摘要
露地菊(Chrysanthemum morifolium)是园林绿化观赏植物中常用的花卉之一。其显著的特点是植株低矮,覆盖力强,繁殖容易,管理粗放,适应范围广,抗逆性强,花色丰富,花多而密,花期长,观赏价值高。在我国东北盐碱地区以及城市盐渍化严重的地区,大部分栽植的露地菊受盐碱的影响则表现出叶色萎黄、生长不良、花芽分化受阻甚至死亡的现象。本研究旨在利用安全标记的遗传转化方法获得转化植株,培育出抗盐碱能力更强的新品系,丰富盐碱地区花卉种植品种,提高盐碱地区花卉种植业收益水平。
研究利用Gateway技术,以磷酸甘露糖异构酶基因PMI为选择标记,以盐生植物星星草中克隆的耐盐碱相关基因PtDHAR为目的基因,构建pCAMBIA1301-PMI-PtDHAR双元表达载体,并将此载体转入农杆菌菌株EHA105中,通过农杆菌介导的方法,把耐盐基因PtDHAR转入露地菊‘火焰’基因组中。
主要研究结果如下:(1)遗传转化载体的构建。从大肠杆菌中通过PCR克隆PMI基因全长,装入pGEM(?)-T Vector载体中,利用限制性内切酶Xhol单酶切,以PMI-T载体和pCAMBIA1301载体为基础,构建pCAMBIA1301-PMI载体;利用Gateway技术构建pCAMBIA1301-PMI-PtDHAR双元表达载体,并将此载体转入到农杆菌菌株EHA105中,通过PCR验证结果表明pCAMBIA1301-PMI-PtDHAR载体已成功转入农杆菌菌株EHA105中。
(2)露地菊‘火焰’对甘露糖和头孢霉素的敏感性试验。研究结果当甘露糖浓度为10 g/L时即能明显抑制叶片外植体的分化和不定芽的产生,可以作为遗传转化时叶片外植体筛选培养的临界耐受浓度;分化率和不定芽增殖系数随着头孢霉素(Cef)浓度的升高而逐渐下降,300 mg/L的Cef浓度可作为叶片外植体在共培养后抑制农杆菌的抑菌浓度。
(3)露地菊‘火焰’遗传转化体系的建立。以预培养1d的叶片外植体为转化材料,用OD600值为0.4的农杆菌菌液侵染10min,在23-25℃黑暗条件下共培养2d,随后进行2d延迟培养,接下来以300mg/L的Cef浓度为抑菌浓度,采用选择压递增的方式进行筛选,初期筛选培养基的甘露糖浓度为8g/L,后期筛选培养基中甘露糖的浓度增加到10g/L,连续筛选至不定芽产生。把不定芽转入甘露糖浓度为10g/L、Cef浓度为100mg/L的生根筛选培养基中继续筛选,直至长势健壮并生根的抗性植株产生。
(4)转化植株的检测。对获得的9株抗性植株进行PCR检测,结果表明获得了2株转PtDHAR基因植株。
Chrysanthemum morifolium is commonly used in ornamental gardens and flower. The significant features are low power plant, coverage, easy to manage the extensive breeding, wide, and resistant to strong, a great variety, spend more dense, long flowering period, ornamental value. In saline and Northeast China, as well as the city of saline, most intense cultivation of Chrysanthemum morifolium affected by salt of the exhibit leaf chlorosis, bad growth, bud is blocked or even death. This study aims to take advantage of the security token of the transformation method to obtain a conversion plant has produced salt more lines, rich in plants species, improve income in flower growers.
This study draws Gateway technology to phosphomannose isomerase gene PMI is safe for planting in the cloned tenuiflora salt tolerance gene PtDHAR, built for the purpose of gene pCAMBIA1301-PMI-PtDHAR dual-element expression vector, and the carrier to Agrobacterium strain EHA105 by Agrobacterium-mediated method, the salt tolerance gene PtDHAR turn into Chrysanthemum morifolium'Huoyan'notation genome, alkali more lines, on large daisy in the country, the better for landscaping services.
The major findings are as follows:
(1) Building genetic transformation vector. From the Escheriachia coli PMI by PCR cloning, gene-digestion method to PMI-T carrier and carrier-based, pCAMBIA1301 into pCAMBIA1301-PMI carrier; use Gateway technology to build pCAMBIA1301-PMI-PtDHAR dual-element expression vector, and the carrier to Agrobacterium strain EHA105 by PCR validation results showed that pCAMBIA1301-PMI-PtDHAR carrier has successfully transferred to Agrobacterium strain EHA105.
(2) Chrysanthemum morifolium'Huoyan'on Mannose and cephalosporin sensitivity. Study results indicate that leaves explants on Mannose is very sensitive, mannose concentration to 10g/L at apparent suppression leaves explants of differentiation and adventitious buds of produce that can be used as genetic transformation when leaves explant culture of critical tolerance filter concentration; differentiation and proliferation factor as adventitious buds cephalosporin Cef concentration but steady decline,300mg/L Cef concentration can be used as leaf explant cultured in total suppression of Agrobacterium tumefaciens inhibitory concentration.
(3) Established in Chrysanthemum morifolium'Huoyan'genetic transformation system. To pre-training 1d of the leaf explants were transformed material, with a value of 0.4 OD600 Agrobacterium infection 10 min,23-25℃in the dark were cultured under the conditions of 2d, for 2d delayed train to 300mg/L of Cef concentration inhibitory concentration, using selective pressure increments were screened, initial screening medium mannose concentration 8g/L, post-screening medium concentration of mannose increased to lOg/L, continuous screening to produce adventitious buds. To shoot into the mannose concentration of 10g/L, Cef concentration of 100mg/L of rooting medium to filter selection, until the growing robust and resistant plants produce root.
(4) Transformed plants testing. To 9 resistant adult plant which obtains carries on the PCR examination, finally indicated that obtained 2 to transfer the PtDHAR gene adult plant.
引文
[1]Dale P J. Spread of engineered genes to wild relatives.Plant Physiol,1999,100:13-15
[2]Kuiper H A, Kleter G A, Notebon H. Assessment of food safety issues related to genetically modified foods.Plant J,2001,27:503-528
[3]Hohn B, Levy A A, Puchta H. Elimination of selection markers from stransgenic plants. Current Opinion in Biotechnology,2001,12(2):139-143
[4]Scutt C P, Zubko E, Meyer P. Techniques for the removal of marker genes from transgenic plants. Biochim,2002,84:1119-1126
[5]Dominguez A, Fagoaga C, NacarroL. Regeneration of transgenic citrus plants under non-selective conditions results in high-frequency recovery of plants with silenced transgenes. Mol Genet Genom,2002,267:544-556
[6]Joersbo M, Okkels F T. A novel principle for selection of transgenic plant cells:posotive selection. Plant Cell Reports,1996,16(3/4):219-221
[7]Shimomura O, Johnson F H, Saiga Y. Extraction, purification and properties of Aquoria, a bioluminescent protein from the luminous Hydromedusan, Aequorea. J Cell Comp Physiol, 1962,59:223-229
[8]赵华,梁婉琪,杨永华,张大兵.绿色荧光蛋白及其在植物分子生物学研究中的应用.植物生理学通讯,2003,39(2):171-178
[9]Reiner. Genes for ribitol, D-arabitol catabolism in Escherichiacoli:their loci in C strains and absence in K-12 and B strains. J Bacteriol,1975,123:530-536
[10]Weisser P, Kramer R, Sprenger G A. Expression of the Escherichla coli pmi gene, encoding phosphomannose-iso-merase in Zymomonas mobilis,leads to untilization of mannose as a novel growth substrate, which can be used as a selective marker. Applied and Environmental Microbiology,1996,6 (11):4155-4161
[11]李晓兵,陈彩艳,翟文学.培育具有安全选择标记或无选择标记的转基因植物.遗传,2003,25(3):345-349
[12]Jaiwal P K, Sahoo L, Singh N D, Singh R P. Strategies to deal with the concem about marker genes in transgenic plants:some environmentfriendly approaches. Current Science, 2002,83(2):128-136
[13]Lee B T, Matheson N K. Phosphomannoisomerase and phosphoglucoisomerase in seeds of Cassia coluteoides and some other legumes that synthesize galactomannan. Phytochem, 1984,23:983-987
[14]Reed J, Privallel M, Powell L. Phosphomannose isomerase:an efficient selectable marker for plant transformation. In Vitro Cell Dev Boil Plant,2001,37:127-132
[15]Joersbo M,Petersen S G, Okkels F T. Parameters interacting with mannose selection employed for the production of transgenic sugar beet. Physiol Plant,1999,105:109-115
[16]Joersbo M, Mikkelsen J D, Brunstedt J. Relationship between promoter strength and transformation frequencies using mannose selection for the production of transgenic sugar beet. Mol Breed,2000,6 (2):207-213
[17]Boscariol R L, Almeida W A B, Derbyshire M T V C. The use of the PMI/mannose selection system to recover transgenic sweet orange plants(Citrussinensis L.Osbeck). Plant Cell Rep,2003,22:122-128
[18]Joersbo M, Donaldson I, Kreiberg J. Analysis of mannose selection used for transformation of sugar beet. Mol Breed,1998,4(2):111-117
[19]Wang A S, Evans R A, Altendorf P R. A mannose selection systemfor production of fertile transgenic maize plants from protoplasts. Plant Cell Rep,2000,19(7):654-660
[20]Negrotto D, Jolley M, Beer S. The use of phosphomannose-isomerase as a selectable marker to recover transgenic plants (Zea mays L.)via Agrobacterium transformation. Plant Cell Rep,2000,19(8):798-803
[21]Wright M, Dawson J, Dunder E. Efficient biolistic transformation of maize (Zea mays L.) and wheat (Triticum aestivum L.) using the phosphomannose isomerase gene, pmi, as the selectable marker. Plant Cell Rep,2001,20:429-436
[22]Paola L, Ye X, Ingo P. Effective selection and regeneration of transgenic rice plants with mannose as selective agent.Mol Breed,2001,7:43-49
[23]He Zhengquan, FuYaPing, Si Hunmin. Phosphomannose-isomerase (PMI) gene as a selectable marker for rice transformation via Agrobacterium. Plant Sci,2004,166:17-22
[24]Kennedy M O, Otha F C, Burger J T. Peal millet transformation system using the positive selectable marker gene Phosphomannose-isomerase.Plant Cell Rep,2004,22:684-690
[25]Todd R, Tague B W. Phosphomannose isomerase:A versatile selectable marker for Arabidopsis thaliana germ-line transformation. Plant Mol Biol Rep,2001,19:307-319
[26]Kim J Y, Jung M. A New Selection System for Pepper Regeneration by Mannose. J Plant Biotechnol,2002,3:129-134.
[27]Zhang P, Puonti J K. PIG-mediated cassava transformation using positive and negative selection. Plant Cell Rep,2000a,19:1041-1048
[28]Zhang P, Potrykus I, Puonti J K. Efficient production of transgenic cassava using negative and positive selection. Transgen Res,2000b,9:405-415
[29]岳建雄,孟钊红,张炼辉.以甘露糖作为筛选剂的棉花遗传转化.棉花学报,2005,17(1):3-7.
[30]彭世清,陈守才.甘露糖阳性选择系统的建立及在番茄转化中的应用.农业生物技术学报,2005,13(2):141-144.
[31]Boscariol R L, Almeida W A B, Derbyshire M T V C. The use of the PMI/mannose selection system to recover transgenic sweet orange plants (citrussinensis L.Osbeck). Plant Cell Rep,2003,22:122-128
[32]Reustle G M, Wallbraun M, Zwiebel M. Selectable marker systems for genetic engineering of grapevine. Acta Hort,2003,603:485-490
[33]Degenhardt J, Poppe A, Montag J and Szankowski I. The use of the phosphomannose-isomerase/mannose selection system to recover transgenic apple plants. Plant Cell Rep, 2006,25:1149-1156
[34]曾黎辉,徐海峰,王会全,吴少华,朱艺萱.PMI基因作为选择标记的植物表达载体构建及其在雪柑转基因中的应用.农业生物技术学报,2008,16(5):858-864
[35]徐海峰.以PMI基因为选择标记雪柑转基因体系的建立[硕士学位论文].福建农林大学,2007
[36]赵可夫,范海.盐生植物及其对盐渍生境的适应生理.北京:科学出版社,2005
[37]Niu X, Bressan R A, Hasegawa P M. Ion Homeostasis in Nacl Stress Environments. Plant Physiol,1995,109:735-742
[38]郑世英,陈吉美.植物的抗盐生理.德州高专学报,2000,16(4):39-40
[39]方允中,郑荣梁.自由基生物学的理论与应用.北京:科学出版社,2002:30-31
[40]陈洁,林栖凤.植物耐盐生理及耐盐机理研究进展.海南大学学报(自然科学版),2003,21(2):176-182
[41]Greenway H, Munns R. Mechanisms of salt tolerance in nonhalophytes. Annu. Rev plantphysiol,1980,31:149-190
[42]Englard S, Seifter S. The Biochemical Functions of Ascorbic Acid. Annu Rev Nutr,1986, 6:365-406
[43]Padh H. Vitamin C, Newer Insights into Its Biochemical Function. Nutr Rev,1991,49:65-70
[44]Foyer C H, Lelandais M, Edwards E A and Mullineaux P M. The Role of Ascorbate in Plants Interaction with Photosynthesis and Regulatory Significance. Am Society Plant Physiol,1991,:131-144
[45]Yabuta Y, Motoki T, Yoshimura K, Takeda T, Ishikawa T, Shigeoka S. Thylakoid Membrane-bound Ascorbate Peroxidase is A Limiting Factor of Antioxidative Systems Under Photo-oxidative Stress. Plant,2002,32:915-925
[46]Winkler B S, Orselli S M, Rex T S. Purification, Cloning and Expression of Dehydroascorbic Acid-reducing Activity from Human Neutrophils:Identification as Glutaredoxin. Free Radical Biol Med,1994,17:333-349
[47]Ito A, Hayashi S, Yoshida T. Purification and Characterization of Cytochrome P-450 Induced by Benz (a) anthracene in Mouse Skin Microsomes. Biochem Biophys Res Commun,1981,101(2):591-598
[48]Hara T, Minakami S. On Functional Role of Cytochrome b5. Biochem J,1971,69:325-330
[49]Urano J, Nakagawa T, Maki Y, Masumura T, Tanaka K, Murata N, Ushimaru T. Molecular Cloning and Characterization of A Rice Dehydroascorbate Reductase. FEBS,2000, 466:107-111
[50]Chen Z, Young T E, Ling J, Chang S C, Gallie D R. Increasing Vitamin C Content of Plants Through Enhanced Ascorbate Recycling. Proc Natl Acad Sci USA,2003, 100:3525-3530
[51]Chen Z, Gallie D R. The Ascorbic Acid Redox State Controls Guard Cell Signalling and Stomatal Movement. Plant Cell,2004,16:1143-1162
[52]Chen Z, Gallie D R. Increasing Tolerance to Ozone by Elevating Foliar Ascorbic Acid Confers Greater Protection Against Ozone Than Increasing Avoidance. Plant Physiol, 2005,138:1673-1689
[53]Hernandez J, Cano J, Portillo B, Rubio M, Martinez-Gomez P. Antioxidant Enzymes as Biochemical Markers for Sharka Resistance in Apricot. Biol Plant,2006,50(3):400-404
[54]张晓磊.星星草PtDHAR及PtFer基因的克隆与表达分析[硕士学位论文].东北林业大学,2008
[55]卢钮,刘军,丰震等.菊花育种研究现状及今后的研究方向.山东农业大学学报(自然科学版),2004,35(1):145-149
[56]Courtney-Gutterson N, Napoli C, Lemieux C. Modification of flower color in florist's Chrysanthemum:production of a white-flowering variety through molecular genetic. BioTechnology,1994,12:268-271
[57]Mitiouehkina T Y, Dolgov S V. Regeneration from leaf disks of Chrysanthemum morifoulium Ramat. Acta Hort,1995,420:112-114
[58]任永霞,季静,王萍.农杆菌介导类胡萝卜素合成酶基因LycB转化菊花的研究.吉林农业大学学报,2005,27(3):255-258
[59]龚学臣,季静,王罡.应用农杆菌介导法进行菊花的遗传转化.北方园艺,2005,(2):66-67
[60]Dolgov S V, Mitiouehkina T Y, Rkavtsova E B. Production of transgenic plants of Chrysanthemum morifoulium ramat with gene of Bac. Thuringiensis delta endotox in.Acta Hort,1995,420:46-47
[61]Zheng Z L, Yang Z B, Jang J C. Modification of plant architecturein chrysanthemum by ectopic expression of the tobacco phytochrome B1 gene. Amer Soc Hort Sci,2001, 126(1):19-26
[62]Petty L M, Harberd N P, Carre I A. Expression of the Arabidopsis gai gene under its own promoter causes a reduction in plant height in Chrysanthemum by attenuation of the gibberellin response. Plant Science,2003,164:175-182
[63]Aswath C R, Mo SY, Kim S H. IbMADS4 regulates the vegetative shoot development in transgenic Chrysanthemum(Dendranthema grandiflorum(Ramat)Kitamura). Plant Science, 2004,166:847-854
[64]邵寒霜,李继红,郑学勤.拟南芥LFYcDNA的克隆及转化菊花的研究.植物学报,1999,41(3):268-271
[65]吕晋慧.根癌农杆菌介导的AP1基因转化菊花的研究[博士学位论文].北京林业大学,2005
[66]Takatsu Y, Hayashi M, Sakuma F.Transgene inactivation in Agrobacterium-mediated Chrysanthemum (Dendranthema grandiflorum(Ramat.)Kitamura) transformants. Plant Biotechnology,2000,17(3):241-245
[67]Wordragen M F, Jong J, Sehornagel M J. Rapid screening for host-bacterium interaction in Agrobacterium-mediated gene transfer to chrysanthemum, by using the GUS introngene. Plant Sci,1992,81:207-214
[68]Shinoyama H, Komano M, Nomura Y, Nagai T. Introduction of delta-endotoxin gene of Bacillus thuringiensis to chrysanthemum (Dendran-thema X grandiflorum(Ramat.) Kitamura) for insect resistance. Breeding Science,2002,52(1):43-50
[69]Dolgov S V, Mityshkina T U, Rukavtsova E B, BuryanovY I. Production of transgenic plants of chrysanthemum morifolium ramat with the gene of Bac. Thuringiensis 8-endotoxin. ActaHorticulture,1995,420:46-47
[70]蒋细旺,包满珠.农杆菌介导CrylAc基因转化菊花[J].园艺学报,2005,32(1):65-69
[71]洪波,仝征,李邱华,马超.地被菊花Fall Color体细胞胚途径再生、遗传转化及转基因植株的抗寒性检测.中国农业科学,2006,39(7):1443-1450
[72]吴月亮.根癌农杆菌介导的PEAMT基因转化菊花的研究[博士学位论文].北京林业大学,2006
[73]佟友丽.露地菊耐低温特性研究及遗传转化体系的构建[博士学位论文].东北林业大学,2008
[74]苏军,段榕琦,胡昌泉等.小白菜再生和农杆菌介导转化体系的建立.福建农业学报,2002,17(4):241-243
[75]蒋细旺.根癌农杆菌介导的Bt与GNA基因转化菊花品种的研究[博士学位论文].华中农业大学,2003
[76]郑丽.根癌农杆菌介导SAG12-ipt和3DN-iaal基因转化切花菊及抗早衰研究[博士学位论文].西南农业大学,2003
[77]Kim J Y, Jung M. A New Selection System for Pepper Regeneration by Mannose. Plant Biotechnol,2002,129-134.
[78]梁机.转rolB基因改良毛白杨生根能力的研究[博士学位论文].北京林业大学,2004
[79]Lowe J M, Davey M R, Power J B. A study of some factors affecting Agrobacterium-transformation and plant regeneration of Dendranthema grandiflora Tzvelev(syn.Chrysanthemum morifoulium Ramat.). Palnt Cell,Tissue and Organ Culture, 1993,33:171-180
[80]Urban L A, Sherman J M, Moyer J W. High frequency shoot regeneration and Agrobacterium-mediated ransformation of Chrysanthemum(Dendranthema grandiflora). Paint Science,1994,98:69-79
[81]郝贵霞,朱祯,朱之梯.杨树基因工程进展.生物工程进展,2000,20(4):6-10
[82]王关林,方宏筠.植物基因工程.科学出版社,2002
[83]王火旭,王关林,王晓岩.大白菜AB81高频再生系统的建立及gusA基因瞬时表达的研究.园艺学报,2001,28(1):74-76
[84]傅荣昭,孙勇如,贾士荣.植物遗传转化技术手册.北京:中国科学技术出版社.1994
[85]Joersbo M, Donaldson I, Kreiberg J. Analysis of mannose selection used for transformation of sugar beet. Mol Breed,1998,4(2):111-117
[86]Paola L, Ye X, Ingo P. Effective selection and regeneration of transgenic rice plants with mannose as selective agent. Mol Breed,2001,7:43-49
[87]Negrotto D. The use of phosphomannose-isomerase as a selectable Marker to Recover Transgenic Maize Plants (Zea mays L) via Agrobacterium Transformation. Plant Cell Rep, 2000,19:797-803
[88]黄霞,黄学林,李哲.影响根癌农杆菌介导的香蕉基因转化早期的主要因素.中山大学学报,2002,41(5):68-72
[89]周思军,李希臣,刘昭军,刘丽艳,杨庆凯.大豆农杆菌介导转化系统的优化研究.东北农业大学学报,2001,32(4):313-319
[90]Schaffer R, Ramsay N, Samach A. The late elongated hypocotyls mutation of Aarbidopsis disrupts circadian rhythms and thephotoperiodic control of flowering. Cell,1998, 93:1219-1229
[91]洪波.逆境诱导转录因子DREB1A基因转化地被菊花的研究[博士学位论文].中国农业大学,2005
[92]王欢.根癌农杆菌介导PCFH基因转化菊花的研究[硕士学位论文].北京林业大学, 2007
[93]周涌.菊花再生体系的建立及根癌农杆菌介导DAD1基因转化体系的优化[硕士学位论文].湖南农业大学,2007