摘要
土壤盐碱化是影响农业生产和生态环境的严重问题,苜蓿既是改良土壤、保持水土的重要植物,同时又是重要的饲料作物。苜蓿转基因研究进行的较早、广泛也较成功。采用传统的育种方法,选育耐盐品种进展极为缓慢。随着分子生物学的发展,本课题研究是利用农杆菌介导法,将抗旱耐盐基因转入苜蓿中,从而可以提高苜蓿的耐盐碱性。
本研究主要以苜蓿的无菌子叶作为外植体,诱导体细胞胚发生。通过对培养基成分等一些影响因素的优化,完成苜蓿愈伤组织诱导、愈伤组织的分化、出苗以及生根的整个再生过程。利用根癌农杆菌介导法对其进行抗旱耐盐基因遗传转化的研究,以期获得转基因植株并对其进行有关耐盐碱性生理指标的检测,取得的主要结果如下:
1、本实验分别优化了苜蓿子叶节各个时期的培养基成分:(MS+2,4-D2.0mg/L+KT 0.25mg/L,蔗糖3%,pH5.8)培养基可诱导苜蓿可诱导苜蓿愈伤组织;在(MS+NAA 0.05mg/L+6BA 0.5mg/L,蔗糖3%,pH5.8)和MS、1/2MS(蔗糖1.5%,pH5.8)培养基上正常发育、萌发成苗以及生根。
2、本实验利用农杆菌介导法将BADH和CMO双价耐盐基因导入苜蓿中,探讨了不同的侵染时间、不同的菌液的浓度等条件对转化效果的影响。结果表明,浓度(OD值)在0.3-0.5左右,侵染25分钟时的愈伤组织的侵染效果最好,转化率较高,并采用40mmol/L的含盐培养基对其进行筛选,转化过程中得到了抗性植株。
3、对得到的抗性植株提取其叶片DNA进行PCR检测以及PCR-southern鉴定,结果表明,PCR检测为阳性的两个单株均有与阳性对照大小完全一致的杂交带出现,与PCR检测结果完全吻合,而非转基因植株没有杂交信号出现,说明PCR产物确实是BADH基因,而不是苜蓿基因组中的同源序列,从而证明外源基因BADH和CMO已成功的整合到基因组中。
4、本实验对转基因阳性植株进行了耐盐性生理指标检测,分别为甜菜碱含量的对比测定、细胞膜透性、游离脯氨酸含量、丙二醛含量、超氧化物歧化酶活性以及过氧化物酶活性。结果表明:甜菜碱、膜透性脯氨酸含量和丙二醛含量随着盐胁迫的增强在增加,且都高于无盐胁迫下的值,而SOD,POD活性则正相反,随着盐胁迫的减弱其活性逐渐减小,并且随着胁迫天数的增加逐渐下降。对转化苗在浓度为1.0%的碱性盐水浇灌条件下,定期测其生理指标,并与非转化苗的指标作对比,发现其甜菜碱的含量有所增加,但是其细胞膜透性、丙二醛含量有着明显的降低,而超氧化物歧化酶活性以及过氧化物酶活性有明显的提高,但对于脯氨酸二者则没有显著的差别。
综上所述:外源基因BADH和CMO已有效地整合到转基因阳性植株基因组中,并成功的获得了表达,从而使转基因阳性植株的抗旱耐盐碱性得到了提高。
The salting of soil is the serious problem that affects the agricultural production and the ecological environment.The alfalfa can improve soil,maintains the water and it's a kind of important grass crops.The transgenic alfalfa research carries on widely and successful.It is extremely slowly to use traditional breeding method to selective salt tolerance variety.(Along with the development of molecular biology,) In order to improve the salt tolerance of alfalfa,we transfer the salt tolerance gene into alfalfa by agrobacterium-mediated transformation.
This research mainly takes explant from sterile cotyledon of alfalfa to induce somatic embryogenesis.Throught optimization of culture medium main influencing factors,completed the entire regenerative process which contains embryogenic callus induction,differentiation, seedling emergence and radiate.It is proven initially the resistance to drought and salt genes had transfer to alfalfa plant,and has carried on the physiological laboratory value to it.The primary coverage is as follows:
First:This experiment separately optimized the culture medium ingredient of alfalfa at each time:(MS+2,4-D 2.0mg/L+KT 0.25 mg/L,sugarcane 3%,pH5.8) medium can induce somatic embryogenesis;(MS+NAA 0.05mg/L+ 6BA 0.5 mg/L,sugarcane 3%,pH5.8)和MS、1/2MS(sugarcane 1.5%,pH5.8) medium can nomal development and germination.
Second:This experiment transfer BADH and CMO gene to alfalfa by Agrobacterium-mediated.In this research,we discuss the transformation effect on different invades dyes time,different density of bacterium.The result indicated that the density(the OD value) about 0.3-0.5,infest 25 minute time to injury the organization the effect to be best.Using culture medium contains 40mmol/L salt-bearing to carry on screening。
Third:Through the PCR and the PCR-Southern blotting,The results showed that,PCR testing positive for the two plants have positive control the size and entirely consistent with a hybrid,and fully consistent PCR test results,but the non-transgene adult plant has not hybridized the signal to appear,so it explained that the PCR product is truly BADHgene,but is not genome team's homologous sequence in the alfalfa.It proved that BADH and CMO bivalent genes had transfer to genome of alfalfa.
Forth:The physiological index contains:contrast to betaine content,membrane permeability, dissociation praline content,MDA content,SOD activity and POD activity.The experimental result indicated that the content of betaine,dissociation praline and MDA are increasing along with the salty coercion.Under 1.0%salt water irrigation,contrast with non-transformation plant, betaine content has increased,but membrane permeability and MDA content has obvious reduction.However the SOD,POD activeness activity are on the contrary.Proline has no remarkable changes.
引文
[1]张振霞.符义坤,储成才.豆科牧草基因工程研究进展[J].遗传,2002.24(5):607-612.
[2]苏加楷、中国苜蓿育种的回顾与展望.www.hbsdfix.com/sanli/zhuanjia/41.htm
[3]陈少良,李金克,尹伟伦,等.盐胁迫条件下杨树组织及细胞中钾、钙、镁的变化[fJ].北京林业大学学报,2002,24:84-88.
[4]刘风荣,陈火英,刘杨,等.盐胁迫下不同基因型番茄可溶性物质含量的变化[J].植物生理与分子生物学学报,2004,30(1):99-104.
[5]惠红霞,许兴,李守明.款胁迫抑制光合作用的可能机}}！.[J].生态学杂志,2004.23(1):5-9.
[6]江良驹,刘友良.尤花果耐款机理的研究一款逆境下脯氨酸和可溶性蛋白的积祟[J].南京农业大学学报,1989.12(4):124-125.
[7]陈月艳,孙国荣,阎秀峰Na2C03胁迫下对螺旋藻生长及抗氧化酶活性的影响[J].植物学通报,1998.15(3):43-47.
[8]于海武,李莹.植物耐盐性研究进展北华大学学报(自然科学版)2004.65(3).
[9]龚明.盐胁迫下人麦和小麦等叶片脂质过氧化伤害与超微结构变化的关系[J].植物学报,1989,31(11):841-846.
[10]马焕成,王沙生.胡杨膜系统的盐稳定性及盐胁迫下的代谢训节[J].两南林学院学报,1998,18(1):15-23.
[11]郑海雷,林鹏.培养盐度对海莲和木榄幼苗膜保护系统的影响[J].厦门大学学搬,1998,37(2):278-282..
[12]许卉,赵丽萍.植物抗盐性机理及评价滨州学院学报2005.21(3).
[13]翟凤林,曹鸣庆.植物的耐盐性及其遗传改良[M].北京:农业出版社.1989.23-35.
[14]Fostad O,Pedersen PA.Container-grown Tree Seedling Response to Sodium Chloride Applications in Different Substrates[J].Environmental Pollution,2000,109(2):203-210.
[15]Chartzoulakis K,KlapakiG.Response of Two Greenhouse Pepper Hybrids to NaCl Salinity During Different Growth Stages[J].Scientia Horticulturae,2000,86(3):247-260.
[16]Grieve GM,Guzy MR,Poss JA,et al.Screening Eucalytus Clone for Salt Tolerance[J].Hortscience.1999.34(5):867-870.
[17]Troncoso A.Matte C,Cantos M,et al.Evalumion of Salt Tolerance of in Vitro-grown Grapevine Rootstock Varieties[J].Vitis.1999.38(2):55-56.
[18]杨茁萌,黄军.石定燧,等.鲁梅克斯K-1杂交酸模耐盐性的初步研究[J]_中国草地2000.(1):26-30.
[19]汀良驹.马凯.姜卫兵.等NaCl胁迫下石榴和桃植株Na+和K+含量与耐盐性的研究[J].园艺学报.1999.22(4):336-340.
[20]王衍安,李际红,张友朋.等.耐盐杨树新无性系选育续报[J].山东林业科技,1997.(4):1-7.
[21]孟康敏.绒毛白蜡等树种耐盐力研究[J].辽宁林业科技,1999,(3):42-46.
[22]苑增武,张孝民.毛齐来,等大庆地区主要造林树种耐盐碱能力评价[J].防护林科技2000,(1):15-17.
[23]张立钦,郑勇平.吴纪良,等.黑杨派新无性系水培苗对盐胁迫反应的研究[J].浙江林学院学报,2000.17(2):121-125.
[24]拿际红.张友鹏.刘国兴,等.3个杨树新无性系耐盐力测定初报[J].山东林业科技,2001,(1)10-13.
[25]陶晶.东北主要杨树抗盐机理及抗性品种选育的研究[D].哈尔滨:东北林业大学.2002.
[26]刘岩,彭学贤.谢友菊.等.植物抗渗透胁迫基因工程研究进展[J].生物工程进展,1997,17(2):31-38.
[27]GaxiolaR.A Novel and Conserved Salt Introduced Protein is an Important Determination of Salt Tolerance in Yeast[J].Emboi.1992.11(6):3157-3164.
[28]Ramon Serrano.FrancisoA.Vicente Moreno,et al.Genetic Engineering of Salt and Drought Tolerance with Yeast Regulating Genes[J].Horticulture,1999.78(1):261-269.
[29]李秋莉,杨桦,高晓蓉,刘大伟,安利佳.植物甜菜碱合成酶的分子生物学和基因工程.生物工程进展,2002.(1):84-86.
[30]张京译.苜蓿的组织培养.国外畜牧学(草原与牧草)[J].1986(1):15-16.
[31]姚爱兴.苜蓿组织培养研究进展.牧草与饲料[J].1991(1):10-13.
[32]李先芳,王文静等.苜蓿组织培养研究进展.郑州牧业工程高等专科学校学报[J].2001,21(2):105-108.
[33]Saunders J W.et al.Production of alfafa plants from callus culture.Crop Science[J].1972,12:804-808.
[34]李聪,熊德邵等.苜蓿愈伤组织再生植株研究.中国草地[J].1989(3):51-55.
[35]危晓薇,蔡丽娟等.紫花苜蓿组织培养及其再生植株.新疆农业科学,1999(2):73-75.
[36]何茂泰.白静仁等.野生黄花苜蓿花药愈伤组织的诱导和植株再生.植物生理通讯[J].1988(6):44.
[37]时永杰.周丽霞.几种豆科牧草的组织培养.四川草原[J].1997(3):27-29.
[38]McCoy TJ.et al.Regeneration of diploid alfafa plants from cells grown in suspension culture.Plant Science Letters[J].1977,(10):59-66.
[39]安利佳,张俊敏等.天蓝苜蓿单细胞培养植株再生.实验生物学报[J].1992,24(2):119-125.
[40]赵剑,杨文杰.高压静电场(HVEF)对苜蓿叶片愈伤组织诱导的影响.生物物理学报[J].1996,12(3):517-521.
[41]张晓东,淋延安.r射线对苜蓿离体培养与植株再生的影响.核农学报[J].1992,6(3):139-146.
[42]金洪,王学敏等.紫花苜蓿组织离体培养的形态建成及其调控.内蒙古农业大学学报,[J].2000,21(1):81-84.
[43].Reish B,Bingham E T.The genetic control of bud formation from callus cultures of diploid aIfalfa Plant Sci Iett.1980,20:71-77.
[44].Brown D C W,Atanassov A.Role of genetic background in somatic embryogenesis in Medicago.plant cell tiss organ Culture.1985.4:111-112.
[45].Chen T H H,Marowitch J A,Thompson B G,Gentoypic effects on somatic embryogenesis and plant regeneration from callus cultures of alfalfa Plant cell tiss organ culture.1987,8:73-81.
[46].Dijak M,Brown D C w.Patterns of direct and indirect embryogenesis from mesophyll protoplasts of Medicago Sativa.Plant cell Tiss organ Culture.1987.9:121-131.
[47].王金发编著。细胞生物学,北京科学出版社,2003
[48].付顺华,梁锦锋,季宗富.植物转基因研究进展[J].云南林业科学.2003.1:70-71.
[49].高必达.植物抗病基因工程研究进展[J].湖南农学院.
[50].管延安.李根英,隋新霞,等.小麦转基因研究进展[J].山东农业科学2002,5:43-46.
[51]刘冬青.棉花转基因研究进展[J].江西农业学报,2003,15(2):40-43.
[52].柳武革,薛庆中.蛋白酶抑制剂及其在抗虫基因工程中的应用[J].生物技术通报,2000,1:20-25.
[53].徐铮李.可食疫苗的开展现状与市场前景.中国制药信息2000,16(12)34-35.
[54].耿德贵.王义勤等.利用转基因植物生产口服疫苗的研究进展.生物工程进展,2002,22(1):19-22.
[55].刘艳军、杨静慧、畅恩芹、刘玉东、柳树梧.提高农杆菌基因转化率方法的研究.两南农业大学学报,2003.25(2):144-146
[56].徐备晖、夏光敏、贺晨霞.农杆菌转化系统研究进展.生命科学,2002,14(4):223-225.
[57].权瑞党、尚悔、张举仁.农杆菌介导的玉米自交系愈伤组织转化条件的优化.植物生理与分子生物学学报,2003,29(3):245-250
[58]王关林、方宏筠主编.植物基因工程.
[59].叶健明、唐克轩、沈大棱.植物转基因方法概述.生命科学.1999,11(2):58-60.
[60].权瑞党、尚梅、张举仁.农杆菌介导的玉米自交系愈伤组织转化条件的优化.植物生理与分子生物学学报,2003,29(3):245-250.
[61].王关林、方宏筠主编.植物基因工程.
[62].叶健明、唐克轩、沈大棱.植物转基因方法概述.生命科学:1999,11(2):58-60.
[63].Centers for Disease.Control Morbidity Weekly Report 39(Centers for Disease Control Atlanta),1990,l-26.
[64].Neurath A R,Kent S B H.Strick N.Location and chemical synthesis of a pre-S gene coded immunodominant epitope of hepatitis B Virus.Science,1984,224:392-394.
[65]Cheng K C.Moss B.Selective synthesis and secretion of particles composed of the hepatitis B Virus middle surface directed by a vaccinia virus:induction of antibodies to pre-S and S epitopes.J Virol-1987.61:1286-1290.
[66].高寿征.病毒性肝炎防止研究(第一版)。北京出版社,31-35.
[67].Mason H S.Lam D M K,Arntzen C J.Expression of hepatitis B Surface antigen in transgenic plants.Proc Natl Acad Sci,USA,1992.89:11745-11749/
[68].刘玉乐,王晋芳,丘并生,等.人乙肝病毒表面抗原基因在转基因烟草中的表达.中国科学(B辑),1993,23(3):252-255.
[69]赵春晖,王荣,赵长生,等。含与不含前导序列的乙肝病毒表面抗原基因在转基因番茄中表达的研究.农业生物技术学报,2000,8(2):190-193.
[70]Ehsani P,Khabiri A Domansky N N.Polypeptides of hepatitis B surface antigen produced in transgenic potato.Gene,1997.190:107-111.
[71]Kapusta J,Modelska A,FiglerowiczM,et al.Aplant-drived edible vaccine against hepatitis B virus.FASEB J.1999,13:1796-1799.
[72]Thanavala Y,Yang Y F,LyonsP,et al.Immunogennicity of transgenic plant-derivedd hepatitis B surface antigen.Proc Natl Acad Sci USA,1995.92:3358-3361.
[73].刘德虎.利用转基因植物生产药用蛋白.生物技术通报,1999,(4)1-5.
[74]Arcioni S.Damiani F,Pezzoti M,et al.Alfalfa Lucerne(Medicago spp).Bajaj Y Biotechnology in Agriculture and Forestry.New York:Springeer,1990.197-241.
[75]Chabaud M,Clotilde L.Cornne M.et al.Transformation of barrel medic(Medicago truncatula Gaertn.)by Agrobecterium tumefaciens and regeneration via somatic embryogenesis of transgenic plants with the MtENOD12 nodulin promoted fused to the gus reporter gene.Plant Cell Rep.1996,305-310.
[76]李聪.熊德邵、耿华珠.苜蓿愈伤组织再生植株的研究.中国草地.1989.(3):51-56.
[77]杨燮荣,和根福,周荣仁.苜蓿组织培养再生植株.植物生理学通讯.1981,(5):33-34.
[78]Sauders J W.Bingham E T.Production of alfalfa plants from callua tissue.Crop Sic.1972,12:804-808.
[79]王关林,方宏筠主编.植物基因工程原理与技术[M].北京,科学出版社.1998.
[80]Reish B,Bingham E T.The genetic control of bud formation from callus cultures of diploid alfalfa Plant Sci lett,1980,20:71-77.
[81]黎裕,王天宇,刘成等.玉米抗旱品种的筛选指标研究 植物遗传资源学报2004,5(3):210-21.
[82]乔亚科,李桂兰,王文颇等.野生、栽培大豆愈伤组织耐盐性与植株盐胁迫下丙二醛(MDA)含量的关系。大豆科学2003,5,22(2).
[83]赵惠玲.稀土对玉米幼苗抗盐性作用的研究 山西农业科学2005,33(2):34-36.
[84]杨国虎.玉米抗旱性的鉴定指标及遗传育种研究进展.[J]甘肃农业科技,2002,10.
[85]郑霞,韦小敏.季良越等.玉米体细胞抗盐突变体的筛选及耐盐性鉴定.[J]河南农业大学学报,2004,6:38(2):26-29.
[86]刘祥久.姜敏,张喜华等.用开苞导入法将抗旱耐盐基因导入玉米自交系的初步研究.[J]杂粮作物,2005,25(3):134-135.
[87]万超文,邵桂花,陈一舞.盐胁迫下大豆耐盐性与籽粒化学品质的关系.[J]中国油料作物学报,2002,24(2):67-72.
[87]王洪新,胡志昂,钟敏.盐渍条件下野生大豆群体的遗传分化和生理适应:同工酶和随机扩增多态DNA研究.植物学报,1997,39:34-42.
[89]石德成,盛艳敏.不同盐浓度的混合盐对羊草苗的胁迫效应.[J]植物学报,1998,40(12):1136-1142.
[90]乔亚科,李桂兰,王文颇等 野生、栽培大豆愈伤组织耐盐性与植株盐胁迫下丙二醛(MDA)含量的关系.[J]大豆科学2003.5.22(2)
