农杆菌介导法

1、农杆菌介导转化法农杆菌介导转化法 农杆菌是普遍存在于土壤中的一种革兰氏农杆菌是普遍存在于土壤中的一种革兰氏阴性阴性细菌，细菌，它能在自然条件下趋化性地感染大多数双子叶植物的受伤它能在自然条件下趋化性地感染大多数双子叶植物的受伤部位，并诱导产生部位，并诱导产生冠瘿瘤或发状根冠瘿瘤或发状根。农杆菌农杆菌感染柳树感染柳树产生产生冠瘿瘤冠瘿瘤原理：原理： 根癌农杆菌和发根农杆菌细胞中分别含有根癌农杆菌和发根农杆菌细胞中分别含有TiTi质粒和质粒和RiRi质粒质粒，其上有一段，其上有一段T-DNAT-DNA，农杆菌通过侵染植物伤口，农杆菌通过侵染植物伤口进入细胞后，可将进入细胞后，可将T-DNAT-DN

2、A插入到植物基因组中。插入到植物基因组中。 因此，农杆菌是一种因此，农杆菌是一种天然天然的植物遗传转化体系。人的植物遗传转化体系。人们将们将目的基因目的基因插入到经过改造的插入到经过改造的T-DNAT-DNA区，借助农杆菌的区，借助农杆菌的感染实现感染实现外源基因外源基因向植物细胞的转移与整合，然后通过向植物细胞的转移与整合，然后通过细胞和组织培养技术，再生出转基因植株。细胞和组织培养技术，再生出转基因植株。 农杆菌介导法起初只被用于双子叶植物中，近年来，农杆菌介导法起初只被用于双子叶植物中，近年来，农杆菌介导转化在一些单子叶植物（尤其是水稻）中也农杆菌介导转化在一些单子叶植物（尤其是水稻）中

3、也得到了广泛应用。得到了广泛应用。Story冠瘿瘤病冠瘿瘤病：双子叶植物经常发生，因肿瘤着生地面在近地面的根茎交界处，形似帽状而得名。1907年， Smith & Townsent 农杆菌诱发冠瘿瘤病。1947年， Braun et al. 证实俩者的关系，但发现有的菌株不 致病。提出了假说：tumour-inducing principle,TIP.肿瘤 诱导因子 。60s , 肿瘤组织中含高浓度的氨基酸（octopine , nopaline） 总称冠瘿碱（opine) 。 Petit et al.证实肿瘤组织 合成的冠瘿碱取决于菌株，而且菌株能专一地利用 冠瘿碱作为菌株生存的唯一

4、的碳源和氮源。(证实了TIP)1974年，Zaenen et al, Schell, Van Larebeke et al. 从致瘤农杆菌中分离出一类巨大的质粒 (tumor inducing plasmid)，称为Ti质粒。 Ti=TIP1977年，Chilton et al.分子杂交技术证实肿瘤细胞中存 在外源的DNA ,与Ti质粒的DNA有同源性，是整 合到了植物染色体的农杆菌质粒DNA片段， T- DNA (transferred DNA),其内有致瘤和冠瘿 碱合成酶等基因。1981年，Ooms et al.发现Ti质粒上有致瘤区(virulence region)，Vir区。Ti质粒

5、是根癌农杆菌细胞核外存在的一种环状双链DNA分子，长度约200kb，平均周长54.175 .4 um，分子质量为（90150）106 Da。在温度低于30的 条件下，Ti质粒可稳定地存在于根癌农杆菌细胞内。Ti质粒Ti质粒除上述上述诱导受侵染的植物组织产生冠瘿瘤 外，还具有以下几种重要功能：赋予根癌农杆菌附着于植物细胞的能力；赋予根癌农杆菌分解代谢冠瘿碱的能力；根癌农杆菌的寄主植物范围；决定所诱导的冠瘿形态和冠瘿碱的成分；参与寄主细胞合成植物激素吲哚乙酸和一些细胞分裂素 的代谢活.1) Ti质粒的结构来自于不同野生型根癌农杆菌的Ti质粒可根据其产生的冠瘿碱类型分为三类：章鱼碱(octopine

6、)类 胭脂碱(nopaline)类 农杆碱（agropine） 类。Ti质粒携带着既能分解又能合成这些化合物的酶类和相应基因，然而冠瘿碱合成基因却不能在根癌农杆菌中表达，它们只有进入植物细胞后才能表达，Ti质粒上的冠瘿碱分解基因产物却能分解冠瘿碱，为宿主细胞提供能源、氮源和碳源。长度：160-250 kb6大功能区：1)致癌区，这个区主要合成植物 生长素和细胞分裂素；2)冠瘿碱合成区；3)冠瘿碱分解区；4)Ti质粒接合转移区(tra)；5)毒性区（Vir）；6)DNA复制区（Rep）。在致癌区和冠瘿碱合成区的两侧存在着一个24 bp直接重复序列，由这三部分所构成的DNA区域叫做T-DNA，插入

7、植物染色体中的Ti质粒片段只有T-DNA。由于T-DNA插入植物细胞染色体中的位置不相同的，因此植物染色体上可能并没有可供T-DNA插入的专一性DNA序列。T-DNA脂碱型根癌农杆菌Ti质粒中T-DNA的左右两侧是一段24bp的重复序列,构成T-DNA的边界序列(border sequence),分别称为左边界(left border,LB)和右边界(right border,RB).在某些章鱼碱型根癌农根癌农杆菌Ti质粒中T-DNA是以两个分开的独立片段形式存在，即T-DNA左边区段和T-DNA右边区段。研究表明，插入在T-DNA边界序列之间的任何DNA都可被转到植物染色体中。因此Ti质粒可

8、用做外源目的基因的载体。T-DNA 区域中的这些基因只有在T-DNA插入到植物基因组后才能激活表达.植物生长素和细胞分裂素,可调节植物细胞的生长和发育,它们的过量表达刺激植物细胞大量快速增长而形成冠瘿。冠瘿碱也是在细胞内合并成分泌出来的,构成根癌农杆菌生长所须的碳源和氮源.Vir区（Vir-region），即毒性区，其长度约为35kb。它们控制根癌农杆菌附着于植物细胞和Ti质粒进入细胞有关部位，与感染后冠瘿形成有关。Vir区位于T-DNA区左侧，包含义个毒性遗传点（virA、vir、virC、 vir、vir和virG）。vir基因的控制着的转移。virvirvirC virG virvirA

9、Vir区区植物细胞受伤后，细胞壁破裂，分泌物中含有高浓度的创伤诱导分子（酚类化合物）：如乙酰丁酮（acetosyringone，AS）和-羟基酰丁香酮（-hydroxacetosyringone，OH-AS）。根癌农杆菌对这一类物质具有趋化性，在植物细胞表面附着后，受这些创伤诱导分子的刺激，Ti质粒vir区毒性基因被激活和表达。目前已经发现9种信号因子，均为水溶性酚类化合物。其中乙酰丁香酮（acetosyringone，AS）和羟基乙酰丁香酮（OH-AS）的作用较强，儿茶酚、原儿茶酚、没食子酸、焦性没食子酸、二羟基苯甲酸、香草酚和对羟基苯酚处理农杆菌时也对Vir区的基因表达起促进作用。双子叶植

10、物在在农杆菌侵染时可以形成大量的信号因子，而使T- DNA可以成功的转入；而单子叶植物需要加入外源酚类物质，才能激活Vir区的基因，达到转基因的目的。最先激活表达的是virA基因，它编码感受蛋白，位于细菌细胞膜的疏水区，可接受环境中的信号分子。在virA蛋白的激活下，virG基因表达，virG蛋白经磷酸化由非活性态变为活化状态，进而激活vir区其他基因表达。virA基因virG基因其中virD基因产物virD1蛋白是一种DNA松弛酶，它可使DNA从超螺旋型转变为松弛型状态；而virD2蛋白则能切割已呈松弛态的T-DNA，2个边界产生缺口，使单链T-DNA得以释放。VirE基因所表达的virE2

11、蛋白是单链T-DNA结合蛋白。可使T-DNA形成1个细长的核酸蛋白复合物（T-复合体），以此保护T-DNA不被包内外的核酸酶降解。最先激活表达的是virA基因，它编码感受蛋白，位于细菌细胞膜的疏水区，可接受环境中的信号分子。在virA蛋白的激活下，virG基因表达，virG蛋白经磷酸化由非活性态变为活化状态，进而激活vir区其他基因表达。其中virD基因产物virD1蛋白是一种DNA松弛酶，它可使DNA从超螺旋型转变为松弛型状态；而virD2蛋白则能切割已呈松弛态的T-DNA，2个边界产生缺口，使单链T-DNA得以释放。VirE基因所表达的virE2蛋白是单链T-DNA结合蛋白。可使T-DNA

12、形成1个细长的核酸蛋白复合物（T-复合体），以此保护T-DNA不被包内外的核酸酶降解。T-复合体依次穿过根癌农杆菌和植物细胞膜及细胞壁。并进入植物细胞核，最终整合进入植物核基因组。T-DNA的转移机理比较复杂，依赖于T-DNA区和vir区共同参与，涉及多个基因表达及一系列蛋白质和核酸的相互作用。Table1 Summary of vir Gene Products Locus Size(kb) ORFsa Proteins Size(kDa) Locationb FunctionvirA2.0190Mplant signal sensor, protein kinaseVirG1.0130Ct

13、ranscriptional activatorVirD4.5416,47,21,75C/M?T-DNA border endonuclease (VirD1 and VirD2); pilot protein?nuclear localization?(VirD2)VirC2.0226,23C?processing of T-DNA(VirC1)VirE2.027, 60.5C/M?single-strand DNA-binding protein (VirE2) virB9.51126,12,11,87,23,32,5.5,25,32,48,38MT-DNA transfer appara

14、tus?T-DNA加工和转移加工和转移 Vir基因表达调控的问题，知道基因表达调控的问题，知道VirA和和VirG基因诱导其它基因诱导其它Vir基基因的表达，当因的表达，当VirD操纵元的被诱导表达后，其中的操纵元的被诱导表达后，其中的VirD1和和VirD2蛋白质具有核酸内切酶的活性蛋白质具有核酸内切酶的活性(Yanofsky MF. 1986)，能够在能够在T-DN A边界重复序列的特异位点切开边界重复序列的特异位点切开T-DNA单链，因此单链，因此T-DNA往往以单链形式进入植物细胞。但是在植物细胞中也发往往以单链形式进入植物细胞。但是在植物细胞中也发现双链现双链T-DNA分子。分子。切

15、刻位点切刻位点(nick site)可作为可作为DNA从从5向向3合成的起始位合成的起始位点，新的点，新的DNA链合成后，链合成后，T-DNA单链即被替换单链即被替换(displacement)释放出来释放出来(图图2)。当然这种缺刻也可通。当然这种缺刻也可通过重组系统把过重组系统把T-DNA双链从双链从Ti质粒上解离下来。质粒上解离下来。缺失研究也表明：右边界重复序列缺失后，T-DNA不能转移，而左边界重复序列的缺失会稍稍降低T-DNA转移频率(Timmerman B1988)，这进一步说明T-DNA单链是在从右边界到左边界5向3替换合成中释放出来的。右边界的缺失，使得右边界的缺失，使得DN

16、A合成不能起始，因而合成不能起始，因而T-DNA不不能释放，所以能释放，所以T-DNA不能转移到植物细胞。而左边界的缺不能转移到植物细胞。而左边界的缺失，仅会影响失，仅会影响DNA合成过程的终止，可能会降低导致合成过程的终止，可能会降低导致T-DNA单链释放，而不会明显影响单链释放，而不会明显影响T-DNA单链的形成。单链的形成。冠瘿也是在冠瘿碱胞内合并成分泌出来的,被冠瘿碱分解基因分解成根癌农杆菌生长所须的碳源和氮源。冠瘿碱分解区Agrobacterium-mediated transformation Advantages:1. Cleaner inserts2. Higher co-ex

17、pression3. More flexible in tissue types(in planta transformation) almost any type of tissues or cells 4. Mostly widely used system ,both monocot and dicot5. Easy in manipulation ,Disadvantages 1. Only good for nuclear transformation2. Limited in host range,genotype-dependent3. Detrimental to some t

18、issuesIntroduction to AgrobacteriumMoloecular mechanisms of T-DNA transfer into the plant genome Factors influencing the T-DNA delivery Regeneration and selection of transgenic plantsTypesA.tumefaciens : the causative agent of plant disease crowng gall.A.rhizogenes : provokes hair roots. Both crown

19、gall and root hair are neoplastic diseases (plant tumors), both gram-negative soil bacterium. Based on the opines produced in the tumors,Ti plasmids are divided in four groups,while the Ri plasmids fall in three groups.Ti plasmidsu Octopine: pTiAch5,pTiAg57,or pTi19955 Nopaline: pTiC58,pTi1D135,pTi2

20、23 Leucinnopine: pTi542,pTiAT4D,L-succinamopine: pTiEU6,pTiAT181 Ri plasmidsu Mannopine: pRi8196,pRiTR7 Agropine: pRi1855,pRi15834,pRiA4 Cucumopine: pRi1659 Strains: Agrobacterium Strains and the origins of Agrobacterium strains(see attached table 1 and 2)refernce plasmid1:238-253(1978)Examples:C58

21、(C58 chromosome,pTi C58)ABI (C58 chromosome,pTiMp90RK,disarmed TiC58)GV3850 (C58 chromosome,pTiGV3850,disarmed TiC58)A281 (C58 chromosome,pTiBo542)EHA101 (C58 chromosome,pTiEHA101,disarmed pTiBo542 EHA105 (C58 chromosome,pTiEHA105,disardisarmed pTiBo542)T37 (T37 chromosome,pTi37)A208 (C58 chromosome

22、,Pti37)Ach5 (Ach5 chromosome,pTiAch5)LBA4404 (Ach5 chromosome,TiAL4404,disarmed pTiAch5) Strains: Agrobacterium Strains and the origins of Agrobacterium strains(see attached table 1 and 2)refernce plasmid1:238-253(1978)Examples:C58 (C58 chromosome,pTi C58)ABI (C58 chromosome,pTiMp90RK,disarmed TiC58

23、)GV3850 (C58 chromosome,pTiGV3850,disarmed TiC58)A281 (C58 chromosome,pTiBo542)EHA101 (C58 chromosome,pTiEHA101,disarmed pTiBo542 EHA105 (C58 chromosome,pTiEHA105,disardisarmed pTiBo542)T37 (T37 chromosome,pTi37)A208 (C58 chromosome,Pti37)Ach5 (Ach5 chromosome,pTiAch5)LBA4404 (Ach5 chromosome,TiAL44

24、04,disarmed pTiAch5)Molecular mechanisms of T-DNA transfer into plant genome Steps in T-DNA delivery1) host perception2) pre-infection attachment 3) induction and initiation of virulence genes 4) synthesis and assembly of the promiscuous pilus 5) processing and delivery of the T-DNA 6) T-DNA integra

25、tion1)host perception: chemical signals produced naturally by plant roots serve chemoattractants such as sucrose,glucose and fructose as well as amino acids like valine and arginine are good chemoattractants.plant isofolavomoids such as formoneton and coumestrol initiating the transcription of chrom

26、osomal genes of Agrobacterium involved in facilitating root colonization.most of the Vir genes except for genes of the virb and vire operons are required for Agrobacterium motility.2).pre-infection attachment a) Agrobacterium must attach to its host cells before the T-DNA is processed and readied fo

27、r transfer,following chemotaxis to the host plant .b) attachment is independent of Ti or Ri plasmid.c) A.tumefaciens adherence to plant cells is saturable .d) The attachment sites are thought to be specific receptors (2,000 such receptors on carrot suspension culture cells)where only live bacterium

28、binds.e) Each plant cell has enough attachment sites for several bacterlum,but plant cells are transformed by only one or a few bacterium .No loss of competence of plant cell causes by the firsrt agrobacterum infection.f) Chromosomal genes involved in attachment chva,chcb,exoc:synthesis of a cyclic

29、-1,2-glucan, implicated in plant cell binding. cel:cellulose synthesizing genes.agrobacterium cells forms large aggregates microscopically, seen as clumps tetheted by cellulose fibrils. pscA:exopolisaccharide systhesis and secretion leading to the systhesis of cellulose fibrils. att: root colonizati

30、on3) induction and initiation of virulence genes a)virA: autophosphorilating protein respond to the environmental signals by transferring a phosphate group to VirG protein.The induction of vir genes expression is sensitive to temperature(nopaline strain(t37,c58)octopine strains(ach5,lba4404)(see fig

31、ure)b)agrobacterium growth phase the best agrobacterium growth phase is mid to late log phase.c)Vir gene induction(agrobacterium induction medium) presence of phenolic compounds(5-200 umol); low temperature(15-25with a pick at 19); acidic medium(ph5.1-5.7); presence of monosaccharides(d-galaturinic

32、acid at 100u m,d-glucuronic acid 10mm,d-glucose10mm)phosphate starvation(from absence to0.1mm phosphate);often used media for bacterium growth and induction:mg/l or ab minimal(see the formula of mg/l and ab)d)mutant VirA or VirG (constitutive expresslon of VirA or VirG )highly increased expression o

33、f other vir gene, such as VirBAs, pH, as well as temperature dependency E)Multiple copy of VirG,VirD and VirB operons super-binary vector(see map) 2)Inoculation and co-culture conditionsa)enhancing attachmentssurfacts:pluronic acid,silwet(l77), soap et alb) Agrobacterium cell densityc)inoculation an

34、d co-culture time periodd)Temperatur 20 to 27e) inoculation and co-culture mediumf)feeder layers tobacco suspension cells g) vacuum infiltration microwoundimgb)Agrobacterium cell density in general, higher density increases T-DNA delivery ,but causes plant cell death .Depending on the explant type a

35、nd species. The Agrobacterium cell densities can be arranged from 10 7to 3x10 9. OD660 1=3x10 9 cells/mlexample: wheat immature embryos 0.51.0 rice embryogenic callus 1.01.5 corn immature embryos 1.0 soybean 0.30.6 cotton 0.30.6c)inoculation and co-culture time period depending on species, explants,

36、the oprimal inoculation time period varies.Examples: Tobacco leaf: second10min cotton hypocit: 13min peanut leaf: 10min soybean cotyledon mode : 1030min rice ECcs and IEs: 5min corn IEs: 5min wheat IEs and ECs: 60min Wheat suspension cells: 15minCo-culture time varies from 2 to 3 days, usually 3days

37、e)inoculation and co-culture medium in general, inoculation and co-culture medium are the same.Since Agrobactereium vir induction requires minimal salts, try lower salts such as 1/5x or 1/10 x; inoculation and co-culture medium should contain induction chemicals such as AS and sugar; auxins such as

38、2,4-D and picloram enhance T-DNA delivery ,and cytokinins such as BA and TDZ inhibit the T-DNA delivery.3)explants and pretreatment of explantsa)types of explants dicot species: more explants available for infection ,such as leaf, cotyledon, stems, seed explants, meristem, mature or immature embryio

39、s, embtyogenic callus et al. monocot species: less explants for infection .the main explants are immature embryos or the callus derived from. most regenerable explants are usually used for transformation; Avoid the explants are sensitive to Agrobacterium infection, such as microspores and protoplast

40、 cells transformation competence varies among explants intre/intra apecies.Example:intra-species: rice preculture immature embryos and embryogenic callus inter-species: embryogenic callus between rice,corn and wheat.b)physiology conditions of the explants wounding vs no wounding almost all dicot exp

41、lants requires wounding ,while monocot explants dont need wounding before during inocuationsonication-mediated transformation bombardment before infection freshly prepared explants usually more competent than the precultured explants. Including auxins such as 2,4-d and picloram in the preculture med

42、ium enhances T-DNA delivery. Avoiding cytokinins, especially TDZ in the pre-culture medium. DNA-demethylation:5-azacytidine. c)pretreatments: Desiccation:air dry of embryogenic callus of sugarcane. Osmotic treatment:corn freshly isolated immature embryos. Cold vs warm treatments:sometimes,it works!

43、4)efficiency of T-DNA delivery and plant cell recoveryOnce sufficient T-DNA delivery is achieved with an explant, the conditions favor the plant cell recovery and subsequenrly cell eivision and development are crucial for generating stable transgebic plants. An efficient T-DNA delivery system is prerequisite for an efficient stable transformation ayatem in most of the circumstances. Agrobacterium is always toxic to plant cells. a)strategies for enhancing the plant cell recovery i