摘要
刺槐(Robinia pseudoacacia L.),为豆科Leguminosae刺槐属(Robinia L.)落叶乔木,原产北美。中国于1877年引入栽培,主要包括刺槐R.pseudoacaciaL.和毛刺槐R.hispida L.两种,常见于中部和东部,由于其生长迅速、木材坚韧、纹理细致、有弹性、耐水湿、抗腐朽、根系发达,具根瘤,有一定的抗旱、抗烟、耐盐碱能力(国家林业局国有林场和林木种苗工作总站,2001),现已成为华北、西北等地区优良的保持水土、防风固沙、改良土壤的优质树种,深受群众欢迎(荀守华等,2009)。刺槐还具有花香浓郁、抗逆性强、病虫害少、价格低等优点,且易于栽培、成活率高、绿化效果好,适宜于景观绿化与经济用途(张亚玲等,2009)。
然而,近年来,生态环境等各方面的影响使刺槐也面临一些严重的病虫害:病害——紫纹羽病和烂皮病等;虫害——白蚁、叶蝉、蚧、小皱蝽(叶)、豆荚螟虫、麦蛾、小蜂(种子)等。而因为以下种种原因,刺槐的转基因工程研究进展较缓慢。其一,一直以来,木本植物的遗传转化成功几率都比较低,而刺槐为大型木本植物,基因组庞大,外源基因很难插入并表达,即使有少数转化成功的株系,在以后的继代培养中也容易发生基因丢失或基因沉默等现象,这是木本植物转基因研究中迄待解决的问题(荀守华等,2009);其二,刺槐的遗传转化工作缺乏再生频率高、重复性好的受体系统;其三,缺乏优良的目的基因的克隆和启动子的构建;其四,转化频率低、实验重复性差,没有形成第一代工程品种(郑进等,2005)。针对上述刺槐的广泛用途及其病虫害特点,更兼于豆科植物是被子植物第二大科,本实验展开了刺槐离体转化受体系统的建立及基因转化的研究,拟建立一个高效的刺槐转化受体系统,并进一步开展抗病虫害的转基因研究,旨在为刺槐及其他豆科植物的抗病虫害方面提供借鉴及良好的应用价值。
本实验首先以生长良好的刺槐植株的带腋芽茎段为外植体,研究了在刺槐高频再生体系的建立过程中不同基本培养基、不同激素浓度及其配比对愈伤组织的诱导、芽的分化及生根的影响;然后在得到高频再生体系的基础上,通过农杆菌介导法转化Bt Cry1A抗虫基因,以GUS染色组织分析为依据探讨了影响转化效率的各种因素,建立了高效、可重复的基因转化体系,为刺槐目的基因的导入打下了基础。试验取得如下研究结果:
1.建立了刺槐高频再生基因转化受体系统。在刺槐离体茎段为外植体诱导愈伤组织的过程中,芽的分化以MS+6-BA 0.3mg/L+NAA 0.04mg/L+蔗糖30g/L+琼脂9g/L条件下分化率最高,每个外植体的平均分化芽数可达到7.3个;诱导生根以MS+NAA0.04mg/L+KT 0.05mg/L+蔗糖30g/L,效果最佳,生根率最高可达93.3,平均生根率达86.7%;在移栽生根苗时,先在温室中驯化炼苗15~20天后移入大田,成活率可达83%以上。
2.以GUS染色检测为依据初步建立了刺槐高效遗传转化体系。以农杆菌菌株LBA4404介导Bt Cry1A基因的转化,首先确定了转化培养中最佳的抑菌素及其浓度和选择压:头孢霉素(Cef)400mg/L即可有效地抑制农杆菌菌株LBA4404的生长;卡那霉素(Km)50mg/L为选择压较适宜。并进一步优化了转化体系:以离体茎段为外植体,预培养1-2天,OD值为0.5~0.7的农杆菌LBA4404菌液浸染外植体以8~10分钟为宜;共培养时间3~4天较适合;共培养培养基中添加AS 30mg/L,GUS+阳性发生率可达最高值。共培养后转入附加Cef 400mg/L的培养基中培养,15天后转入附加Km 50mg/L的培养基中筛选抗性植株。
Robinia pseudoacacia L,a member of Robinia in Leguminosae was originated from North America.It possesses many important features,such as rapid growth,strong.and elastic woody fibers,anti-adversity,well-nodulated root system and so on,so that it becomes widespread in north and northwest of China for soil and water Conservation. Robinia pseudoacacia is also an indispensable species for urban avenue virescence due to its rich fragrance,easy planting,and valuable appearance for public attention.
Robinia pseudoacacia ever featured in strong resistance to infection of pathogenic organisms.However,in recent years,it has been found to suffer some serious diseases, such as violet root rot and skin ulceration caused by pathogenic microorganisms,and invasion from bugs small wrinkle(leaf),pod borer,Angoumois grain moth,small bees (seeds).
Though transgenic engineering has been successfully used to introduce advantageous genes into some important plant species to improve resistance to attacking from pests and diseases,it has been not available in Robinia pseudoacacia mainly due to lacking a well-established receptor system.
In present study,we used the growing-well stems of Robinia pseudoacacia with axillary's buds as explants.Firstly,we studied the influence of several factors on differentiation of buds and roots,including different media,different hormones and different concentration ratio of some hormones mixtures.And then,based on availability of a high frequency regeneration system,we tested the effects of various factors on transformation efficiency of Bt CrylA gene mediated by Agrobacterium tumefaciens with histochemical assays of GUS.The transformation procedure established here was proved to be reproducible and effective,and opened up a possibility of more beneficial genes to be introduced into Robinia pseudoacacia.Detailed experiments and data were listed as the following
(1) Establishment of a high frequency regeneration system,of Robinia pseudoacacia:we found that the most effective medium to induce callus is MS basic medium containing 0.3mg/L of 6-BA(6-benzyladenine),0.04mg/L of NAA (naphthalene acetic acid) and 30g/L of sucrose and 9g/L of agar,and that the mean bud differentiation rate is 80%,the mean number of buds differentiated from an explant reaches up to 7.3,and MS medium supplemented with 0.04mg/L ofNAA,0.05mg/L of KT(kinetin) and 30g/L of sucrose is very efficient to improve rooting.The highest rooting frequency is 93.3%and the mean rooting frequency is 86.7%.Our data showed that the plants cultivated in tubes should be acclimated in green house for 15~20 days before planting into outdoor soils,making the surviving rate reach higher than 83%.
(2) Establishment of a high efficient transformation system of Robinia pseudoacacia:in the transformation of Bt CrylA gene mediated by Agrobacterium tumefaciens strain LBA4404,the most suitable bacteriostatic agent is 400mg/L Cef(cephalosporin) and the selecting concentration is 50mg/L of Km(kalamycin ). Based on further optimization of necessary factors to improve the transformation frequency by GUS histochemical assays,we established an efficient transformation system of Robinia pseudoacacia:including:(1) stems should be pre-cultured in-vitro for 1~2 days;(2),Agrobacterium tumefaciens was inoculated till OD600 reached 0.5~0.7;(3) they were co-cultured for 3~4 days.Our data also suggested adding 30mg/L of Acetosyringone(AS) into the co-cultured medium could dramatically improve transformation frequency;(4) the co-cultured explants were transferred to MS medium containing 400mg/L of Cef;(5) After about 15 days,they were transferred to the same medium with 50mg/L of Km to select Km-resistant shoots.
引文
[1]曹帮华,龙庄如,梁玉堂.石林刺槐微体快繁的研究[J].山东农业大学学报,1993,24(5):52-61.
[2]曹帮华,龙庄如,冯宪修.刺槐微体快速繁殖速度及产率计算[J].山东林业科技,1995,(2):14-17.
[3]高艳明,李建设,高娜,曹君迈等,刺槐组织培养繁殖技术[J],林业科技,2005年1月,第30卷第1期:9-10
[4]郭军战,舒庆艳.四倍体刺槐组织培养中的外植体选择和消毒研究[J].西北林学院学报,2002年17卷第1期
[5]国家林业局国有林场和林木种苗工作总站.中国木本植物种子.北京:中国林业出版社,2001
[6]顾红雅,陈章良等.植物基因与分子操作[M].北京大学出版社,1995
[7]郝贵霞,朱祯,朱之悌.毛白杨遗传转化系统的优化研究[J].植物学报,1999,41(9):936-940
[8]何光源,杨广笑,刘勇.植物基因工程[M].清华大学出版社,2007
[9]黄茶英,刘青林.激素、通气和pH值对四倍体刺槐和二乔刺槐离体生长的影响[J].中南林学院学报23(5):38-41
[10]胡晓婷.宋福平.冯书亮.杜立新.宋健,Bt分子生物学研究进展[J].华北农学报2007(22):6-9
[11]及华,温春季,李瑞先.刺槐优良无性系组织培养快速繁殖[J].河北林业科技,1993,(1):11-12.
[12]及华.刺槐玻璃苗愈伤组织化再生正常植株[J].河北林学院学报,1994,19(2):102-104.
[13]贾士荣,曹东孙.转基因植物[J].植物学通报1992(9):3-15
[14]贾士荣.农杆菌介导的植物遗传转化[M].北京:中国农科院生物技术中心出版,1995
[15]李洪清,李美茹等.影响农杆菌介导的木薯基因转化因素的研究[J].实验生物学报,1999,32(4):391-396
[16]李浚明.植物组织培养教程[M].北京:中国农业大学出版社,2003
[17]李晋涛.DNA芯片技术及其在基因表达检测中的应用[J].生物技术,1999, 9(4):30-33
[18]李卫,郭光新,郑国錩.根癌农杆菌介导遗传转化研究的若干新进展[J].科学通报,2000,45:798-807
[19]李小玉 四倍体刺槐高频再生体系的建立及农杆菌介导的甜菜碱醛脱氢酶基因转化的研究,2002年硕士学位论文,甘肃农业大学
[20]梁小友,米景九,朱玉贤,等.双抗(抗病毒及抗虫)植物表达载体的构建及番茄的化鉴定[J].植物学报,1994,36(11):849-854
[21]雷呈,武玉暄,孙天洲等.宽叶刺槐组织培养和快繁技术研究[J].安徽农业科技,2003,31(4):653-654.
[22]刘曼.LFY类基因在茎端分子组织形成及其活动中的作用,北京大学正学者论文集2001年,378-391
[23]牛正田,张秀梅,罗晓芳.刺槐对3种抗生素敏感性的测定[J].植物生理学通讯,2004,40(1):98-99
[24]宋婉,续九如.果树种质资源鉴定及DNA指纹图谱应用研究进展[J].北京林业大学学报,2000,22(1):76-80
[25]司正霞,彭正峰,周青。刺槐离体快繁技术研究[J].河南林业科技,2004,(2):11
[26]沈峻岭、赵芳、李云等.次生型刺槐遗传转化体系的建立[J].核农学报2006,20(6):477-481
[27]滕长英.山东农业大学研究生论文,1999
[28]王春霞.山东农业大学硕士研究生学位论文,1996
[29]王莉,李春燕,邢震等.培养基激素配比及琼脂用量对四倍体刺槐组培苗生根的影响[J].林业科技,2004,29(2):1-2.
[30]王关林,方宏筠,植物基因工程[M].北京:科学出版社,2002
[31]王启忠.刺槐未授粉子房培养单倍体植株初获成功[J].林业科技通讯,1981(7):5-6.
[32]王启忠.用组织培养繁殖刺槐优良无性系苗木[J].林业科技开发,1988,4:30-33.
[33]王树芝,田砚亭,罗晓芳.刺槐宽叶和四倍体无性系的组织培养[J].植物生理学通讯,1999,35(3):204-205.
[34]王树芝,田砚亭,罗晓芳.刺槐的离体培养研究进展[J].河北林果研究,1999,14(4):368-373.
[35]王文强.农杆菌介导的四倍体刺槐BADH基因高效转化体系的建立和转基因植株的检测.甘肃农业大学硕士学位论文,2003
[36]王晓春.王罡.季静.王萍.用基因枪法将Bt基因转入大豆的研究[J].大豆科学2007,26(2):140-143
[37]王影,黄敏仁.林木原生质体培养研究进展[J].南京林业大学学报,1993,17(2):91-96.
[38]文平,王仁祥.转基因植物研究进展[J].生物学教学,2005年第12期
[39]夏阳,梁慧敏,陈受宜等.四倍体刺槐转甜菜碱醛脱氢酶基因的研究[J].中国农业科学,2004,37(8):1208-1211
[40]薛建平 司怀军 田振东,植物基因工程,2008
[41]奚元龄,颜昌敬.植物细胞培养手册[M].北京:农业出版社,1989
[42]荀守华等,我国刺槐遗传育种现状及发展对策[J].山东林业科技,2009,1:92-96
[43]闫新甫.转基因植物(M)北京:科学出版社,2003
[44]燕丽萍rd29A基因植物表达载体的构建及四倍体刺槐再生诱导研究
[45]燕丽萍,夏阳,王太明等.四倍体刺槐立体茎段高效再生体系,山东林业科技2006,164(3):1-3
[46]颜培强,李丽杰,康宏等.应用RNAi技术培育抗2中病毒病的转基因烟草.中国生物工程杂志2007,27(11):27-31
[47]叶景丰,姜忠灏.四倍体刺槐组培苗生根培养及生根苗移栽研究[J].辽宁林业科技,2004(1):15-16
[48]赵芳,李云.生在调节物质对刺槐复叶离体再生的影响,核农学报,2004,18(3):207-211
[49]张敦伦,李秀娣.组织培养诱导刺槐植株[J].林业科技通讯,1979,(5):6-7.
[50]张汉尧.刘小珍.杨宇明.植物抗虫基因工程研究进展.河南农业科学2005(03):11-15
[51]张松,达克东,曹辰兴等。抗生素对韭菜根尖培养植株再生的影响。核农学报,2003,17(2):101-104
[52]张志宏、方宏筠,景士西等,苹果主栽品种高效遗传转化系统的建立及其.影响因子的研究,遗传学报,1998,25(2):160-165
[53]张亚玲,田小伟,曹锋军等,刺槐在景观绿化中的应用.[J]国土绿化,2009,2:47-48
[54]郑进,华中师范大学博士论文,2006
[55]郑进,康薇,洪华珠.刺槐遗传转化研究进展.湖北林业科技,2005(6):37-41
[56]中国森林编辑委员会,中国森林,第3卷。北京:中国林业出版社,2000.
[57]中国科学院上海植物生理研究所,上海市植物生理学会.现代植物生理学实验指南[M].2004
[58]Arrillaga I,Merkle S A.Regenerating plants from in vitro culture of black locust cotyledon and leaf explants[J].Hort Science,1993,28(9):942-945.
[59]Arrillaga I,Tobolski J J,MerkleA.Advances in somatic embryogenesis and plant production of black locust(Robinia pseudocacia L)[J].Plant Cell Reports,1994,13(4):171-175.
[60]Chalupa V.Effect of benzylaminopurine and thidiazuronon in-vitro shoot proliferation of Tilia cordata Mill,Sorbus aucuparia L and Robinia pseudoacacia L[J].Biologia plant,1987,29(6):425-429
[61]Chalupa V.In vitro propagation of willows(Salix spp.),European mountain ash(Sorbus aucuparia L.)and black locust(Robinia pseudoacacia L.[J].Biol.plant.25(4):305-307.
[62]Civinova B,Sladky Z.Stimulation of the regeneration capacity of tree shoot segment explants in vitro.Biologia Plants,1990,32(6):407-413
[63]Chyi YanSan,R A Jorgensen,Goldstein D,et al.Location and stability of Agrobacterium-mediated T-DNA insertions in the Lycopersicon genome[J].Mol.Gen.Genet,1986,204:64-69
[64]Davis J M,keathley D E.Toward efficient clonal propagation of mature black locust tree vsing tissue cultrue[J].Nitrogea Fixing Tree Research Reports,1987,5:57-58.
[65]Davis J M,Keathley DE.Detection and analysis of T-DNA in crown gall tumors and kanamycin-resistant callus of Robinia Pseudoacacia.Can J.For.Tes,1989,19:1118-1123
[66]Deblaere R.Efficient Octopine Ti.plasmid derived vectors for Agrobaeterium mediated gene transfer to plant[J],Nucl Acid Res,1985,13:4777-4788
[67]Deguchi M,Gyokusen Kand Saito A.Propagation of plantlets by the culture of multiple shoots and roots induced from callus originating in the cambium of Robinia hispida L[J].J.Jpn.For.Soc.1994 (76):346-354.
[68] Hamilto CM, Frary A,Lewis C. Stable transfer of intact high molecular weight DNA into plant chromosomes [J].Proc Natl Acad Sci USA,1996 (93): 9975-9979
[69] Han K H, Keathley D E.Isolation and culture of protoplasts from callus tissue of black locust(Robinia pseudoacacia L.)[J]. Nitrogen Fixing Tree Research Reports,1988,6:68-70
[70] Han K H, Keathley D E,Gordon M P.Cambial tissue culture and subsequent shoot regeneration from mature black locust(Robinia pseudoacacia)[J]. Plant Cell Reports,1993,12(4):185-188.
[71] Han K H, Shin D I, Keathley D E. Tissue culture responses of explants taken from branch sources with different degrees of juvenility in mature black locust(Robinia pseudoacacia)trees[J]. Tree physiology,1997,17(10):671-675
[72] Holford P, Newburg H J. The effects of antibiotics and their breakdown products on the in vitro growth of antirrhinum majus[J]. Plant Cell Rept, 1992,11(2):93-94.
[73] Howe G T, Goldfarb B,Strauss S H. Agrobacterium-mediated transformation of hybrid poplar suspension cultures and regeneration of transformed plants[J]. Plant Cell Tissue Org Cult,1994,36: 59-71
[74] Huang FH, Li X Y 1994.Effects of concentration of aacetosyringone and Agrobacterium tunefaciens on on GUS gene transformation e ffieiency of PoPulus[J]. In Vitro, 30: 67
[75] Hu QJ,Han YF.A study on induction of plantlets from leaves of Robinia pseudoacacia[J].Hereditas,1985, 7 (4): 20-21
[76] Igasaki T, Mohri T, Iehikawa H, Shinohara K. Agrobacterium tumefociens mediated transformation of Robinia Pseudoacacia [JJ. Plant Cell ReP, 2000, 19:448-453
[77] James DJ, Passey A J, BabaraJ. Agrobacterium mediated transformation of the Cultivated stvawberry(Fragaria×Anannassa Duch.) using disarmed binary veetors[J].Plant sci, 1990, 69:79-94
[78] James A Bauml, Thierry Bogaert2, William Clintonl, Gregory R Heckl, Pascale Feldmann2, Oliver Ilaganl, Scott Johnson 1, Geert Plaetinck2, Tichafa Munyikwal, Michael Pleaul, Ty Vaughnl & James Robe. Control of coleopteran insect pests through RNA interference[J]. Nature Biotechnology, 25, 1322-1326 (2007)
[79] Jansson BJ , GardnerRC.Loealized transient expression of GUS in leaf disk following co-cultivation with Agrobacterium[J]. Plant Mol Biol, 1988,14:61-72
[80] Jong ho woo, Myung suk choi,Young goo park.Plant regeneration from callus culture of black locust (Robinia pseudoacacia.L).[J]. Journal of Korean Forestry Society, 1995,84(2):145-150.
[81] Kamlesh Kanwar, Sehgal RN, DeepakSood. Effect of explant type on the micropropagation of Robinia pseudoacacia [J]. Indian Journal of Forestry, 1995,18 1 :47-52
[82] Lin J J, Assadd-garcis N, Kou J.Plant horm one effort of antibiotics on the transformation efficiency of plant tissue by Agrobacterium tumefaciens cells. Plant Sci, 1995, 109: 171-177
[83] Luan Qing-shu, Luo Feng-xia. Shoot tissue culture of Robinia pseudoacacia f. decaisneana [J]. Journal of Forestry Research,2002,13(1) 51-55.
[84] Merkle S A, Wiecko A T. Regeneration of Robinia pseudoacacia somatic embryo genesis [J]. Canadian Journal of Forest Research, 1989,19(2): 285-288
[85] Miguel CM, Oliveira M M.1999. Transgenic almond. Prunus dulcis Mill. Plants obtained by Agrobaeterium mediated transtormation of leaf explants-Plant Cell Reports 18: 387-393
[86] Pang S Z, Sanford JC et al. Agrobacterium mediated genes transfer in Papaya. Hort Sci, 1988, 113:287-291
[87] Redei K, Osvath Bujtas Z. Clonal approaches to growing black loctst(Robinia pseudoacacia) in Hungary: a review. Forestry, 2002,75(5): 547-552.
[88] Mao YB, Cai WJ, Wang JW, Hong GJ, Tao XY, Wang LJ, Huang YP & Chen XY. Silencing a cotton bollworm P450 monooxygenase gene by plant-mediated RNAi impairs larval tolerance of gossypol. Nature Biotechnology 2007 Nov; 25(11): 1307-131