~539
[86]Apse MP, Aharon GS, Snedden WS, et al, Salt tolerance conferred by overexpression of avacuolar Na+/H+antiporter in Arabidops, Science,1999,285:1256~1258
[87]Zhang HX, Blumwald E, Transgenic salt-tolerant tomato plants accumulate salt in foliagebut not in fruit, Nat Biotech,2001,19:765~768
[88]吕慧颖，盐生植物盐角草、番杏Na+>/H+>逆向转运蛋白基因克隆及特性研究，[博士学位论文]，哈尔滨；东北农业大学，2003
[89]Rufloni BC, Daminao F, Massabo, et al, Organogenesis and embryogenesis in LISIANTHUSRUSSELLIANUS HOOK, Acta Horticulture,1990,280:83~88
[90]Massabò F, Ruffoni B, Plant production by somatic embryogenesis in cell suspensioncultures of Lisianthus russellianus Hook, Plant Tissue Culture and Biotech,1996,2（4）:194~198
[91]毛元荣，刘茜，周根余，洋桔梗叶片再生体系的建立，上海师范大学学报(自然科学版)，2004，33(1):92~96
[92]张子学，丁为群，乔华伟，等，洋桔梗的快速繁殖研究，中国林副特产，2005,4:1~3
[93]杨燕燕，陈崇顺，等，洋桔梗高频再生体系的建立及其卡那霉素敏感性测定，江苏农业科学，2007，2:98~100
[94]龚明霞，陈小凤，方锋学，洋桔梗组培苗玻璃化原因及恢复的研究，西南农业学报，2009，22（6）：1718~1721
[95]陈小凤，龚明霞，方锋学，等，自然光及NH4对洋桔梗的玻璃化的影响，广西农业科学，2008，39（4）519~522
[96]Handa T, Genetic transformation of Antirrhinum majus and inheritance of altered phenotypeinduced by Ri T DNA, PIant Science,1992,81:199~206
[97]Semeria L, Vaira AM, Accotto GP, et al, Genetic transformation of Eustoma grandiflorumGriseb by microprojectile bombardment, Euphytica,1995,85:125~130
[98]Ledger SE, Deroles SC, Manson DG, et al, Transformation of lisianthus (Eustomagrandiflorum), Plant Cell Report,1997,16(12):853~858
[99]Simon C, Deroles J, Marie B, et al, An antisense chalcone synthase cDNA leads to novelcolour patterns in lisianthus (Eustoma grandiflorum) flowers, Molecular Breeding,19984(1):55~66
[100]Zaccai M, Lewinsohn E, Pichersky E, Modifying lisianthus traits by genetic engineering,ActaHorticulturae,2001,552:137~142
[101]Mercuri A, Sacchetti A, Benedetti LD, et al, Green fluorescent flowers, Plant Science,2002,162:647~654
[102]Fukuda A, Nakamura A, Tagiri A, et a1, Function intracellular localization and theimportance of salt tolerance of a vacuolar Na+/H+antiporter from rice, Plant and CellPhysiology,2004,45:146~159
[103] Dheeraj V, Sneh L, Singla P, Functional validation of a novel isoform of Na+/H+antiporter from Pennisetum glaucum for enhancing salinity tolerance in rice, Journal ofBiosciences,2007,32:621~628
[104]Chen H, An R, ang JH,et al, Over-expression of a vacuolar Na+/H+antiporter geneimproves salt tolerance in an upland rice. Mol Breeding,2007,19:215~225
[105]Ordogh M, Jambor-Benczur E, Tilly-Mandy A, MICROPROPAGATION OF′ECHO′CULTIVARS OF EUSTOMA GRANDIFLORUM
[106]夏玉凤，以gfp为报告基因研究蛋白亚细胞定位，生物技术通报，2006，（2）：11~13
[107]Scott A, Wyatt S, Tsou PL, et al, Model system for plant cell biology; GFP imaging inliving onion epidermal cells, Biotechniques,1999,26:1125~1132
[108]Stanislawska-Sachadyn A, Sachadyn P, Ihle K, et al, The construction of bifunctional fusionproteins consisting of MutS and GFP, Journal of Biotechnology,2006,121:134~143
[109]KAY R, Chan A, DALY M, Duplication of CaMV35S Promoter Sequences Creates aStrong Enhancer for Plant Genes, Science,1987,236(4806):1299~1302
[110]晏小兰，邱国华，李宝健，启动子对外源基因在转基因植物中表达的影响，中山大学学报，1995，34（2）：60~67
[111]吕萍萍，胡军，陈少良，等，抗盐胡杨Na＋/H＋逆向转运蛋白基因PeNhaD1的功能，植物生理与分子生物学报，2007，33（2）：173~178
[112]Shigaki T, Cheng NH, Pittman JK, et al, Structural determinant of Ca2+transport in theArabidopsis H+/Ca2+antiporter CAX1, BioChem,2001,276:43152~43159
[113]Cheng NH, Jon K, Pittman, et al, Functional Association of Arabidopsis CAX1and CAX3Is Required for Normal Growth and Ion Homeostasis, Plant Physiology,2005,138：2048~2060
[114]Yamaguchi T, Gilad S, Aharon JB, et al, Vacuolar Na+/H+antiporter cation selectivity isregulated by calmodulin from within the vacuole in a Ca2+and pH-dependent manner, PNAS,2005,102(44):16107–16112
[115]张文会，郭磊，郭彦，植物生长激素对野生大豆耐盐性的影响，安微农业科学，2007，35（5）：1296~1297
[116]黄彬城，PSY基因植物表达载体的构建及对洋桔梗转化的研究，硕士学位论文，吉林；吉林大学，2004
[2]Zhu JK, Salt and drought stress signal transduction in plants. Ann Rev Plant Biol，2002，53：247~273．
[3]Zaccai M, Lewinsohn E, Pichersky E, Modifying lisianthus traits by genetic engineering，ActaHorticulturae,2001,552:137~142
[4]陈景明，植物耐盐逆性的研究概况，安徽农业科学，2006，34(14)：3277~3278
[5]Greenway H, Munns R, Mechanisms of salt tolerance in nonhalophytes，Annual Review ofPlant Physiology,1980,31:149~190
[6]Haro R, Banuelos M, Quintero FJ, et a1, Genetic basis of sodium exclution and sodiumtolerance in yeast, Plant Physiol,1993,89:868~874
[7] SANTA-CRUZ A, ACOSTA M, RUS A, et a1, Short-term Salt Tolerance Mechanisms inDifferentially Salt Tolerant Tomar to Species, Plant Physiol Biochem,1999,37(1):65~71
[8]Xiang Dian-jun, Hu Xiang-yang, Zhang Yu, Over-Expression of ICE1Gene in TransgenicRice Improves Cold Tolerance Rice Science,2008,15(3):173~178
[9] Ashraf M, Foolad, MR, Roles of glycine betaine and proline in improving plant abiotic stressresistance, Environmental and Experimental Botany,2007,59(2):206~216
[10]郭北海，甜菜碱醛脱氢酶基因转化小麦及其表达，植物学报，2000，42(3)：279~283
[11]宁顺斌，宋运淳，王玲，等，盐胁迫诱导的植物细胞凋亡—植物抗盐的可能生理机制，实验生物学报，2000，33(3)：245~253
[12]Rubio F, Gassmann W, Schroeder JI, Sodium driven potassium uptake by the plant potassiumtransporter HKT1and mutations conferring salt tolerance, Science,1995,270:1660~1663
[13]Flowers TJ, Troke PF, Yeo AR, The mechanism of salt tolerance in halophytes, Ann RevPlant Physiol,1977,28:89~121
[14]Mansour MF, NaC1alteration of plasma membrane of A lliumcepa epidermal cells,alleviation by calcium, Plant Physiol,1994,145:726~730
[15]Tester M, Davenport R, Na tolerance and Na transport in higher plants, Ann, Bot,2003,91：1~25
[16]Maser P, Eckelman B, Vaidyanathan R, et a1, Altered shoot／root Na distribution andbifurcating salt sensitivity in A rabidopsis by genetic disruption of the Nat transporterAtHKT1. FEBS Letts,2002,531:157~161
[17]张淑红，张恩平，庞金安,植物耐盐性研究进展，北方园艺，2000，3：19~20
[18]Michelet, Boutry M,The plasma membrane H+-ATPase, A highly regulated enzyme withmultiple physiological functions, Plant Physiol,1995,108:1~6
[19]王宝山，赵可夫，邹琦,作物耐盐机理研究进展及提高作物抗盐的对策，植物学通报，1997，14(增刊)：25~30
[20]余叔文，汤章城，植物生理与分子生物学，北京：科学出版社，1998，752~769
[21]许祥明，叶和春，李国凤，植物抗盐机理的研究进展，应用与环境生物学报，2000，6(4)：379~387
[22]康绍忠，新的农业科技革命与21世纪我国节水农业的发展，干旱地区农业研究，l998，l6(1)：ll~l7
[23] Carios GB, Facundo G, Dana EM, et a1, Mitochondrisa are the main target for oxidativedamage in leaves0f wheat (Triticum aestivum L.), pesticide of Experimental Botany,2004,55:403~407
[24] Wu YX, Andreas VT, Physiological Effects0f Azoxystrobin and Epoxiconazole onsenescence and the oxidative Status of Wheat, Pesticide Biochemistry and Physiology,200l,71:l~l0
[25]Salin ML, Toxic oxygen species and protective systems of the ch1oroplast, Physio1.plant,l987,72:681~689
[26]Asada K, Ascorbate peroxidase-a hydrogen pero xide scavenging enzyme in plants, Physio1,Plant,1992,85:235~241
[27]Frid chI, Superoxide dismute, Ann Rev. Biochem, l975,44:l47~l59
[27]陈洁，林栖风，植物耐盐生理及耐盐机研究进展，海南大学学报自然科学版，2003，21(2):177~182
[28]杨素欣，王振镒，盐胁迫下小麦愈伤组织生理生化特性的变化，西北农业大学学报，1999，27(2)：48~51
[29]Ramiro HL, Mariana NM, Celina ML, et a1, Effect of photooxidative stress induced byparaquat in two wheat cultivars with differential tolerance to water, Plant Science,2003, l64：84l~848
[30]蒋明义，荆家海，王韶唐，渗透胁迫对水稻幼苗膜脂过氧化及体内保护酶系统的影响，植物生理学报，l99l，l7(1)：80~84
[31]毛桂莲，许兴，徐兆桢，对盐生理生化研究进展，中国生态农业学报，2004，12(1)：43~46。
[32]李辉，李德芳，陈安国等，植物耐盐研究概况，中国麻业科学，2007，29（4）：227~232
[33]GomezCadenas A, Tadeo FR，PrimoMillo E, Talon M, Involvement of abscic acid andethylene in the responses of citrus seedlings to salt shock，Plant Physio1,1998,103:475~484
[34] Gomez Cadenas A, Arbona V, Jacas J, et al, Abscisic acid reduces leaf abscission andincreases salt tolerance in citrus plants, Plant Growth Regu,2002,21:234~240
[35] Noaman MM, Dvorak J, Dong JM, Genes inducing Salt tolerance in wheat, Lophopyrumelongatum and amphiploid and their responses to ABA under salt stress, Prospects for SalineAgriculture (Series：Tasks for Vegetation Science),2002,37:139~144
[36]Kuiper D, Schuit PJC, Auctal cytokinln concentration in plant tissue as an indicator for saltresistance in cerea1, Plant and Soil,1990:243～250
[37]翟凤林，曹鸣庆，植物耐盐性及其改良，北京:农业出版社，1989:5~7
[38]牛庆杰，李伟，李慧英，等，向日葵耐盐碱材料筛选的新途径，吉林农业科学，1998(3):27~28.
[39]赵连城，刘孟雨，武田和义，等，国外大、小麦种质资源在盐碱地直接筛选可利用材料初报，作物品种资源，1994，4:19~20
[40]翁跃进，马雅琴，耐盐春小麦新品系SW-99-10和SW-99-12，中国种业，2002，2:45
[41]史湘华，殷鸣放，赵辉，等，盐碱地与耐盐碱树种的选育，中国林副特产，2005(2):60~62
[42] Ashraf M. Breeding for salinity Tolerance in Plants, Plant Sci,1994,13:17~42
[43]顾红艳，于福安，魏天权，等，耐盐碱水稻新品种津源85选育及栽培技术，农业科技通讯，2007，1:20~21
[44]张雪艳，郜玉田，抗盐碱优质牧草新品种谷稗，农村科学实验，2008，3:34
[45]张荃，赵彦，张慧，等，海水农业:梦想与现实，山东科学，1993，3(12):1~5
[46]Jones RA, High salt tolerance potential in Lycopersion species during germinateon,Euphytica,1986,35:575~582
[47]King IP，Introgression of salt-tolerance from Thinopyrum bessarabicum into wheat, Newphytol,1997,137:25~83
[48]高玉红，李云，植物离体培养筛选耐盐突变体的研究，核农学报，2004，18(6):448~452
[49]Wang CQ, Song H, Wang XF, et al, Selection of salt-tolerant variant from China pink,Acta-Horticulutrae-Sinica,2001,28(5):469~471
[50]刘凤华，郭岩，陈受宜，等，转甜菜碱脱氢酶基因植物的耐盐性研究，遗传学报，1997，24(1)：54~58
[51]贾庚祥，BADH转化番茄提高耐盐性的研究，[博士学位论文]，北京；中国科学院植物研究所，2002
[52]Kishor, Overexpression of△-pyrroline-5-carboxylate synthetase increases prolineproduction and confers osmotolerance in transgenic plants, Plant Physiol1995,108:1387~1394
[53]刘俊君，彭学贤，王慧中，等，转mtlD／gutD双价转基因水稻的耐盐性，科学通报，2000，45(7)：724~729
[54]Flowers TJ, Garcia M, Koyama M, et a1, Breeding for salt tolerance in crop plants-the roleof molecular biology, Acta Physiologiae Plantarum,1997,19:427~433
[55]Yang SX, Zhao YX, Zhang Q, et a1, HAL1Mediate Salt Adaptation in Arabidopsis Thaliana,Cell Research,2001, ll (2):142~148
[56]Bordas M, Montesinos C, Dabauza M, et a1, Transfer of the Yeast Salt Tolerance Gene HAL1to Cucujmis Melo L, Cultivars and in Vitro Evaluation of Salt Tolerance, TransgenicResearch,1997,6(1):41~50
[57]张荃，王淑芳，赵彦修，等，HALl基因转化番茄及耐盐转基因番茄的鉴定，生物工程学报，2001，17(6)：658~662
[58]Gisbert C, Rus A M, Bolarin MC, et a1, The Yeast HAL1Gene Improves Salt Tolerance ofTransgenic Tomato, Plant Physiol,2000,123(1):393~402
[59]Arrillagada I, Mascarell RG, Gisbert C, et a1, Expression of the Yeast HAL2Gene in TomatoIncreases the in Vitro Salt Tolerance of Transgenic Progenies，Plant Science,1998,136(2):219~226
[60]Gervera M, Ortega C, Navarro A, et a1, Generation of Transgenic Citrus Plants with theTolerance to Salinity Gene HAL2from Yeast, Journal of Horticulture Science&Biotechnology,2000,75(1):26~30
[61]黄绍兴，阎隆飞，高等植物对渗透胁迫的基因表达，农业生物技术学报，1995，3(3)：l~6
[62]刘昀，李冉辉，郑易之，等，大豆PM2蛋白及其结构可提高烟草耐盐性，深圳大学学报理工版，2007，24（1）：95~101
[63]Kazuo T, Kyoko K, A recessive Arobidopsis mutant that grows photoautotrohically undersalt stress shows enhanced active oxygen detoxification, Plant Cell,1999, ll:1195~1206
[64]Mckersie DB, Murnaghan J, Jones SK, et al, Iron-su-peroxide dismutase expression intransgenic in afalfa increases winter survival without a detectable increase in photosyntheticoxidative stress tolerance, Plant Physiol,2000,122:1427~1437
[65]Du J, Zhu Z, Li WC, Over-expression of exotic superoxide dismutase gene MnSOD andincrease in stress resistance in maize, Plant Physiol Mol Biol,2006,32:57~63
[66]Perl AC, Perl-Treves R, Galili S, et a1, Enhanced oxidative stress defense in transgenicpotato expression tomato CulZn superoxide dismutase, Theor Appl Genet,1993,85:568~576
[67]Kasuga M, Liu Q, Miura S, et a1, Improving plant drought, salt, and freezing tolerance bygene transfer of a single stress-inducible transcription factor, Nat Biotech,1999,17:287~291
[68]Yamaguchi-Shinozaki k, Shinozaki K, A novel cis-acting element in all Arabidopsis gene isinvolved in responsiveness to drought, low-temperature or high-salt stress, Plant Cell,1994,6:251~264
[69]Winicov I, Bastola DR, Transgenic overexpression ofthe transcription factor Alfinl enhancesexpression of the endogenous MsPRP2gene in alfalfa an d improves salinity tolerance of theplants, Plant Physiol,1999,120:473~480
[70]Ratner A, Jacoby B, Effect of K+its coumter anion and pH on sodium efflux from barleyroots, J Exp Physiol,1976,148:425~433
[71]Zhu JK, Genetic analysis of plant salt tolerance using arabidopsis，Plant Physiol，2000，124(3):941~948
[72]Shi H, Ishitani M, Kim C, et a1, The Arabidopsis thaliana salt tolerance gene SOS1encodesa putative Na+/H+antiporter, Prec Natl Acad Sci USA,2000,97(12):6896~6901
[73]Watanabe Y, Oshima N, Tamai Y, Co-expression of the Na+/H+antiporter and H-ATPasegenes of the salt tolerant yeast Zygosaccharomyces rouxii in Saccharomyces cerevisiae,FEMS Yeast Res,2005,5(4-5):411~417
[74]Waditee R, Hibino T, Nakamura T, et a1, Overexpression of a Na+/H+antiporer confers salttolerance on a freshwater cyanobacterlum, making it capable of growth in sea water, ProcNatl Acad Sci U S A,2002,99(6):4109~4114
[75]Shi H, Lee B H, Wu S J, et a1, Overexpression of a plasma membrane Na+/H+antiportergene improves salt tolerance in Arabidopsis thaliana, Nat Biotech,2003,21(1):81~85
[76]Gaxiola R A, Rao R, Sherman A, et al, The Arabidopsis thaliana ptoton transporters.AtNHX1and Avpl, can founction in canon detoxify-cation in yest, Proc Natl AcadSci,1999,96:1480~1485
[77]Fukuda A, Nakamura A, Tanska Y, Molecular cloning and expression of the Na+/H+antiporter gene in Oryza sativa.Biochem Bioph Acta,1999,1446:149~155
[78]Hamada A, Shono M, Xia T, et al, Isolation and characterization of a Na+/H+antiporter genefrom the halophyte Atriplex gmelini, Plant Mol Bio1,2001,46:35~42
[79]Apse MP, Aharon GS, Blumwale E, et al, Identification and characterzation of aNaCl-inducible vacuolar Na+/H+antiporterin Beta vulgaris, Physiol Plant,2002,116:206~212
[80]Quintero FJ, Blatt MR, Pardo JM, Functional conservation between yeast and plantendosomal Na+/H+antiporter, FEBS Letter,2000,471:224~228
[81] Barkla BJ, Apse MP, Manolson MF, et al, The plant vacuolar Na+/H+antiport， Symposiaof the Society for Experimental Biology，1994,48:141~153
[82]Garbaino J, Dupont FM，Rapid Induction of Na+/H+Exchanger Activity In Barler RootTonoplast, Plant Physiol,1989,89:1~4
[83]Ballesteros E, Blumwald E, et al, Na+/H+Antiport Activity in Tonoplast Vesicles IsolatedFrom Sunflower Roots Induced By NaCl Stress, Physiologia Plantrum,1997,99:328~334
[84]Blumwald E, Poole, RJ, Na+/H+Antiport In Isolated Tonoplast Vesicles From StorageTissue of Beta Vulgarius, Plant Physiol,1985,78:163~167
[85]Yokoi S, Quintero FJ, Cubero B, et a1, Differential expression and function of Arabidopsisthaliana NHX Na+/H+antiporters in the salt stress response, The Plant Journal,2002,30(5):529~539
[86]Apse MP, Aharon GS, Snedden WS, et al, Salt tolerance conferred by overexpression of avacuolar Na+/H+antiporter in Arabidops, Science,1999,285:1256~1258
[87]Zhang HX, Blumwald E, Transgenic salt-tolerant tomato plants accumulate salt in foliagebut not in fruit, Nat Biotech,2001,19:765~768
[88]吕慧颖，盐生植物盐角草、番杏Na+>/H+>逆向转运蛋白基因克隆及特性研究，[博士学位论文]，哈尔滨；东北农业大学，2003
[89]Rufloni BC, Daminao F, Massabo, et al, Organogenesis and embryogenesis in LISIANTHUSRUSSELLIANUS HOOK, Acta Horticulture,1990,280:83~88
[90]Massabò F, Ruffoni B, Plant production by somatic embryogenesis in cell suspensioncultures of Lisianthus russellianus Hook, Plant Tissue Culture and Biotech,1996,2（4）:194~198
[91]毛元荣，刘茜，周根余，洋桔梗叶片再生体系的建立，上海师范大学学报(自然科学版)，2004，33(1):92~96
[92]张子学，丁为群，乔华伟，等，洋桔梗的快速繁殖研究，中国林副特产，2005,4:1~3
[93]杨燕燕，陈崇顺，等，洋桔梗高频再生体系的建立及其卡那霉素敏感性测定，江苏农业科学，2007，2:98~100
[94]龚明霞，陈小凤，方锋学，洋桔梗组培苗玻璃化原因及恢复的研究，西南农业学报，2009，22（6）：1718~1721
[95]陈小凤，龚明霞，方锋学，等，自然光及NH4对洋桔梗的玻璃化的影响，广西农业科学，2008，39（4）519~522
[96]Handa T, Genetic transformation of Antirrhinum majus and inheritance of altered phenotypeinduced by Ri T DNA, PIant Science,1992,81:199~206
[97]Semeria L, Vaira AM, Accotto GP, et al, Genetic transformation of Eustoma grandiflorumGriseb by microprojectile bombardment, Euphytica,1995,85:125~130
[98]Ledger SE, Deroles SC, Manson DG, et al, Transformation of lisianthus (Eustomagrandiflorum), Plant Cell Report,1997,16(12):853~858
[99]Simon C, Deroles J, Marie B, et al, An antisense chalcone synthase cDNA leads to novelcolour patterns in lisianthus (Eustoma grandiflorum) flowers, Molecular Breeding,19984(1):55~66
[100]Zaccai M, Lewinsohn E, Pichersky E, Modifying lisianthus traits by genetic engineering,ActaHorticulturae,2001,552:137~142
[101]Mercuri A, Sacchetti A, Benedetti LD, et al, Green fluorescent flowers, Plant Science,2002,162:647~654
[102]Fukuda A, Nakamura A, Tagiri A, et a1, Function intracellular localization and theimportance of salt tolerance of a vacuolar Na+/H+antiporter from rice, Plant and CellPhysiology,2004,45:146~159
[103] Dheeraj V, Sneh L, Singla P, Functional validation of a novel isoform of Na+/H+antiporter from Pennisetum glaucum for enhancing salinity tolerance in rice, Journal ofBiosciences,2007,32:621~628
[104]Chen H, An R, ang JH,et al, Over-expression of a vacuolar Na+/H+antiporter geneimproves salt tolerance in an upland rice. Mol Breeding,2007,19:215~225
[105]Ordogh M, Jambor-Benczur E, Tilly-Mandy A, MICROPROPAGATION OF′ECHO′CULTIVARS OF EUSTOMA GRANDIFLORUM
[106]夏玉凤，以gfp为报告基因研究蛋白亚细胞定位，生物技术通报，2006，（2）：11~13
[107]Scott A, Wyatt S, Tsou PL, et al, Model system for plant cell biology; GFP imaging inliving onion epidermal cells, Biotechniques,1999,26:1125~1132
[108]Stanislawska-Sachadyn A, Sachadyn P, Ihle K, et al, The construction of bifunctional fusionproteins consisting of MutS and GFP, Journal of Biotechnology,2006,121:134~143
[109]KAY R, Chan A, DALY M, Duplication of CaMV35S Promoter Sequences Creates aStrong Enhancer for Plant Genes, Science,1987,236(4806):1299~1302
[110]晏小兰，邱国华，李宝健，启动子对外源基因在转基因植物中表达的影响，中山大学学报，1995，34（2）：60~67
[111]吕萍萍，胡军，陈少良，等，抗盐胡杨Na＋/H＋逆向转运蛋白基因PeNhaD1的功能，植物生理与分子生物学报，2007，33（2）：173~178
[112]Shigaki T, Cheng NH, Pittman JK, et al, Structural determinant of Ca2+transport in theArabidopsis H+/Ca2+antiporter CAX1, BioChem,2001,276:43152~43159
[113]Cheng NH, Jon K, Pittman, et al, Functional Association of Arabidopsis CAX1and CAX3Is Required for Normal Growth and Ion Homeostasis, Plant Physiology,2005,138：2048~2060
[114]Yamaguchi T, Gilad S, Aharon JB, et al, Vacuolar Na+/H+antiporter cation selectivity isregulated by calmodulin from within the vacuole in a Ca2+and pH-dependent manner, PNAS,2005,102(44):16107–16112
[115]张文会，郭磊，郭彦，植物生长激素对野生大豆耐盐性的影响，安微农业科学，2007，35（5）：1296~1297
[116]黄彬城，PSY基因植物表达载体的构建及对洋桔梗转化的研究，硕士学位论文，吉林；吉林大学，2004