摘要
大豆是现代农业十分重要的农作物之一,富含蛋白质、植物油和多种对人体有益的成分,是人类食品中主要的植物蛋白来源,同时也是重要的工业原料和牲畜饲料。随着科技的发展和人们生活水平的提高,大豆的产量逐渐开始无法满足人们的生活需求,而传统的育种方法又无法有效解决这一矛盾。于是自1988年开始,越来越多的研究人员开始利用基因工程技术,培育具有优良性状的大豆品种,并取得了一定的成就。但大豆的基因转化一直存在着转化率低、重复性差等问题。因此,建立高效、稳定的大豆转化技术体系仍是目前国内外学者研究的热点之一。本实验采取目前最常用的农杆菌介导的转化方法,以多个大豆品种的子叶节外植体为受体材料,进行了大豆遗传转化实验。并且针对转化过程中的重要步骤实施了一系列优化措施,旨在通过提高农杆菌侵染效率、丛生芽的诱导效率等,进一步提高大豆再生效率和转化效率,建立一种稳定性好、转化率高的大豆转化技术体系,为今后的大豆分子育种和基因功能研究打下良好的基础。
本实验主要研究成果如下:
1、高再生能力的大豆基因型筛选。实验采用含PBI121质粒的农杆菌EHA105和GV3101侵染7个大豆品种的子叶节外植体,发现不同大豆基因型之间再生效率差异明显,并从中筛选出中黄10号、文丰7号两个再生效率较高的大豆品种。
2、降低了组织培养过程中的褐化现象。通过对共培培养基进行半胱氨酸(L-cy)浓度的梯度试验,发现L-cy浓度为400 mg?L-1时,能有效地抑制子叶节外植体的组织褐化现象,提高了外植体的成活效率。
3、降低了残留农杆菌污染。本实验通过优化共培后的除菌方法,配合除菌剂的使用,大大降低了残留农杆菌污染的比率,有效地解决了农杆菌介导转化体系容易造成侵染后的农杆菌残留污染的现象,保证了转化实验的顺利进行。
4、提高了丛生芽的诱导效率。通过在恢复期去掉子叶节外植体第一组新芽的方法,大大提高了外植体诱发的丛生芽数量,从而提高了再生效率;并以此降低了丛生芽发育的顶端优势,提高了筛选效率。
5、Thidiazuron(TDZ)的活化效果。TDZ是一种高效的细胞分裂素,本实验在丛生芽诱导阶段添加了适量的TDZ,结果发现诱导的活性细胞数量得到了大幅度增加。
6、生根培养基的优化。通过对多品种的生根诱导实验,确定了生根诱导高效稳定的生根培养基配比及IBA的最适浓度1.0mg?L-1,并且选出了容易发根的大豆品种中黄10号和文丰7号。
7、优化炼苗移栽步骤。本实验对再生植株的炼苗移栽步骤作了一系列优化,解决了幼嫩再生植株无法快速适应外界环境的问题,提高了移栽成活效率,为之后的检测与研究创造了条件。
8、转化植株的获得。通过对7个品种的大豆进行优化转化实验,最终获得了43株含有转化外源基因的转基因再生植株,平均转化效率为1.76%,效率最高的EHA105侵染大豆中黄10转化实验中,最终转化效率达到4.61%,高于目前大豆转化的平均水平。
Soybean is one of the most important crops, which is rich in protein, vegetable oil and a variety of useful physiological chemical compositions of human. Soybean is not only a major human food source of vegetable protein, is also an important materials for industry and animal feed. With the technology and people's living standards improve, soybean production began to not content the needs of people's lives. Traditional breeding methods and can not effectively solve the problem of soybean’s gap between supply and demand. So that, more and more researchers have tried to overcome these problems use genetic engineering technology since 1988, and has achieved some success. But, compare with other plants, the transformation frequency of soybean is still low relative, and the repeatability is not good. Actually a few of cases of transgenic soybeans were successful due to the low transformation rate.Establish an efficient and stable system for soybean transformation technology is still one of the hot topic for the scientists.
In this study,we use Agrobacterium-mediated transformation method for transforming 7 soybeans, the cotyledon-node explant was uesd for the transgenic receptor. In order to improve transformation efficiency, this experiment was conducted to compare the efficiency of several factors during transformation. This will lay a good foundation for the gene function research and molecular breeding of soybean.
The main results of this research as follows
1. 7 soybean genotypes were used for transformation.The Agrobacterium tumefaciens strain was EHAl05 and GV3101,which carried binary vector PBI121.The result in this research had shown that regeneration frequency was higher of“zhonghuang10”and“wenfeng7”.
2. To reduce the browning effect,we added L-cy in the co-cultivation medium.The result indicate that though the experiments for concentration gradient of L-cy,the suitable L-cy cencentration is 400mg/L for the zhonghuang13 soybean.The browning rate has dropped by more than 90%。
3. Reduce the level of residual Agrobacterium contamination.We design of three methods to wash the cotyledon-node explant,after the co-cultivation,and finally chose one methods that the lever of residual Agrobacterium was lowest.
4. Improve the efficiency of shoot induction.We chose some of cotyledon-node explant that just shooting after the co-cultivation,cut off the new shoot,and then transfer them to a new medium.The result indicate that the efficiency of shoot induction was most increased.
5. We add Thidiazuron(TDZ) in the shoot induction medium.And found TDZ is more effective than 6-BA at activated cell development.
6. Duing the root regeneration stage,We had determined the optimal concentration of IBA,and found that the genotypes has some infulence on the rooting ability.
7. We also did some research on transplanting, and improve the efficiency of the survival of transplanted.It is made some good provide for the molecular detection.
8. Through the soybean transformation experiments,we finally obtained 43 transgenic plants,increases in transformation efficiency to an average of 1.7%, which the most is 4.61%. Conversion efficiency is higher than the current average level of soybean transformation.
引文
[1]郝瑞莲,卢厂远.大豆优质高蛋白育种研究的探讨.中国农学通报, 2004, 20(1): 96-9
[2]陈锦清,黄锐芝.油料作物基因.中国生物工程杂志, 2004, 24(5): 24-29
[3]贾士荣,曹冬孙.转基因植物.植物学通报, 1992, 9(2): 3-15
[4] M. B. DE, E. L. HERRERA, M. M. VAN. Expression of foreign genes in regenerated plants and their progeny. EMBOJ, 1984, 3: 1681-1689
[5] D. E. MCCABE, W. F. SWAIN, B. J. MARTINELL, et al. Stable transformation of soybean (Glycine max) by particle acceleration. Biotechnology, 1988, 6: 923–926
[6] M. A. W. HINCHEE, D. V. CONNORWARD, A. C. NEWELL, et al. Production of transgenic soybean plants using Agrobacterium-mediated DNA transfer. Bio/Technology, 1988, 6, 915–922
[7]崔广荣植物转基因方法及特点和转基因沉默现象.安徽技术师范学院学报, 2003, 17(1): 37-41
[8] J. KIM, C. E. LAMOTE, E. HACK. Plant regeneration in vitro from primary leaf nodes of soybean seedling. Plant Physiol.1990, 136: 664-669
[9] J. A. TOWNSEND, L. A. THOMAS. An improved method of Agrobacterium-mediated transformation of soybean cells.Patent, 1993, WO94: 0262
[10] R. DI, V. PURCELLX, G. B. COLLINS, et al. Production of transgenic soybean lines expressing the bean pod mottle virus coat protein precursor gene. Plant Cell Reps. 1996. 15: 746-750
[11] Z. ZHANG, A. Q. XING, P. STASWICK, et al. The use of glufosinate as a selective agentin Agrobacterium mediated transformation of soybean.Plant Cell Org Tiss Cult. 1999, 56(1): 37-46
[12] T. E. CLEMENTE, B. G.LAVALLE, A. R. HOWE, et a1. Progeny analysis of glyphosate- selected transgenic soybeans derived from Agrobacterium-mediated transformation.Crop Sci. 2000. 40: 797~803
[13]程林海,孙毅,刘少翔.大豆生物工程研究进展.植物学通报.2001,18(3):367-370
[14]张晓娟,方小平,罗丽霞等. BA和TDZ对诱导大豆胚轴植株再生的影响.中国油料作物学报. 2000, 22(1), 24-26
[15] S. SATO, C. NEWELL, K. KOLACZ.Stable transformation via particle bombardment in tow different soybean regeneration systems:Plant Cell Rep, 1993, 12: 408-413 [ 1 6] L. HAIKUN, Y. CHAO, W. ZHIMING. Efficient Agrobacterium tumefaciens-mediated transformation of soybeans using an embryonic tip regeneration system: Planta, 2004, 219: 1042–1049
[17]宋雯雯,刘宝辉,杨明亮等.农杆菌介导法将SPS基因导入大豆的研究.大豆科学. 2008, 27(3): 387-390
[18]马丽萍,胡正,张保缺等.一种快速、高效的大豆农杆菌转化技术.中国农业科学. 2008, 41(3): 661-668
[19]王庆中.转BoTMT基因大豆的获得及鉴定.北京:中国农业科学院. 2006
[20] W. A. PARROTT, I. M. HOFFMAN, D. F. HILDEBRAND, et al, Recovery of primary transformants of spybean. Plant Cell Rep: 1989, 7: 615-617
[21] B. MAZUR, E. KREBBERSX, S. TINGEY. Gene discovery and product development for grain quality traits.Science. 1999. 285: 372-375
[22] J. J. FINER, M. D. MCMULLEN. Transformation of soybean via particle bombardment of embryogenic suspension culture tissue.In Vit ro Cell Dev Boil, 1991, 27 (4) : 1752182
[23]邓向阳,卫志明,许智宏.大豆主栽品种体细胞胚胎发生的影响因素及再生植株.实验生物学报: 2000, 33, 69-78
[24]王晓春,王罡,季静.农杆菌介导的大豆体细胞胚遗传转化影响因子的研究.大豆科学. 2005, 24(1): 21~26
[25]王晓春,王罡,季静等. 2,4-D对大豆体细胞胚胎发生及增殖的影响.大豆科学. 2009, 28(6): 1112-1114
[26] K. N. KAO, W. A. KELLER, R. A. MILLER. Experimental cell Research. 1970.63: 338~340
[27] W. ZHIMING, X. ZHIHONG. Plant regeneration from protoplast of soybean (Glycine max L. ). Plant Cell Rep.1988, 7: 348-351
[28] S. K.DHIR, S. DHIR, M. J. WIDHOLM. Regeneration of fertile plants from protoplasts of soybean (Glycinemax (L.)Merr.):genotypic differences in culture response. Plant Cell Reps. 1992, 11: 285~289
[29]张贤泽,小松田隆夫.大豆原生质体经体细胞胚胎再生植株.中国科学(B辑). 1993, 23(2):154-158
[30]姬月梅.大豆高效转基因技术体系的建立及NaH逆向转运蛋白TaNHX2基因的导入研究. 2008, 4-5
[31] S. SANIMAH, Q. NAIRA, K. K. HYE, H. C. YOUNG, et al. Metabolic changes in Agrobacterium tumefaciens-infected Brassica rapa, Journal of Plant Physiology 2009, 166, 1005-1014
[32]王连铮.大豆致瘤及基因转移研究.中国科学. B辑. 1984,2: 137-141
[33]李洪泉,李红卫.根癌农杆菌Chry5对我国栽培大豆子叶致瘤作用的研究.大豆科学. 1994, 13(2): 112-115
[34] H. LIU, C, YANG, Z. WEI. Efficient Agrobacterium tumefaciens-mediated transformation of soybeans using an embryonic tip regeneration system.Planta, 2004, 219: 1042—1049
[35] M. C. BYRNE. Strain and cultivar specificity in the Agrobacterium soybean interaction. PlantCell Tissue Organ Cult: 1987, 8(11), 3-15
[36] P. M. Olhoft, E. L. Flagel, C. M. Donovan, et al. Efficient soybean transformation using hygromycin B selection in the cotyledonary-node method.Planta, 2003, 216: 723-735
[37]陈秀华,柏锡,潘欣等.转cry1Iem基因大豆的培育及抗虫性检测.大豆科学.2009, 28(6): 959-963
[38] D. OWENS, L. OWELL, E. DEAN. Cress Genotypic Variability of Soybean Response to Agrobacterium Strains Harboring the Ti or Ri Plasmids.Plant Physiology, 1985, 77: 87—94
[39]刘海坤,卫志明.大豆遗传转化研究进展.植物生理与分子生物学学报, 2005, 31(2): 126-134
[40]李宝健.应用农杆Ti质粒系统将外源基因导入籼稻的研究.中国科学. 1990, 2: 144-149
[41] T. E. CLEMENTE, J. B. LAVALLE, R. A. HOWE. Progeny analysis of glyphosate-selected transgenic soybeans derived from Agrobacterium-mediated transformation. Crop Sci, 2000, 40: 797-803
[42]刘金华,王丕武,武丽敏.脯氨酸、硝酸银对农杆菌转化大豆的影响.大豆科学. 2003, l(22): 36-40
[43]汲逢源,王戈亮,许亦农.抗氧化剂对农杆菌介导的大豆下胚轴GUS基因瞬时表达的影响.植物生态学报. 2006, 30(2): 330-334
[44]黄丹莹,江贵波.植物组织培养褐变产生的因素及对策.广西轻工业, 2006, 5: 31-32
[45]王栋,买合木提克衣木,玉永雄等.植物组织培养中的褐化现象及其防止措施.黑龙江农业科学. 2008, (1): 7-10
[46]李海燕,刘淼,武小霞等.大豆转化过程中的褐化现象研究.作物杂志. 2010, 1: 33-35
[47]王萍,张淑珍,李文滨等.大豆不同基因型胚尖不定芽的诱导及对抗生素的敏感性.作物杂志. 2010. 2: 50-53
[48]姬月梅,张银霞,宋晓华.农杆菌介导大豆遗传转化的研究进展.农业科学研究. 2009, 30(1): 47-50
[49] N.TRICKH, J. FINERJ. Sonication-assisted Agrobacte Hum-mediated transformation of soybean [Glycine max(L.)Merril1]embryogenic suspension culture tissue.Plant Cell Report. 1998. 17: 482~487
[50] M. T. KLEIN, E. D. WOLF, R. WU,e t al. High-velocity microprojectiles for delivering nucleic acids into living cells. Nature, 1987, 327(7): 70-73
[51] P. G.MAUGHAN, R. PHILIP, M. J. CHO, et al. Biolistic transformation, expression, and in-heritance of bovine beta-casein in soybean (Glycine Max).In Vitro Cellular and Developmental Bioloy. 1999. 35: 344~349
[52] ZH.HU, ZH.LIU, C. X.LANG, et al.Several parameters involved in soybean transformation by particle bombardments.Acta Agriculturae Zhejian Gensis, 1999, 11 (5): 242-244
[53] P. A. SIMMONDS, P. A. DONALDSON. Genotype screening for proliferative embryogenesis and biolistic transformation of short-season soybean genotypes.Plant Cell Reports.2000. 19. 485-490
[54]王萍,王罡,吴颖等.影响大豆基因枪遗传转化因子的研究.农业生物技术学报. 2002. 10(3): 36-37?
[55]李保山.基因枪法在植物转基因中的应用.科技信息. 2008, 17: 648-649
[56]王晓春,刘尚前,罗永华.大豆再生体系和遗传转化研究进展.大豆科学. 2009, 28(4): 731-735
[57]赵锦,刘孟军,蒋洪恩.转基因技术及其在植物育种中的应用.生物技术, 2002, 12(6): 42-43
[58] C. PAUL, E. MURPHY JEAN, S. SWAIN, F. WILLIAM. Stable transformation of soybean by electroporation and root formation from transformed callus.Proc Natl Acad Sci USA, 1987, 84, 3962-3966
[59] S. K. DHIR, S. DHIR, P. STURTEVANT ANN, et al.Regeneration of transformed shoots from electroporated soybean (Glycinemax (L.) Merr. ) protoplasts.Plant Cell Reports, 1991, 10: 97-101
[60]王关林,方宏筠.植物基因工程原理与技术.北京:科学出版社, 1998: 61
[61] W. LIN, J. ODELL, M. SCHREINER. Soybean protoplasts culture and direct gene uptake and pression by cultured soybean protoplasts. Plant Physiol, 1987. 84: 56-61
[62]雷勃钧,尹光初,卢翠华等.外源DNA直接导入大豆的研究.大豆科学, 1991, 10(1):58-62
[63]崔言.大豆利用花粉管通道技术导入抗病虫目的基因的研究.哈尔滨:东北农业大学, 2000
[64]刘德璞,袁鹰,唐克轩等.大豆花粉管通道技术转化雪花莲凝集素(GNA)基因.分子植物育种, 2006, (5): 68-71
[65]武小霞,刘伟婷,刘琦等.利用花粉管通道法将抗虫基因(cry V)转入大豆的研究.大豆科学. 2010, 29(4): 565-574
[66]李海燕.大豆转基因的研究进展.生物技术. 2007, 17(6): 83-86
[67]刘佳,刘志华,徐广惠等.抗草甘膦转基因大豆(RRS)对根际微生物和土壤氮素转化的影响.农业环境科学学报. 2010, 29(7): 1341-1345
[68]林树柱,曹越平,卫志明.根癌农杆菌介导的大豆遗传转化.生物工程学报. 2004, 20(6): 817-820
[69]王振华,杨德光,张辉等.大豆遗传转化技术在转基因大豆研究中的应用.生物技术通报. 2010, 10: 60-66
[70]于洋,侯文胜,韩天富.农杆菌介导大豆遗传转化技术的研究进展.大豆科学. 2010. 29(4): 696-701
[71] A. BRASILEIRO, F. ARAGLO. Marker genes for in vitro selection of transgenic plants.Plant Biotechnology. 2001, 3(3): 113-121
[72]陈肖英,叶庆生,刘伟. TDZ研究进展.亚热带植物科学, 2003, 32(2): 59-63
[73]卜学贤,陈维伦.试管植物的玻璃化现象.植物生理学通讯, 1987, (5): 13-1.
[74] M. SUJATHA, M. SAILAJA. Stable genetic transformation of castor (Ricinus communis L.)via Agrobacterium tumefaciens-mediated gene transfer using embryo axes from mature seeds. Plant Cell Rep. 2005. 23: 803-810
[75] P. M.OLHOFT, D. A. SOMER. L-cysteine increases Agrobacterium-mediated T-DNA delivery into soybean cotyledonary-node cells. Plant Cell Rep, 2001, 20: 706-711
[76]杜升伟,刘业丽,姚丙晨等.大豆转化体系的优化和Dof4基因转入大豆的研究.大豆科学. 2010, 29(3): 399-402
[77]王广科,李明春,李晋川等.高山被孢霉Δ6-脂肪酸脱氢酶基因转化大豆的研究.成都医学院学报. 2010, 5(2): 93-96
[78]王全伟,李新玲,张海玲等.转Na+/H+逆向转运蛋白基因(nhaA)大豆植株的获得及耐盐性分析.分子植物育种. 2010, 8(4): 701-707
[79]应珊,何晓薇,王秀荣等.影响农杆菌介导的大豆转化效率的因素研究.分子植物育种. 2008, 6(1): 32-40
[80]张艳,南相日,满为群等.大豆离体培养及高频再生基因型的筛选.植物生理学通讯. 2010, 46(11): 1135-1139
[81]张洁,张东旭,商蕾.转基因技术在大豆育种上的应用与研究. 2008, 23(增): 133-138
[82]李文霞,宁海龙,吕文河等.农杆菌介导大豆子叶节转化系统的优化.中国农业科学. 2008, 41(4): 971-977
[83]郭东全,杨向东,包绍君等.转CryIA和CpTI双价抗虫基因大豆的获得与稳定表达.中国农业科学. 2008, 41(10): 2957-2962
[84]王昌陵,赵军,李英慧等.转录因子ABP9转化大豆(Glycine max L.)及遗传转化条件优化.中国农业科学. 2008, 41(7): 1908-1916
[85]段莹莹,赵琳,陈李淼等.农杆菌介导的大豆子叶节和下胚轴转化方法的比较及优化.大豆科学. 2010, 29(4): 590-593
[86]张东旭,张洁,商蕾等.大豆胚尖再生体系的研究.河北农业大学学报. 2008. 31(4): 7-13.
[87]王升吉,吴元华,王洪岩等.大豆不同外植体组织培养及再生研究.沈阳农业大学学报, 1999, 30(3): 255-259
[88]武小霞,李静,刘伟婷等.大豆农杆菌子叶节转化菌株适宜生长时期及浸染浓度的研究.东北农业大学学报. 2010. 41(1): 1-6
[89]王翠亭,卫志明.重要禾谷类粮食作物的遗传转化.植物生理学通讯. 2001. 38(2): 105-110
[90] Y. H. ISHIDA, S. SATTO, Y. OHTA, et al. High effieieney transformation of maize mediated by Agrobaeterium tumefaeiens. Nature Biotechnology. 1996. 14(6): 745-750
[91]薛仁镐.农杆菌介导大豆萌动种子遗传转化的影响因素研究.安徽农业科学. 2008, 36(7): 2666-2667
[92]武小霞,李静,王志坤等.乙酰丁香酮浓度和共培养pH对大豆再生频率的影响.东北农业大学学报. 2010, 41(5): 1-4
[93] Z. ZHAO, W. GU, T. CEIL, et a1. High throughput genetic transformation mediated by Agrobacterium tumefaciens in maize. Mol. Breed. 2001. 8: 323-333
[94]刘金华,王丕武,武丽敏等.脯氨酸、硝酸银对农杆菌转化大豆的影响.大豆科学. 2003. 22(1): 36-39
[95] K. ATTILA, L. DONGXUE, I. ARIEF, et al. Agrobacterium rhizogenes-mediated transformation of soybean to study root biology.Nature Protocols. 2007. 2(4): 948-952
[96] Y. KANEDA, Y. S. TABCI, A. NISHIMURA, et al. Combination of thidiaznron and basal media with low salt concentrations increases the frequency of shoot organogenesis in soybeans(Glycinemax (L.) Merr. ).Plant Cell Rep. 1997. 17: 8-12
[97]张晓娟,方小平,罗丽霞等. TDZ和BA对诱导大豆胚轴植株再生的影响.中国油料作物学报. 2000, 22(1): 24-26