Zinc finger nuclease and homing endonucleasae-mediated assembly of multigene plant transformation

Breakthrough Technologies

Zinc Finger Nuclease and Homing

Endonuclease-Mediated Assembly of Multigene

Plant Transformation Vectors1[OA]

Vardit Zeevi2,Zhuobin Liang2,Uri Arieli,and Tzvi Tz?ra*

Department of Molecular,Cellular,and Developmental Biology,University of Michigan,Ann Arbor,Michigan 48109(V.Z.,Z.L.,T.T.);and Department of Life Sciences,Ben-Gurion University of the Negev,Beer Sheva 84105,Israel(U.A.,T.T.)

Binary vectors are an indispensable component of modern Agrobacterium tumefaciens-mediated plant genetic transformation systems.A remarkable variety of binary plasmids have been developed to support the cloning and transfer of foreign genes into plant cells.The majority of these systems,however,are limited to the cloning and transfer of just a single gene of interest. Thus,plant biologists and biotechnologists face a major obstacle when planning the introduction of multigene traits into transgenic plants.Here,we describe the assembly of multitransgene binary vectors by using a combination of engineered zinc ?nger nucleases(ZFNs)and homing endonucleases.Our system is composed of a modi?ed binary vector that has been engineered to carry an array of unique recognition sites for ZFNs and homing endonucleases and a family of modular satellite vectors.By combining the use of designed ZFNs and commercial restriction enzymes,multiple plant expression cassettes were sequentially cloned into the acceptor binary https://www.sodocs.net/doc/623827917.html,ing this system,we produced binary vectors that carried up to nine genes.Arabidopsis(Arabidopsis thaliana)protoplasts and plants were transiently and stably transformed,respectively,by several multigene constructs,and the expression of the transformed genes was monitored across several generations.Because ZFNs can potentially be engineered to digest a wide variety of target sequences,our system allows overcoming the problem of the very limited number of commercial homing endonucleases.Thus,users of our system can enjoy a rich resource of plasmids that can be easily adapted to their various needs,and since our cloning system is based on ZFN and homing endonucleases,it may be possible to reconstruct other types of binary vectors and adapt our vectors for cloning on multigene vector systems in various binary plasmids.

To date,biotechnological improvement of plant species has largely been limited to the introduction of single novel traits into the genomes of target plants. However,many agronomic traits may depend on complex interactions between several proteins,and biotechnological improvement of a particular species may thus require the delivery and expression of whole,complex metabolic pathways(Halpin et al., 2001;Daniell and Dhingra,2002;Lyznik and Dress, 2008;Naqvi et al.,2010).In addition,transgenic mod-i?cation of commercially important plant species also calls for the development of novel tools for the re-moval,addition,and replacement of existing trans-genes in plant cells(Hanin and Paszkowski,2003; Porteus,2009;Moon et al.,2010;Weinthal et al.,2010). Thus,two of the major challenges that are still to be addressed in plant biotechnology are the development of a technology to combine several transgenic traits in a single plant by stacking a number of genes in the same chromosomal locus into a single multigene array and the successive manipulation of this array by genome editing(Dafny-Yelin and Tz?ra,2007;Lyznik and Dress,2008;Taverniers et al.,2008;Naqvi et al.,2010). The main strategies for the introduction of multiple genes into plant cells include retransformation,co-transformation,sexual crossing,and transformation of multigene constructs(for review,see Dafny-Yelin and Tz?ra,2007;Naqvi et al.,2010).While proven useful for the production of transgenic plants with novel traits,re-transformation,cotransformation,and sexual crossing approaches all suffer from several?aws(Dafny-Yelin and Tz?ra,2007;Naqvi et al.,2010).Retransformation and sexual crossing,for example,are time consuming and rely on the use of different selectable marker genes for each transformation/crossing cycle.In cotransfor-mation,it is virtually impossible to predict the number of insertions and the distribution of the inserted genes across the plant genome.In addition,cotransformation may result in complex integration patterns that may hinder the use of such plants for commercial purposes, for which single and well-characterized integration events are required.While sexual crosses may be sim-pli?ed by using marker-free transgenic plants,the deliv-ery of multiple genes as a single,well-de?ned,multigene

1This work was supported by the United States-Israel Binational Agricultural Research and Development Fund(grant https://www.sodocs.net/doc/623827917.html,–4150–08to T.T.).

2These authors contributed equally to the article.

*Corresponding author;e-mail tz?ra@bgu.ac.il.

The author responsible for distribution of materials integral to the ?ndings presented in this article in accordance with the policy described in the Instructions for Authors(https://www.sodocs.net/doc/623827917.html,)is: Tzvi Tz?ra(tz?ra@bgu.ac.il).

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https://www.sodocs.net/doc/623827917.html,/cgi/doi/10.1104/pp.111.184374

array would perhaps be the simplest and most reliable method for the production of multigene transgenic plants.Furthermore,multigene arrays may also offer the advantage of simplifying successive manipulations of multigene assays in transgenic plants by genome-editing technologies(Porteus,2009;Weinthal et al., 2010).Yet,while multigene constructs have been suc-cessfully used in several studies(Bohmert et al.,2000, 2002;Wu et al.,2005;Zhong et al.,2007;Fujisawa et al., 2009),the assembly of multigene constructs remains chal-lenging,being nearly impossible to achieve by tradi-tional cloning methods(Dafny-Yelin and Tz?ra,2007; Naqvi et al.,2010).

Only a handful of dedicated vector assembly systems have been developed in the past several years for the assembly of multigene transformation vectors(Cheo et al.,2004;Sasaki et al.,2004;Karimi et al.,2005;Chen et al.,2006,2010;Wakasa et al.,2006).Most of these vector assembly systems have a rather limited capacity and hence have been utilized for the delivery of only a small number(i.e.up to?ve)of transgenes in a single array.Noteworthy here is the assembly system of Lin et al.(2003),in which a combination of the Cre/loxP recombination system and two homing endonucleases is used for successive cloning of a potentially in?nite number of genes onto a transformation-competent arti-?cial chromosome-based vector.Lin et al.(2003)used their system for the delivery of eight different genes from two independent T-DNA molecules that were launched from a single binary vector into the rice(Oryza sativa)genome.However,a crucial limitation of the ingenious approach of Lin et al.(2003)was that,in contradiction to classical cloning by restriction enzymes, once assembled into the binary vector,new DNA frag-ments can no longer be removed or replaced by others. Another important contribution to the?eld is the unique multiple-round in vivo site-speci?c assembly system of Chen et al.(2010)that enables in vivo recombination-based assembly of multigene vectors. The system was used to construct several multitrans-gene binary vectors and led to the production of transgenic plants with eight transgenes(Chen et al., 2010).A number of other recombination-based cloning strategies have also been developed for the construc-tion of multigene plant transformation vectors(Cheo et al.,2004;Sasaki et al.,2004;Karimi et al.,2005;Chen et al.,2006,2010;Wakasa et al.,2006).However,here too,the irreversible nature of the recombination-based reactions does not enable the modi?cation of existing binary vectors,and users of such systems may be required to completely rebuild their transformation vectors to give different gene combinations.Multigene transformation vectors have also been assembled by using both traditional cloning methods.Wu et al. (2005),for example,combined the use of type II restriction enzymes and Gateway-mediated cloning to construct several binary vectors with up to10 different expression cassettes and used the combina-tion to reconstitute the docosahexaenoic acid biosyn-thetic pathway in Indian mustard(Brassica juncea).Fujisawa et al.(2009)combined the use of type II restriction enzymes and homing endonucleases to construct a seven-transgene-long T-DNA molecule, which they applied to genetically modify the carote-noid biosynthesis pathway in Brassica napus.Other examples include the engineering of soybean(Glycine max),potato(Solanum tuberosum),rice,Arabidopsis (Arabidopsis thaliana),and several other plant species by using four-to?ve-transgene-long transformation vectors(for review,see Naqvi et al.,2010;Peremarti et al.,2010).While proven successful for the metabolic engineering of various plant species,such vectors were mostly custom designed to their speci?c tasks, and their modi?cation for other metabolic pathways and multigene traits may prove to be dif?cult or even impossible to achieve.

A versatile and modular system for the assembly of multigene binary vectors has been developed by Goderis et al.(2002),who exploited a set of homing endonucle-ases to construct a vector system facilitating the succes-sive cloning of independent plant expression cassettes. The principles of the assembly system and successor satellite(pSAT)vector system were previously repor-ted by Tz?ra et al.(2005).An important advantage of this method,over the approaches described above,is its modularity.Plant expression cassettes can easily be removed or replaced from existing binary vectors. Nevertheless,the capacity of the vector system and its successors is limited by the very small number of commercially available homing endonucleases.

We have recently shown that zinc?nger nucleases (ZFNs),engineered restriction enzymes that can be designed to bind and cleave long stretches of DNA sequences(Mani et al.,2005),can be used for molec-ular cloning,and we have used such enzymes for the construction of dual-gene binary vectors(Zeevi et al., 2008).In this paper,we describe a modular binary vector assembly system that represents a fundamental improvement over the original design of Goderis et al. (2002)in that it supports the construction of multigene transformation vectors not only by homing endonu-cleases but also by designed ZFNs.We describe the design of our system and demonstrate its use by cloning nine different DNA fragments onto a modi?ed binary transformation vector.We also show that such vectors can be used for the production of multigene transgenic plants and for the transient expression of multiple genes in plant protoplasts.The advantage of using ZFNs for the construction of multigene trans-formation systems is discussed.

RESULTS

Design of the Binary Vector and Novel pSATs

We followed the basic design of the pAUX(Goderis et al.,2002)and its successor pSAT(Chung et al.,2005; Tz?ra et al.,2005;Dafny-Yelin et al.,2007;Dafny-Yelin and Tz?ra,2007)family of plasmids to facilitate the

Multigene Binary Vectors

assembly of multiple genes into a single binary plas-mid.In the pSAT system,functional plant expression cassettes are individually cloned into different pSAT plasmids(e.g.gene B in pSAT2;Fig.1A),and an expression cassette from each type of pSAT plasmid can be cloned onto pPZP-RCS1-or pPZP-RCS2-based (Goderis et al.,2002)Agrobacterium tumefaciens binary plasmids by using matching homing endonucleases (Fig.1A).As was the case for the original set of pAUX plasmids,the original set of pSAT plasmids was com-posed of seven different versions,in which the expres-sion cassettes were?anked by Asc I(pSAT1),Asc I and I-Ppo I(pSAT2),I-Ppo I(pSAT3),I-Sce I(pSAT4),I-Ceu I (pSAT5),PI-Psp I(pSAT6),and PI-Tli I(pSAT7).Here,we subsequently expanded this set to include pSAT10, pSAT11,and pSAT12,in which the expression cassettes were?anked by ZFN10,ZFN11,and ZFN12(333?nger ZFNs),respectively(e.g.gene A in pSAT12;Fig. 1A).We also constructed pRCS11.1,which is a modi?-cation of pPZP-RCS1that was engineered to include,in addition to the original recognition sites of Asc I,I-Ppo I, I-Sce I,I-Ceu I,PI-Psp I,and PI-Tli I,recognition sites to

the Figure1.General features of the ZFN-and homing endonuclease-mediated multigene binary vector assembly system.A,The

general structure of a typical pSAT vector is exempli?ed by pSAT6,in which the promoter is?anked by the unique Age I and Nco I

sites,the terminator is?anked by the unique Xba I and Not I sites,the gene of interest is cloned into an extended MCS,and the

entire plant expression cassette is?anked by recognition sites for ZFNs(e.g.ZFN12in pSAT12)or homing endonuclease(e.g.

PI-Psp I in pSAT6).The ZFN and homing endonuclease recognition sites are shown on the binary https://www.sodocs.net/doc/623827917.html,ing ZFNs and

homing endonucleases,up to nine expression cassettes can be transferred from the pSAT vectors into the T-DNA region of

pRCS11.1.LB,Left border;P,promoter sequence;RB,right border;T,terminator sequence.B,Structure and scale of the pRCS11.1

[1.HYG][2.N][3.AMP][4.GUS][5.BAR][6.PAP1][10.EYFP-CHS][11.DsRed2-P][12.ChrD-RFP]nine-transgene-long plant transfor-

mation binary vector.The general structure and direction of each expression cassette is shown.Promoters and terminators are as

follow:35S,double CaMV35S;Rbcs,Rubisco small subunit;ocs,octopine synthase;hs,hsp18.1heat shock.Proteins are as

follow:DsRed2-P,P protein of SYNV fused to DsRed2;ChrD-RFP,chromoplast-speci?c carotenoid-associated protein ChrD fused

to RFP;EYFP-CHS,chalcone synthase fused to EYFP;PAP1,Arabidopsis transcription factor production of anthocyanin pigment1;

BAR,Basta resistance-encoding gene;AMP,ampicillin resistance;N,N protein of SYNV;HYG,hygromycin resistance.

Zeevi et al.

ZFN10,ZFN11,and ZFN12(Fig.1A).This system was subsequently used for the construction of several multi-gene binary plasmids,as described below.

Construction of Multigene Vectors by Exploiting ZFNs We constructed a set of pSAT plasmids carrying a wide variety of genes.Table I lists the different pSAT plasmids constructed,the genes they carry,and the enzymes used for their cloning into pRCS11.1.We started by cloning the constitutive expression cassette of the endoplasmic reticulum-bound chalcone syn-thase(CHS)gene(Pelletier and Shirley,1996)tagged with enhanced yellow?uorescent protein(EYFP) from pSAT10.EYFP-CHS by using ZFN10to produce pRCS11[10.EYFP-CHS].Next,we added the constitu-tive expression cassette of the phospho(P)protein of Sonchus yellow net rhabdovirus(SYNV;Goodin et al., 2001,2002)tagged with DsRed2from pSAT11.DsRed2-P by using ZFN11,to produce pRCS11.1[10.EYFP-CHS] [11.DsRed2-P].

The key to the versatility of the original pSAT/pRCS2 multigene assembly system lies in the ability it offers not only to add but also to remove and replace DNA fragments by homing endonucleases during the con-struction of the various binary plasmids(Tz?ra et al., 2005).Thereafter,we demonstrated that ZFNs too can also be used to remove and replace DNA fragments from existing binary constructs.We started by adding the plasmid backbone from pSAT12.MCS into pRCS11.1 [10.EYFP-CHS][11.DsRed2-P]and produced pRCS11.1 [10.EYFP-CHS][11.DsRed2-P][12.AMP].As expected, the resultant plasmid conferred resistance to ampicillin in Escherichia coli cells.We also observed that a DNA preparation of this plasmid from an overnight E.coli culture resulted in much higher DNA yield than that obtained with its progenitor or with other pRCS2-based vectors,most probably due to the presence of the ColE1 origin of replication on the pSAT12.MCS backbone.We then used ZFN12to remove the pSAT12.MCS backbone and replaced it with a constitutive expression cassette of the chromoplast-speci?c carotenoid-associated protein ChrD from Cucumis sativus(Vishnevetsky et al.,1996, 1999)tagged with red?uorescent protein(RFP)from pSAT12.ChrD-RFP to produce pRCS11.1[10.EYFP-CHS] [11.DsRed2-P][12.ChrD-RFP].As expected,this plas-mid no longer conferred resistance to ampicillin in E.coli cells.We tested the expression of the three reporter genes from pRCS11.1[10.EYFP-CHS][11.DsRed2-P][12. ChrD-RFP]in Arabidopsis protoplasts.As expected, the EYFP-tagged CHS associated with the rough endo-plasmic reticulum throughout the cell,around the pro-toplasts,and around the nucleus(Fig.2A);DsRed2-P was observed in the cytoplasm clustered around the chloroplasts;and ChrD-RFP,which has been character-ized previously as a chloroplast-associated protein(Ben Zvi et al.,2008),indeed localized in the chloroplasts (Fig.2B).The clustering of DsRed2-P was probably due to the tendency of DsRed2to aggregate in living cells. We next added a fourth plant expression cassette for the SYNV nucleocapsid(N)protein and produced pRCS11.1[2.N][10.EYFP-CHS][11.DsRed2-P][12.ChrD-RFP].Interaction of the SYNV N protein with the SYNV P protein is required to translocate it into subnuclear compartments in Nicotiana benthamiana cells(Goodin et al.,2001,2002).Indeed,nuclear localization and subnuclear compartmenting of the DsRed2-P signal was observed in pRCS11.1[2.N][10.EYFP-CHS][11. DsRed2-P][12.ChrD-RFP]-infected protoplasts(Fig.2, F and K)but not in pRCS11.1[10.EYFP-CHS][11. DsRed2-P][12.ChrD-RFP]-infected protoplasts(Fig.2, B and I).Our data thus show that designed ZFNs can be used for the construction of multigene vectors and that such vectors can be used to drive the simultaneous expression of several genes in plant cells.

Assembly of Multigene Binary Vectors for

Stable Transformation

pRCS11.1[2.N][10.EYFP-CHS][11.DsRed2-P][12.ChrD-RFP]carried four pairs of repetitive elements(i.e.cau-li?ower mosaic virus[CaMV]dual35S promoters and CaMV35S terminators).We next tested whether the four transgenes and two additional genes(i.e.a hygromycin resistance gene driven by the control of the octopine synthase promoter and terminator and the Basta resis-tance gene driven by the control of the Rubisco small subunit promoter and terminator)can be stably trans-formed and expressed in transgenic plants.Many com-monly used binary vectors have been designed with the selection marker cassette cloned next to the T-DNA’s left border(van Engelen et al.,1995;Hellens et al.,2000).We elected to clone the hygromycin-resistant expression cassette near the T-DNA’s right border.This design,

Table I.Genes,vectors,and enzymes used for the construction of multigene vectors,in order of their assembly in pRCS11.1 pSAT Protein Enzyme(s)Phenotype

10EYFP-CHS ZFN10Decoration of endoplasmic reticulum by yellow?uorescence 11DsRed2-P ZFN11Decoration of the cytoplasm and nucleus by red?uorescence 12ChrD-RFP ZFN12Decoration of the chloroplasts by red?uorescence

2N Asc I-I-Ppo I Directing DsRed2-P into subnuclear compartments

1HYG Asc I Hygromycin resistance

3AMP I-Ppo I Bacterial ampicillin resistance gene and origin of replication 5BAR I-Ceu I Basta resistance

4GUS I-Sce I Heat shock-induced GUS expression

6PAP1PI-Psp I Activation of anthocyanin expression

Multigene Binary Vectors

while having the potential to produce transgenic plants with truncated T-DNAs,allowed us to examine whether repetitive elements have a negative impact on the T-DNA structure during transformation.To achieve the above design,we ?rst produced pRCS11.1[1.HYG][2.N][10.EYFP-CHS][11.DsRed2-P][12.ChrD-RFP],in which the hygromycin-resistance gene expression cassette was cloned into the Asc I site of pRCS11.1[3.N][10.EYFP-CHS][11.DsRed2-P][12.ChrD-RFP].We then added the plasmid backbone from pSAT3.MCS into the I-Ppo I site to facilitate future cloning and subsequent isolation of T-DNA-plant junction sequences by plasmid rescue and the Basta hygromycin-resistant expression cassette into the I-Ceu I site of pRCS11.1[3.N][10.EYFP-CHS][11.DsRed2-P][12.ChrD-RFP].Finally ,we used the seven-transgene-long pRCS11.1[1.HYG][2.N][3.AMP][5.BAR][10.EYFP-CHS][11.DsRed2-P][12.ChrD-RFP]binary plasmid (Fig.3A)to produce transgenic Arabidopsis plants.Confocal microscopy analysis revealed that nine of our 27transgenic lines exhibited yellow and red ?uo-rescence expression patterns similar to the patterns of protoplasts infected with pRCS11.1[2.N][10.EYFP-CHS][11.DsRed2-P][12.ChrD-RFP](data not shown).Molec-ular analysis of two randomly selected transgenic lines revealed that PCR ampli?cation of their T-DNA regions was similar to that of the seven-transgene-long binary vector (Fig.3A);these ?ndings indicate that our transgenic lines carried all the T-DNA mol-ecule-encoded transgenes within their genome.

We then allowed several randomly selected trans-genic lines to mature and set seed.Shown in Figure 4are examples of hygromycin-and Basta-resistant seedlings.Collectively,our data indicate that the T-DNA-encoded seven transgenes were stably expressed and inherited.

Production of Nine-Transgene-Long Transgenic Plants

We constructed a nine-transgene-long binary vector by adding the GUS and PAP1(for Arabidopsis transcription factor production of anthocyanin pigment;Borevitz et al.,2000;Ben Zvi et al.,2008)expression cassettes into the I-Sce I and PI-Psp I sites of pRCS11.1[1.HYG][2.N][3.AMP][5.BAR][10.EYFP-CHS][11.DsRed2-P][12.ChrD-RFP].In the ?nal binary vector,pRCS11.1[1.HYG][2.N][3.AMP][4.GUS][5.BAR][6.PAP1][10.EYFP-CHS][11.DsRed2-P][12.ChrD-RFP],GUS expression is controlled by the hsp18.1heat shock promoter (Takahashi and Komeda,1989),while PAP1expression is controlled by the CaMV dual 35S promoter.We used this vector to produce over 30hygromycin-resistant transgenic Arab-idopsis plants,of which about half exhibited PAP1-related reddish hypocotyls and cotyledons during germination.We further characterized these lines and determined whether they are likely to harbor the full T-DNA molecule by monitoring DsRed2-P expres-sion,which was located next to the left border.Twelve T0plants of the PAP1transgenic plants also expressed DsRed2-P ,which indicated that they are likely to harbor at least one full T-DNA copy.Molecular anal-ysis of several lines revealed that PCR ampli?cation of several regions on their T-DNA was similar to that of the nine-transgene-long binary vector,as demon-strated,for example,in Figure 3B.These observations indicate that the plants carried the entire T-DNA molecule-encoded genes.

We allowed seven of the transgenic lines to mature and set seed and determined the inheritance and sta-bility of the T-DNA in the next generations.T0plants of lines L6,L7,and L10exhibit a plausible

single-insert

Figure 2.Multiple gene expression in protoplasts from triple-and quadruple-gene long binary vectors.EYFP-CHS expression was targeted to the rough endoplasmic reticulum (shown in yel-low in A and E),while ChrD-RFP was targeted to the chloroplasts (shown in red in B,F,I,and K)and overlapped with the chloroplast auto?uorescence (shown in blue in C,G,J,and L).DsRed2-P was targeted to the cyto-plasm (shown in red in B and I)and was redirected into the nucleus in the presence of free N protein to form subnuclear aggregates (shown in red in F and K).D and H present merged signals of EYFP ,DsRed2,RFP ,and plastid auto?uorescence.I and J are magni?cations of B and C;K and L are magni?cations of F and G.All panels are single confocal sections.

Zeevi et al.

segregation pattern when grown on a hygromycin-containing medium (Table II).T1seedlings of lines L2,L6,L7,and L10,T2seedlings that derived from homo-zygous (i.e.L6-1)and heterozygous (i.e.L6-2and L10-2)parents,exhibited resistance to Basta,as did T2seed-lings derived from lines L2and L3(Table II;Fig.5A).Confocal microscopy analysis revealed that all T0lines and their progeny (Table II)exhibited red and yellow ?uorescence,as shown,for example,for line L6-1(Fig.5,C–F).Analysis of several T2lines revealed that they all expressed heat shock-induced GUS expression (Fig.5G).GUS expression was not observed in untreated plants.In addition,all T1,T2,and T3hygromycin-resistant plants exhibited PAP1expression (Table I),as shown,for example,for L3-3-1(Fig.5B)and L6-2-1-1(Fig.5F)plants.The segregation analyses suggest that a single transgene locus was obtained in some of the analyzed lines (i.e.L6,L7,and L10).

We further analyzed four transgenic lines to dem-onstrate the stability and integrity of the T-DNA inserts in T3(lines L2and L10)and T4(lines L6and L7)hygromycin-resistant plants by PCR ampli?cation of overlapping fragments of their T-DNA region (Fig.6).When combined with segregation analysis,our molecular data further supported the notion that our transgenic plants carry full T-DNA inserts.No

less

Figure 3.PCR analysis of multigene binary T-DNAs in transgenic plants.A,PCR analysis of seven-transgene-long T-DNA inserts in a binary plasmid (P)and two transgenic plants (L1and L2).B,PCR analysis of nine-transgene-long T-DNA inserts in a transgenic plant (L6)and in a binary plasmid (P).The gen-eral structure and the expected sizes (in kb)of the PCR bands are shown.Abbreviations are as in Figure

1.

Figure 4.Phenotypic analysis of seven-transgene-long transgenic Arabidopsis plants.A,Hygromycin resistance in two independent transgenic T1Arab-idopsis plants (L1and L2).w.t.,Wild-type plants.B,Basta resistance in a hygromycin-resistant trans-genic Arabidopsis line (left).Wild-type plants are shown on the right.

Multigene Binary Vectors

important,our data clearly show that multigene-expressing plants can be produced using our multi-gene transformation vectors.

DISCUSSION

We demonstrated that by using engineered ZFNs, we can overcome the limitation imposed by the small number of commercial homing endonucleases to the construction of multigene binary vectors.We used several ZFN-and homing endonuclease-constructed binary vectors for transient and stable genetic transfor-mation of plant cells.We then showed that transgenic plants that had been stably transformed by nine-transgene-long T-DNA stably expressed the cloned genes across several generations.To the best of our knowledge,our report is one of just a small number of studies in which a very large number of independently expressed genes were delivered using a single T-DNA molecule into plant cells and,moreover,is the only

Table II.Analysis of transgenic Arabidopsis plants across several generations

%%% L1+c+100ND d

L1-1+++++ND

L2++100+

L2-1+++++100+

L2-1-1++

L2-1-2++

L2-1-3++

L2-2+++++100+L2-2-1++

L3++100ND

L3-1+++++100

L3-3+++++100+L3-3-1+ND

L6++75+

L6-1+++++100+

L6-1-1++100

L6-1-1-1++

L6-1-2++ND

L6-1-3++ND

+L6-2+++++75+

L6-2-1++75

L6-2-1-1++

L6-2-2++100

L6-2-2-1++ L7++75+

L7-1+++++75ND

L7-1-1+ND75

L7-1-1-1++

L7-1-2+ND75

L7-1-3+ND100

+L7-2+++++75ND

L7-2-1+ND

L7-2-2+ND75 L8++100ND L8-1+++++ND

L10++75+

L10-1+++++100ND

L10-1-1+ND

L10-1-2+ND

L10-2+++++

75+

L10-2-1++

L10-2-2++

L10-3+++++ND

L10-4+++++ND

L10-5+++++ND

a Percentage of hygromycin-resistant plantlets growing on hygromycin-containing germination medium.

b Several hygromycin-resistant plantlets were transferred to soil and sprayed with Basta.c+,Positive phenotype.d ND,Not determined.

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report in which a multigene vector has been assembled by a modular,step-by-step construction method (for review,see Naqvi et al.,2010).Thus,our approach represents an important technical leap in the construc-tion of multigene plant transformation vectors.

There are three main advantages of our system over other multigene construction systems,as detailed be-low.First,we demonstrated that ZFNs can be used in a manner similar to the technique that relies on homing endonucleases and that ZFNs can be exploited not only for adding but also for replacing DNA fragments from existing multigene binary vectors.Thus,in con-trast to many other multigene vector assembly systems (Lin et al.,2003;Karimi et al.,2005;Wakasa et al.,2006;Chen et al.,2010),our system has the dual advantage of being modular and of enabling the modi?cation of existing plant expression cassettes at any stage during the construction of the transformation vector.In addi-tion,the cloning capacity of existing pRCS11.1-based binary vectors can be increased by reengineering their multiple cloning sites (MCSs)for additional features (which may include,for example,recognition sites for new ZFNs and homing endonucleases or even sites for Cre/lox and Gateway recombination systems)not only prior to but also during the process of assembling multigene vectors.This can be achieved by cloning an expanded ZFN-recognition MCS into the growing pRCS11.1-based binary vectors,which can then be used for successive additions of new expression cas-settes.This feature further adds to the modularity of our ZFN-based vector assembly system,as compared with the more rigid design of other binary vector assembly systems.

Second,because ZFNs can be designed to target and digest an extremely large number of target sequences (Maeder et al.,2008),novel ZFNs can be developed and used to expand our nine-gene-long system beyond its current capacity .Since most pSAT vectors share a similar basic structure (Tz?ra et al.,2005),it is easy to convert existing pSAT vectors into novel vectors by creating pSAT backbones with new ZFN recognition sites.Furthermore,since we used semipalindromic ZFN recognition target sequences,it may be feasible to adapt existing ZFNs (i.e.those that have been devel-oped for targeting experiments in various nonplant eukaryotic cells [Urnov et al.,2005;Carroll et al.,2008;Zimmerman et al.,2008;Geurts et al.,2009;Takasu et al.,2010])for cloning purposes.Indeed,we have previously shown that ZFNs can be expressed and puri?ed to the level of molecular biology reagents by using relatively simple expression and puri?cation steps (Zeevi et al.,2010;Tovkach et al.,2011).While speci?c modi?cations may be required to adapt our expression and puri?cation system for novel ZFNs,the simplicity of the process and the availability of dedi-cated ZFN assembly and expression vectors for bacte-rial expression and for plant genome editing (Tovkach et al.,2010;Zeevi et al.,2010)may further facilitate

the

Figure 5.Phenotypic analysis of nine-transgene-long transgenic Arabidopsis plants.A,Basta resistance in three independent transgenic lines.Clock-wise from bottom left:offspring of T0L10plants,offspring of T1L6-1plants,offspring of T1L7plants,and wild-type plants.B,PAP1phenotype in offspring of T2L3-3-1plants (right).Wild-type plants are shown on the left.C to F,EYFP-CHS expression (in yellow),ChrD-RFP and DsRed2-P expression (in red),plastid auto?uorescence (in blue),and merged signals in leaf of T1L6-1plants.All panels are single con-focal sections.G,Heat shock-induced GUS expression in transgenic leaves.Clockwise from top left:samples from L2-1-1,L2-2-1,L6-1-1,L10-2-1,L10-1-1,and L6-2-2.H,PAP1phenotype in seedlings of offspring of T2L6-2-2plants (left).Seedlings of wild-type plants are shown on the right.

Multigene Binary Vectors

use of our system for the assembly of multigene vector systems.

Third,our system supports the use of a very large family of pSAT-and pAUX-based plasmids.The pSAT family of plasmids is composed of plasmids that have been designed to facilitate (1)the overexpression of target genes under the control of various promoters and terminators (Chung et al.,2005;Tz?ra et al.,2005);(2)the analysis of protein-protein interactions by using the bimolecular ?uorescence complementation and multicolor bimolecular ?uorescence complementation assays (Citovsky et al.,2006;Lee et al.,2008);(3)the fusion of target genes to various auto?uorescence proteins (Tz?ra et al.,2005);(4)RNA interference-mediated down-regulation (Dafny-Yelin et al.,2007);(5)the expression of ZFNs (Tovkach et al.,2009,2010);(6)Gateway-mediated gene cloning (Tz?ra et al.,2005;Chakrabarty et al.,2007);and (7)the expression of epitope-tagged proteins (T.Tz?ra,unpublished data).Thus,users of our system can enjoy a rich resource of plasmids that can be easily adapted to their various

needs.Furthermore,since our cloning system is based on ZFN and homing endonucleases,it may be simple to transfer,or reconstruct,the ZFN and homing endo-nuclease MCS from pRCS11.1into any other type of binary vectors and adapt our pSAT set of vectors for cloning on multigene vector systems in various binary plasmids.Such binary plasmids can,for example,be based on the transformation-competent arti?cial chro-mosome (Liu et al.,1999;Lin et al.,2003)and BIBAC (Hamilton,1997;Frary and Hamilton,2001)vectors,which can facilitate the cloning and transfer of an extremely large number of independent plant expres-sion cassettes.

An important feature of our multigene vector as-sembly system lies in the unique structure of the ?nal array of transgenes,in which each expression cassette or gene is ?anked by pairs of unique ZFNs or homing endonucleases.This structure may facilitate genomic editing of multitransgene arrays in transgenic plants by harnessing the cell’s nonhomologous end-joining (NHEJ)DNA-repair pathway.Both homing

endonu-Figure 6.PCR analysis of T-DNA inserts in off-spring of transgenic Arabidopsis plants.A,Struc-ture of the nine-gene-long T-DNA molecule and sizes and locations of the overlapping ampli?ed PCR fragments.Abbreviations are as in Figure 1.B,PCR analysis of binary plasmid and T3(lines L2and L10)and T4(lines L6and L7)hygromycin-resistant plants.Note the two distinct fragments in lane c,produced using a single pair of primers,due to the presence of repetitive elements on the nine-gene-long T-DNA molecule.The molecular marker (M)and expected sizes of the PCR frag-ments are given in kb.

Zeevi et al.

cleases and ZFNs have been used for gene targeting in plant cells(i.e.site-speci?c mutagenesis,gene addi-tion,deletion,and/or replacement;Salomon and Puchta,1998;Chilton and Que,2003;Tz?ra et al., 2003;Cai et al.,2009;Shukla et al.,2009;Townsend et al.,2009).Yet,while most ZFN-mediated genome-editing systems rely on homologous recombination between the donor DNA and the target genome,the presence of repetitive elements(i.e.promoter and terminator sequences)in multitransgene arrays may hinder the homologous recombination-mediated ge-nome editing of such structures.Thus,genome editing of multitransgene arrays(i.e.removal,replacement,and addition of expression cassettes by NHEJ)can poten-tially be achieved by transient ZFN expression in target cells,with or without the addition of a donor DNA molecule(Weinthal et al.,2010).Indeed,both I-Sce I-and I-Ceu I-mediated transgene addition(Salomon and Puch-ta,1998;Chilton and Que,2003;Tz?ra et al.,2003)and ZFN-mediated transgene deletion(Petolino et al.,2010) have been reported in plant cells.In addition,we have recently demonstrated that ZFN-and NHEJ-mediated gene replacement is also feasible in plant species(D. Weinthal,T.Taylor,and T.Tz?ra,unpublished data). Worth noting is the fact that by?anking each expression cassette with semipalindromic sequences,the editing process may be technically simpli?ed,since,in contrast to most homologous recombination-mediated gene-replacement methods,it will require the expression of just one ZFN monomer and not pairs of ZFNs.Also worth noting is that other types of engineered en-zymes,such as engineered meganucleases(Gao et al., 2010)and TALENs(Cermak et al.,2011;Mahfouz et al., 2011),have also been used for genome editing in plant cells.Developing procedures for the expression and pu-ri?cation of engineered meganucleases and TALENs for cloning purposes will further extend the repertoire of enzymes suitable for the assembly of multigene vector systems and their successive manipulation in plant cells.

The production of transgenic plants often results in a wide range of transgene-related phenotypes,a phe-nomenon that is typically attributed to a transgene positional affect(Matzke and Matzke,1998).Thus, obtaining plants with superior transgenic performance (e.g.high and stable expression levels during the plant life span and across several generations)often calls for the production and screening of a large number of transgenic plants.Increasing the number of transgenes in plants is likely to increase the number of transgenic plants that need to be produced,screened,and se-lected for the desired performances.This procedure may be time consuming and labor intensive,especially if the different transgenes are scattered across the genome.Linking several transgenes to the same ge-nomic location may assist in the screening process and in maintaining superior clones across several genera-tions.The tendency of multiple DNA molecules,de-rived from the cotransformation of several different Agrobacterium strains or from cobombardment of sev-eral plasmids,to integrate into the same genomic locus (De Neve et al.,1997)has been utilized as a viable method for the production of single-locus multigene transgenic plants(for review,see Naqvi et al.,2010; Peremarti et al.,2010).While this approach has been successfully used to produce transgenic plants in which up to11transgenes are cointegrated into the same locus(Chen et al.,1998;Zhu et al.,2008;Naqvi et al.,2009),it is impossible to predict the physical organization and the arrangement of cotransformed DNA molecules in the transgenic plants.Indeed,it has been suggested that future progress in multigene transformation may depend not only on driving the transgenes under the speci?c combinations of pro-moter and terminator sequences but also on organizing the transgenes in a predetermined pattern(Peremarti et al.,2010).Our modular system can thus be ef?ciently adapted for the analysis of various regulatory elements and gene organization patterns by shuf?ing between different pSAT vectors.

We observed a variety of phenotypes among our different transgenic lines,as was evident,for ex-ample,by differences in hygromycin resistance(Fig. 3A)and GUS expression(Fig.5G).We also observed that transgenic lines in which all the transgenes were expressed across several generations could be obtained(Table II).Yet,we could not identify a clear correlation between the phenotypes of different transgenes on a given T-DNA or between different lines.Thus,for example,lines that exhibited strong resistance to hygromycin did not necessarily exhibit high levels of PAP1or GUS expression,while plants that exhibited high levels of PAP1expression did not yield high intensity of their?uorescence genes.Sim-ilarly,Fujisawa et al.(2009)reported that they could not associate the expression level of individual trans-genes with the performance of three transgenic lines; similarly,they could not determine the ef?ciency of a speci?c promoter in a given multigene array due to differences in the gene expression levels,which did not correlate with their promoter types.Simi-larly,Chen et al.(2010)reported phenotypic variation not only between the same trait in different trans-genic lines but also between individual traits,driven by the same promoter in a given multigene array,in a selected transgenic line.It thus seems that not only may the expression levels of individual genes vary among different lines but also that similar promoters,driving different genes,may behave dif-ferently in a particular multigene cluster.We are currently applying our multigene vector assembly system to construct a set of multigene binary vectors in which identical expression cassettes will be cloned to different organizations.We will use our vectors to produce collections of independent transgenic plants that will be subjected to gene expression anal-ysis in an attempt to produce the necessary data that will assist in de?ning the putative rules for the organization of regulatory elements within a given construct.

Multigene Binary Vectors

MATERIALS AND METHODS

Construction of pSAT Vectors

To construct pSAT12.MCS,we PCR ampli?ed the pSAT6.MCS(Tz?ra et al., 2005)backbone by using5#-ATAAGAATGCGGCCGCGTAAGTGTTGGTGC-TGTAAGTATGGATGCAGTAATCATGGTCATAGCTGTTTCC-3#and5#-GAC-GCACCGGTAGCACCAACACTTACGTTGGTGCTGGCACTGGCCGTCGTTT-TACAACG-3#and ligated the Age I-Not I-digested PCR product to the Age I-Not I 1.2-kb fragment from pSAT6.MCS(Tz?ra et al.,2005).The construction of pSAT10.MCS and pSAT11.MCS was described previously(Zeevi et al.,2008). pRCS11.1was constructed by cloning the Sma I fragment of self-annealed pair of primers5#-TCCCCCGGGTTCCCACAAACTTACTTGTGGGAAAGCAC-CAACACTTACGTTGGTGCTCCCGGGGGA-3#and5#-TCCCCCGGGAGCA-CCAACGTAAGTGTTGGTGCTTTCCCACAAGTAAGTTTGTGGGAACCCGG-GGGA-3#,which encode the ZFN11and ZFN12sites,and the Kpn I fragment of self-annealed pair of primers5#-GGGGTACCTGCATCCATGTAAGTATGGATG-CAGGTACCCC-3#and5#-GGGGTACCTGCATCCATACTTACATGGATGCAG-GTACCCC-3#,which encode ZFN10into the same sites of pRCS2.The ChrD-RFP expression cassette was transferred from pSAT6A.ChrD-RFP(Citovsky et al., 2006)as an Age I-Not I fragment into pSAT12.MCS to produce pSAT12.ChrD-RFP. The construction of pSAT10.EYFP-CHS and pSAT11.DsRed2-P has been de-scribed previously(Zeevi et al.,2008).To produce pSAT2.N,the nucleocapsid protein(N)of the SYNV expression cassette was transferred as an Age I-Not I fragment from pSAT3.N(Tz?ra et al.,2005)into pSAT2.MCS(Tz?ra et al.,2005). pSAT5A.RbcsP.BAR was constructed by transferring the BAR-encoding sequence as an Xma I-Xho I fragment from pSAT1A.ocsP.BAR(Chung et al.,2005)into the same sites of pSAT5A.RbcsP.MCS(Chung et al.,2005).pSAT4.hsP.GUS was constructed by transferring the Nco I-Bam HI GUS-coding sequence from pRTL2-GUS into pSAT4.hsP.MCS(Tovkach et al.,2009).pSAT6A.PAP1was constructed by transferring the Kpn I-Bam HI PAP1-coding sequence from pCHS3-PAP1 (Borevitz et al.,2000)into pSAT6A.MCS(Chung et al.,2005). Assembly,Expression,and Puri?cation of ZFNs The coding sequences of Arabidopsis(Arabidopsis thaliana)ZFN10,ZFN11, and ZFN12were assembled from overlapping oligonucleotides and cloned into the bacterial expression vector pET28-XH to produce pET28-ZFN10, pET28-ZFN11,and pET28-ZFN12.The protocol for the molecular assembly of ZFNs from overlapping oligonucleotides,and their expression and puri?ca-tion,have been described previously in detail(Zeevi et al.,2010).Brie?y,the ZFN DNA-binding coding sequences were assembled by a single PCR from a mixture of ZFN backbone primers and ZFN?nger-speci?c primers.Each PCR product was then cloned into pET28-XH,where it was fused with the Fok I endonuclease domain and a6xHis tag.For expression in Escherichia coli cells, the ZFN expression vectors were transferred into BL21GOLD(DE3)PlysS cells(Stratagene).The cells were cultured,harvested,and lysed,and the extracted proteins were puri?ed on nickel-nitrilotriacetic acid agarose beads (Qiagen)as described previously(Zeevi et al.,2010).Eluted ZFNs were stored at220°C in50%glycerol.Alternatively,we used the Expressway in vitro protein synthesis system(Invitrogen)for in vitro expression of ZFNs. Construction of Binary Vectors

For ZFN-mediated digestion of pSAT expression cassettes and binary plasmids,about200ng each of acceptor and donor plasmids was digested with0.05to1m L of puri?ed enzyme in10m M Tris(pH8.8),50m M NaCl,1m M dithiothreitol,100m M ZnCl2,50m g mL21bovine serum albumin,and100m g mL21tRNA in a total reaction volume of20to30m L.The reaction was preincubated for30min at room temperature,followed by the addition of MgCl2to a?nal concentration of5m M.The digestion reaction mixture was further incubated for2to40min at room temperature.For homing endonu-clease-mediated digestion of pSAT expression cassettes and binary plasmids, we followed the recommended reaction conditions for each enzyme.Cleaved fragments were separated by gel electrophoresis,puri?ed with a GFX Gel Band Puri?cation Kit(Amersham),dephosphorylated with shrimp alkaline phosphatase(Fermentas),ligated with T4ligase(New England Biolabs),and transferred to chemically competent DH5a E.coli cells by using standard molecular biology protocols.The order in which the different pSAT expression cassettes were assembled into pRCS11.1is described in“Results.”Binary vectors were transformed into chemically competent EHA105Agrobacterium tumefaciens cells as described previously(Tz?ra et al.,1997).pET28ZFN expression plasmids and the pSAT and binary plasmids described in this study are available upon request.

Protoplast Transfection and Production of

Transgenic Plants

The Tape-Arabidopsis Sandwich(Wu et al.,2009)method was used for protoplast isolation and transfection by multigene binary vectors.Transfected protoplasts were cultured in W5solution on1%bovine serum albumin-coated six-well plates for16to24h at24°C to allow expression of the transfected DNA.Transgenic Arabidopsis plants were produced by using the standard ?ower-dip transformation method(Clough and Bent,1998).Transgenic plants were selected on hygromycin selection medium.For analysis of resistance to Basta,hygromycin-resistant seedlings were transferred to soil,allowed to grow,and then sprayed with commercial Basta.

Confocal Microscopy

Protoplasts and plant tissues were viewed directly with a confocal laser-scanning microscope(TCS SP5;Leica).EYFP was excited with an argon laser at514nm,and?uorescence was monitored between525and540nm.DsRed2 and RFP were excited with a helium-neon laser at561nm,and?uorescence was monitored between570and630nm.Chlorophyll?uorescence was monitored above660nm.

Received July30,2011;accepted November11,2011;published November14, 2011.

LITERATURE CITED

Ben Zvi MM,Negre-Zakharov F,Masci T,Ovadis M,Shklarman E,Ben-Meir H,Tz?ra T,Dudareva N,Vainstein A(2008)Interlinking showy traits:co-engineering of scent and colour biosynthesis in?owers.Plant Biotechnol J6: 403–415

Bohmert K,Balbo I,Kopka J,Mittendorf V,Nawrath C,Poirier Y, Tischendorf G,Trethewey RN,Willmitzer L(2000)Transgenic Arabi-dopsis plants can accumulate polyhydroxybutyrate to up to4%of their fresh weight.Planta211:841–845

Bohmert K,Balbo I,Steinbu¨chel A,Tischendorf G,Willmitzer L(2002) Constitutive expression of the b-ketothiolase gene in transgenic plants:

a major obstacle for obtaining polyhydroxybutyrate-producing plants.

Plant Physiol128:1282–1290

Borevitz JO,Xia Y,Blount J,Dixon RA,Lamb C(2000)Activation tagging identi?es a conserved MYB regulator of phenylpropanoid biosynthesis.

Plant Cell12:2383–2394

Cai CQ,Doyon Y,Ainley WM,Miller JC,Dekelver RC,Moehle EA, Rock JM,Lee YL,Garrison R,Schulenberg L,et al(2009)Targeted transgene integration in plant cells using designed zinc?nger nucleases.

Plant Mol Biol69:699–709

Carroll D,Beumer KJ,Morton JJ,Bozas A,Trautman JK(2008)Gene targeting in Drosophila and Caenorhabditis elegans with zinc-?nger nucleases.Methods Mol Biol435:63–77

Cermak T,Doyle EL,Christian M,Wang L,Zhang Y,Schmidt C,Baller JA, Somia NV,Bogdanove AJ,Voytas DF(2011)Ef?cient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting.Nucleic Acids Res39:e82

Chakrabarty R,Banerjee R,Chung S-M,Farman M,Citovsky V, Hogenhout SA,Tz?ra T,Goodin M(2007)PSITE vectors for stable integration or transient expression of auto?uorescent protein fusions in plants:probing Nicotiana benthamiana-virus interactions.Mol Plant Microbe Interact20:740–750

Chen L,Marmey P,Taylor NJ,Brizard JP,Espinoza C,D’Cruz P,Huet H, Zhang S,de Kochko A,Beachy RN,et al(1998)Expression and inheritance of multiple transgenes in rice plants.Nat Biotechnol16: 1060–1064

Chen QJ,Xie M,Ma XX,Dong L,Chen J,Wang XC(2010)MISSA is a highly ef?cient in vivo DNA assembly method for plant multiple-gene transformation.Plant Physiol153:41–51

Chen QJ,Zhou HM,Chen J,Wang XC(2006)A Gateway-based platform for multigene plant transformation.Plant Mol Biol62:927–936

Cheo DL,Titus SA,Byrd DR,Hartley JL,Temple GF,Brasch MA(2004)

Zeevi et al.

Concerted assembly and cloning of multiple DNA segments using in vitro site-speci?c recombination:functional analysis of multi-segment expression clones.Genome Res14:2111–2120

Chilton M-D,Que Q(2003)Targeted integration of T-DNA into the tobacco genome at double-stranded breaks:new insights on the mechanism of T-DNA integration.Plant Physiol133:956–965

Chung SM,Frankman EL,Tz?ra T(2005)A versatile vector system for multiple gene expression in plants.Trends Plant Sci10:357–361 Citovsky V,Lee LY,Vyas S,Glick E,Chen MH,Vainstein A,Gafni Y, Gelvin SB,Tz?ra T(2006)Subcellular localization of interacting pro-teins by bimolecular?uorescence complementation in planta.J Mol Biol 362:1120–1131

Clough SJ,Bent AF(1998)Floral dip:a simpli?ed method for Agrobacterium-mediated transformation of Arabidopsis thaliana.Plant J16:735–743

Dafny-Yelin M,Chung S-M,Frankman EL,Tz?ra T(2007)pSAT RNA interference vectors:a modular series for multiple gene down-regulation in plants.Plant Physiol145:1272–1281

Dafny-Yelin M,Tz?ra T(2007)Delivery of multiple transgenes to plant cells.Plant Physiol145:1118–1128

Daniell H,Dhingra A(2002)Multigene engineering:dawn of an exciting new era in biotechnology.Curr Opin Biotechnol13:136–141

De Neve M,De Buck S,Jacobs A,Van Montagu M,Depicker A(1997) T-DNA integration patterns in co-transformed plant cells suggest that T-DNA repeats originate from co-integration of separate T-DNAs.Plant J11:15–29

Frary A,Hamilton CM(2001)Ef?ciency and stability of high molecular weight DNA transformation:an analysis in tomato.Transgenic Res10: 121–132

Fujisawa M,Takita E,Harada H,Sakurai N,Suzuki H,Ohyama K, Shibata D,Misawa N(2009)Pathway engineering of Brassica napus seeds using multiple key enzyme genes involved in ketocarotenoid formation.J Exp Bot60:1319–1332

Gao H,Smith J,Yang M,Jones S,Djukanovic V,Nicholson MG,West A, Bidney D,Falco SC,Jantz D,et al(2010)Heritable targeted mutagen-esis in maize using a designed endonuclease.Plant J61:176–187 Geurts AM,Cost GJ,Freyvert Y,Zeitler B,Miller JC,Choi VM, Jenkins SS,Wood A,Cui X,Meng X,et al(2009)Knockout rats via embryo microinjection of zinc-?nger nucleases.Science325:433 Goderis IJ,De Bolle MF,Franc?ois IE,Wouters PF,Broekaert WF, Cammue BP(2002)A set of modular plant transformation vectors allowing?exible insertion of up to six expression units.Plant Mol Biol 50:17–27

Goodin MM,Austin J,Tobias R,Fujita M,Morales C,Jackson AO(2001) Interactions and nuclear import of the N and P proteins of Sonchus yellow net virus,a plant nucleorhabdovirus.J Virol75:9393–9406 Goodin MM,Dietzgen RG,Schichnes D,Ruzin S,Jackson AO(2002) pGD vectors:versatile tools for the expression of green and red?uo-rescent protein fusions in agroin?ltrated plant leaves.Plant J31:375–383 Halpin C,Barakate A,Askari BM,Abbott JC,Ryan MD(2001)Enabling technologies for manipulating multiple genes on complex pathways.

Plant Mol Biol47:295–310

Hamilton CM(1997)A binary-BAC system for plant transformation with high-molecular-weight DNA.Gene200:107–116

Hanin M,Paszkowski J(2003)Plant genome modi?cation by homologous recombination.Curr Opin Plant Biol6:157–162

Hellens R,Mullineaux P,Klee H(2000)Technical Focus.A guide to Agrobacterium binary Ti vectors.Trends Plant Sci5:446–451

Karimi M,De Meyer B,Hilson P(2005)Modular cloning in plant cells.

Trends Plant Sci10:103–105

Lee LY,Fang MJ,Kuang LY,Gelvin SB(2008)Vectors for multi-color bimolecular?uorescence complementation to investigate protein-protein interactions in living plant cells.Plant Methods4:24

Lin L,Liu YG,Xu X,Li B(2003)Ef?cient linking and transfer of multiple genes by a multigene assembly and transformation vector system.Proc Natl Acad Sci USA100:5962–5967

Liu YG,Shirano Y,Fukaki H,Yanai Y,Tasaka M,Tabata S,Shibata D (1999)Complementation of plant mutants with large genomic DNA fragments by a transformation-competent arti?cial chromosome vector accelerates positional cloning.Proc Natl Acad Sci USA96:6535–6540 Lyznik LA,Dress V(2008)Gene targeting for chromosome engineering applications in eukaryotic cells.Recent Pat Biotechnol2:94–106 Maeder ML,Thibodeau-Beganny S,Osiak A,Wright DA,Anthony RM, Eichtinger M,Jiang T,Foley JE,Winfrey RJ,Townsend JA,et al(2008)

Rapid“open-source”engineering of customized zinc-?nger nucleases for highly ef?cient gene modi?cation.Mol Cell31:294–301

Mahfouz MM,Li L,Shamimuzzaman M,Wibowo A,Fang X,Zhu JK (2011)De novo-engineered transcription activator-like effector(TALE) hybrid nuclease with novel DNA binding speci?city creates double-strand breaks.Proc Natl Acad Sci USA108:2623–2628

Mani M,Kandavelou K,Dy FJ,Durai S,Chandrasegaran S(2005)Design, engineering,and characterization of zinc?nger nucleases.Biochem Biophys Res Commun335:447–457

Matzke AJ,Matzke MA(1998)Position effects and epigenetic silencing of plant transgenes.Curr Opin Plant Biol1:142–148

Moon HS,Li Y,Stewart CN Jr(2010)Keeping the genie in the bottle:transgene biocontainment by excision in pollen.Trends Biotechnol28:3–8

Naqvi S,Farre′G,Sanahuja G,Capell T,Zhu C,Christou P(2010)When more is better:multigene engineering in plants.Trends Plant Sci15:48–56 Naqvi S,Zhu C,Farre G,Ramessar K,Bassie L,Breitenbach J, Perez Conesa D,Ros G,Sandmann G,Capell T,et al(2009)Transgenic multivitamin corn through bioforti?cation of endosperm with three vitamins representing three distinct metabolic pathways.Proc Natl Acad Sci USA106:7762–7767

Pelletier MK,Shirley BW(1996)Analysis of?avanone3-hydroxylase in Arabidopsis seedlings:coordinate regulation with chalcone synthase and chalcone isomerase.Plant Physiol111:339–345

Peremarti A,Twyman RM,Go′mez-Galera S,Naqvi S,Farre′G,Sabalza M, Miralpeix B,Dashevskaya S,Yuan D,Ramessar K,et al(2010)Pro-moter diversity in multigene transformation.Plant Mol Biol73:363–378 Petolino JF,Worden A,Curlee K,Connell J,Strange Moynahan TL, Larsen C,Russell S(2010)Zinc?nger nuclease-mediated transgene deletion.Plant Mol Biol73:617–628

Porteus MH(2009)Plant biotechnology:zinc?ngers on target.Nature459: 337–338

Salomon S,Puchta H(1998)Capture of genomic and T-DNA sequences during double-strand break repair in somatic plant cells.EMBO J17: 6086–6095

Sasaki Y,Sone T,Yoshida S,Yahata K,Hotta J,Chesnut JD,Honda T, Imamoto F(2004)Evidence for high speci?city and ef?ciency of mul-tiple recombination signals in mixed DNA cloning by the multisite Gateway system.J Biotechnol107:233–243

Shukla VK,Doyon Y,Miller JC,DeKelver RC,Moehle EA,Worden SE, Mitchell JC,Arnold NL,Gopalan S,Meng X,et al(2009)Precise genome modi?cation in the crop species Zea mays using zinc-?nger nucleases.Nature459:437–441

Takahashi T,Komeda Y(1989)Characterization of two genes encoding small heat-shock proteins in Arabidopsis thaliana.Mol Gen Genet219: 365–372

Takasu Y,Kobayashi I,Beumer K,Uchino K,Sezutsu H,Sajwan S, Carroll D,Tamura T,Zurovec M(2010)Targeted mutagenesis in the silkworm Bombyx mori using zinc?nger nuclease mRNA injection.

Insect Biochem Mol Biol40:759–765

Taverniers I,Papazova N,Bertheau Y,De Loose M,Holst-Jensen A(2008) Gene stacking in transgenic plants:towards compliance between def-initions,terminology,and detection within the EU regulatory frame-work.Environ Biosafety Res7:197–218

Tovkach A,Zeevi V,Tz?ra T(2009)A toolbox and procedural notes for characterizing novel zinc?nger nucleases for genome editing in plant cells.Plant J57:747–757

Tovkach A,Zeevi V,Tz?ra T(2010)Validation and expression of ZFNs and in plant cells.Methods Mol Cell Biol649:315–336

Tovkach A,Zeevi V,Tz?ra T(2011)Expression,puri?cation and charac-terization of cloning-grade zinc?nger nuclease.J Biotechnol151:1–8 Townsend JA,Wright DA,Winfrey RJ,Fu F,Maeder ML,Joung JK,Voytas DF(2009)High-frequency modi?cation of plant genes using engineered zinc-?nger nucleases.Nature459:442–445

Tz?ra T,Frankman LR,Vaidya M,Citovsky V(2003)Site-speci?c inte-gration of Agrobacterium tumefaciens T-DNA via double-stranded inter-mediates.Plant Physiol133:1011–1023

Tz?ra T,Jensen CS,Wangxia W,Zuker A,Altman A,Vainstein A(1997) Transgenic Populus:a step-by-step protocol for its Agrobacterium-mediated transformation.Plant Mol Biol Rep15:219–235

Tz?ra T,Tian G-W,Lacroix B,Vyas S,Li J,Leitner-Dagan Y,Krichevsky A, Taylor T,Vainstein A,Citovsky V(2005)pSAT vectors:a modular series of plasmids for auto?uorescent protein tagging and expression of multiple genes in plants.Plant Mol Biol57:503–516

Multigene Binary Vectors

Urnov FD,Miller JC,Lee YL,Beausejour CM,Rock JM,Augustus S, Jamieson AC,Porteus MH,Gregory PD,Holmes MC(2005)Highly ef?cient endogenous human gene correction using designed zinc-?nger nucleases.Nature435:646–651

van Engelen FA,Molthoff JW,Conner AJ,Nap JP,Pereira A,Stiekema WJ (1995)pBINPLUS:an improved plant transformation vector based on pBIN19.Transgenic Res4:288–290

Vishnevetsky M,Ovadis M,Itzhaki H,Levy M,Libal-Weksler Y,Adam Z, Vainstein A(1996)Molecular cloning of a carotenoid-associated protein from Cucumis sativus corollas:homologous genes involved in carote-noid sequestration in chromoplasts.Plant J10:1111–1118 Vishnevetsky M,Ovadis M,Vainstein A(1999)Carotenoid sequestration in plants:the role of carotenoid-associated proteins.Trends Plant Sci4: 232–235

Wakasa Y,Yasuda H,Takaiwa F(2006)High accumulation of bioactive peptide in transgenic rice seeds by expression of introduced multiple genes.Plant Biotechnol J4:499–510

Weinthal D,Tovkach A,Zeevi V,Tz?ra T(2010)Genome editing in plant cells by zinc?nger nucleases.Trends Plant Sci15:308–321

Wu FH,Shen SC,Lee LY,Lee SH,Chan MT,Lin CS(2009)Tape-

Arabidopsis Sandwich:a simpler Arabidopsis protoplast isolation method.Plant Methods5:16

Wu G,Truksa M,Datla N,V rinten P,Bauer J,Zank T,Cirpus P,Heinz E,Qiu X (2005)Stepwise engineering to produce high yields of very long-chain polyunsaturated fatty acids in plants.Nat Biotechnol23:1013–1017

Zeevi V,Tovkach A,Tz?ra T(2008)Increasing cloning possibilities using arti?cial zinc?nger nucleases.Proc Natl Acad Sci USA105:12785–12790 Zeevi V,Tovkach A,Tz?ra T(2010)Arti?cial zinc?nger nucleases for DNA cloning.Methods Mol Biol649:209–225

Zhong R,Richardson EA,Ye ZH(2007)Two NAC domain transcription factors,SND1and NST1,function redundantly in regulation of second-ary wall synthesis in?bers of Arabidopsis.Planta225:1603–1611

Zhu C,Naqvi S,Breitenbach J,Sandmann G,Christou P,Capell T(2008) Combinatorial genetic transformation generates a library of metabolic phenotypes for the carotenoid pathway in maize.Proc Natl Acad Sci USA105:18232–18237

Zimmerman KA,Fischer KP,Joyce MA,Tyrrell DL(2008)Zinc?nger proteins designed to speci?cally target duck hepatitis B virus covalently closed circular DNA inhibit viral transcription in tissue culture.J Virol 82:8013

Zeevi et al.

儿童歌谣大全

儿童歌谣大全 各位读友大家好，此文档由网络收集而来，欢迎您下载，谢谢 篇一：儿童歌谣歌词大全 [小螺号]儿歌歌词 小螺号，嘀嘀嘀吹海鸥听了展翅飞小螺号，嘀嘀嘀吹浪花听了笑微微小螺号，嘀嘀嘀吹 声声唤船归啰 小螺号，嘀嘀嘀吹 阿爸听了快快回啰。 茫茫的海洋，蓝蓝的海水 吹起了小螺号，心里美吔 [小鸟小鸟] 蓝天里有阳光，树林里有花香 小鸟小鸟，你自由地飞翔 在田野，在草地，在湖边，在山冈 小鸟小鸟迎着春天歌唱 啦啦啦啦啦。爱春天，爱阳光，爱湖水，爱花香小鸟小鸟，我的好朋友让我们一起飞翔歌唱

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鸟儿鸟儿怎样飞？ 拍拍翅膀飞呀飞 谁会游呀，鱼会游 鱼儿鱼儿怎样游？ 摇摇尾巴点点头 谁会跑呀，马会跑马儿马儿怎样跑？四脚离地身不摇。[我叫轻轻] 张友珊词汪玲曲 走路轻轻轻轻 上夜班的阿姨还没醒呀 敲门轻轻轻轻 给邻居叔叔送呀送封信 说话轻轻轻轻 姐姐灯下看书多用心呀 大家夸我是好孩子 给我取个名字叫呀叫轻轻 走路轻轻轻轻 上夜班的阿姨还没醒呀 敲门轻轻轻轻 给邻居叔叔送呀送封信 说话轻轻轻轻 姐姐灯下看书多用心呀

最新The Finger Family 英文儿歌

The Finger Family英文歌词： Daddy Finger, Daddy Finger. Here I am! Here I am! How are you, today? Very well, I thank you. Run away. Run away. Mommy Finger, Mommy Finger, Here I am! Here I am! How are you, today? Very well, I thank you. Run away. Run away. Brother Finger, Brother Finger, Here I am! Here I am! How are you, today? Very well, I thank you. Run away. Run away. Sister Finger, Sister Finger, Here I am! Here I am! How are you, today? Very well, I thank you. Run away. Run away. Baby Finger, Baby Finger, Here I am! Here I am! How are you, today? Very well, I thank you. Run away. Run away. 中文翻译：手指家庭手指爸爸，手指爸爸，我在这里!我在这里! 你今天怎么样? 很好，谢谢你。跑开。跑开。手指妈妈，手指妈妈，我在这里!我在这里! 你今天怎么样? 很好，谢谢你。跑开。跑开。手指哥哥，手指哥哥，我在这里!我在这里! 你今天怎么样? 很好，谢谢你。跑开。跑开。手指妹妹，手指妹妹，我在这里!我在这里! 你今天怎么样? 很好，谢谢你。跑开。跑开。手指宝宝，手指宝宝，我在这里!我在这里! 你今天怎么样? 很好，谢谢你。跑开。跑开。

儿歌歌词大全

《樱桃小丸子》 小小年纪谈起理想一串串 想当专家、想做博士、想出唱片老爸老妈老师老友都夸赞 想来容易，说来简单，做做就难要数一百，先数一二三 要过明天先过好今天 瞄准目标看齐 噼里啪啦，噼里啪啦 做事不偷懒 噼里啪啦，噼里啪啦

学习不怕难 我们脚踏实地地干 瞄准目标看齐 噼里啪啦，噼里啪啦 读书真勤快 噼里啪啦，噼里啪啦 今天学得好 噼里啪啦，噼里啪啦 明天理想能实现 《多啦A梦》 如果我有仙女棒变大变小变漂亮

还要变个都是漫画巧克力和玩具的家 如果我有机器猫我要叫他小叮当 竹蜻蜓和时光隧道能去任何的地方 让小孩大人坏人都变成好人 (hi 大家好,我是小叮当) ang ang ang小叮当帮我实现所有的愿望 躺在草地上幻想想动想西想玩耍 想到老师还有考试一个头就变成两个大好在我有小叮当困难时候求求他 万能笔和时间机器能做任何的事情 让我的好朋友一齐分享他 (啊!救命啊!有老鼠!) ang ang ang 小叮当帮我实现所有的愿望 躺在草地上幻想想动想西想玩耍 想到老师还有考试一个头就变成两个大好在我有小叮当困难时候求求他 万能笔和时间机器能做任何的事情 让我的好朋友一齐分享他 (小叮当永远是你们的好朋友喔!) ang ang ang 小叮当帮我实现所有的愿望

ang ang ang 小叮当帮我实现所有的愿望 《铁臂阿童木》 越过辽阔天空，啦啦啦飞向遥远群星，来吧！阿童木，爱科学的好少年。善良勇敢的啦啦啦铁臂阿童木，十万马力七大神力，无私无畏的阿童木。穿过广阔大地，啦啦啦潜入深深海洋，来吧！阿童木，爱和平的好少年。善良勇敢的啦啦啦铁臂阿童木，我们的好朋友啊， 无私无畏的阿童木。 《小龙人之歌》 天上有，无数颗星星，那颗最小的就是我，我不知道我从哪里来，也不知道我在哪里生。地上有，无数个龙人，那个最小的就是我，我不知道我从哪里来，也不知道我在哪里生。啊----这是我将在妈妈怀 抱里，啊寻遍天涯，去找他 《我是一条小青龙》我头上有只角，我身后有尾巴，谁也不知道，我有多少秘密？我头上有只角，我身后有尾巴，谁也不知道，我有多少秘密。我是一条小青龙（小青龙，小青龙）我有许多小秘密（小秘

56首经典儿歌歌词大全

56首经典儿歌歌词大全 1、做早操 早上空气真叫好，我们都来做早操。 伸伸臂，弯弯腰，踢踢腿，蹦蹦跳，天天锻炼身体好。 2、饭前要洗手 小脸盆，水清请，小朋友们笑盈盈，小手儿，伸出来， 洗一洗，白又净，吃饭前，先洗手，讲卫生，不得病。 3、小手绢 小手绢，四方方，天天带在我身上。 又擦鼻涕又擦汗，干干净净真好看。 4、搬鸡蛋 小老鼠，搬鸡蛋，鸡蛋太大怎么办?一只老鼠地上躺， 紧紧抱住大鸡蛋。一只老鼠拉尾巴，拉呀拉呀拉回家。 5、大骆驼 骆驼骆驼志气大，风吹日晒都不怕。 走沙漠，运盐巴，再苦再累不讲话。 6、螳螂 螳螂哥，螳螂哥，肚儿大，吃得多。飞飞能把粉蝶捕， 跳跳能把蝗虫捉。两把大刀舞起来，一只害虫不放过 7、大蜻蜓 大蜻蜓，绿眼睛，一对眼睛亮晶晶， 飞一飞，停一停，飞来飞去捉蚊蝇。 8、小鸭子 小鸭子，一身黄，扁扁嘴巴红脚掌。 嘎嘎嘎嘎高声唱，一摇一摆下池塘。 9、拍手歌 你拍一，我拍一，天天早起练身体。 你拍二，我拍二，天天都要带手绢。 你拍三，我拍三，洗澡以后换衬衫。 你拍四，我拍四，消灭苍蝇和蚊子。 你拍五，我拍五，有痰不要随地吐。 你拍六，我拍六，瓜皮果核不乱丢。 你拍七，我拍七，吃饭细嚼别着急。 你拍八，我拍八，勤剪指甲常刷牙。 你拍九，我拍九，吃饭以前要洗手。

你拍十，我拍十，脏的东西不要吃。 10 、小螃蟹 小螃蟹，真骄傲，横着身子到处跑， 吓跑鱼，撞倒虾，一点也不懂礼貌 11 、庆六一 儿童节，是六一，小朋友们真欢喜。 又唱歌来又跳舞，高高兴兴庆六一。 12、花猫照镜子 小花猫，喵喵叫，不洗脸，把镜照， 左边照，右边照，埋怨镜子脏，气得胡子翘。 13、蚂蚁搬虫虫 小蚂蚁，搬虫虫，一个搬，搬不动，两个搬，掀条缝， 三个搬，动一动，四个五个六七个，大家一起搬进洞。 14、小青蛙 小青蛙，呱呱呱，水里游，岸上爬， 吃害虫，保庄稼，人人都要保护它。 15、花儿好看我不摘 公园里，花儿开，红的红，白的白， 花儿好看我不摘，人人都说我真乖。 16 、红绿灯 大马路，宽又宽，警察叔叔站中间， 红灯亮，停一停，绿灯亮，往前行。 17 、七个果果 一二三四五六七，七六五四三二一。 七个阿姨来摘果，七个篮子手中提。七个果子摆七样。 苹果、桃儿、石榴、柿子、李子、栗子、梨。 18、睡午觉 枕头放放平，花被盖盖好。 小枕头，小花被，跟我一起睡午觉，看谁先睡着。 19 、吃荸荠 荸荠有皮，皮上有泥。洗掉荸荠皮上的泥，削去荸荠外面的皮，荸荠没了皮和泥，干干净净吃荸荠。 20 、小云骑牛去打油 小云骑牛去打油，遇着小友踢皮球，皮球飞来吓了牛，摔下小云撒了油。 21 、盆和瓶 车上有个盆，盆里有个瓶，乒乒乒，乓乓乓，不知是瓶碰盆，还是盆碰瓶。

幼儿英语歌曲歌词：The finger family

幼儿英语歌曲歌词：The finger family Daddy Finger, Daddy Finger. Here I am! Here I am! How are you, today? Very well, I thank you. Run away. Run away. Mommy Finger, Mommy Finger, Here I am! Here I am! How are you, today? Very well, I thank you. Run away. Run away. Brother Finger, Brother Finger, Here I am! Here I am! How are you, today? Very well, I thank you. Run away. Run away. Sister Finger, Sister Finger, Here I am! Here I am! How are you, today? Very well, I thank you.

Run away. Run away. Baby Finger, Baby Finger, Here I am! Here I am! How are you, today? Very well, I thank you. Run away. Run away. 手指家庭 手指爸爸，手指爸爸，我在这里！我在这里！你今天怎么样？ 很好，谢谢你。 跑开。跑开。 手指妈妈，手指妈妈，我在这里！我在这里！你今天怎么样？ 很好，谢谢你。 跑开。跑开。 手指哥哥，手指哥哥，我在这里！我在这里！你今天怎么样？ 很好，谢谢你。 跑开。跑开。 手指妹妹，手指妹妹，我在这里！我在这里！你今天怎么样？

幼儿儿歌歌词大全

幼儿儿歌歌词大全 1、小白兔 小白兔乖乖，把门开开，快点开开，我要进来，不开不开就不开，妈妈没回来，谁叫也不开。小白兔乖乖，把门开开，妈妈回来，我要进来，快开快开快快开，妈妈回来了，我来把门开。 2、小花狗 小花狗，真叫脏，不洗脚丫就上床。问它为什么？它说忘，忘，忘！ 3、吃饭 小宝宝，坐坐好，妈妈盛饭喂宝宝。细细嚼，慢慢咽，宝宝吃得直叫好。 4、干净 鼻涕擦干净，指甲剪干净，脸儿洗干净，妈妈和和亲一亲。 5、乒乓球 乒乓球,两人打,他给我,我给他,谁都不愿落在自己家. 6、踢毽子 小毽子,小毽子,飞上天, 落下地，我们都来踢踢它,踢不好儿没关系. 7、走夜路 夜色黑,星星闪,小朋友,把家还,突然窜出一只兔,吓得它,满处窜. 8、小盒子 小盒子,作用大,铅笔橡皮装的下, 还有一支小钢笔,装在里面心欢喜, 9、大马路 马路长又宽,我站在中间, 老师过来拜拜手,让我赶快走,说是有危险. 10、木头人

三三三，我们都是木头人， 不许哭来不许笑，还有一个不许动。 11、小皮球,小小篮,落地开花二十一,二五六,二五七, 二八二九三十一;三五六,三五七,三八三九四十一…… 12、小蜜蜂 小蜜蜂，嗡嗡嗡，嗡嗡嗡，大家一起勤劳动， 来匆匆，去匆匆，走得兴味浓，春暖花开不做工， 将来哪里好过冬？嗡嗡嗡，嗡嗡嗡，不学懒惰虫。 13、请你唱个歌吧 小杜鹃,小杜鹃，我们请你唱个歌，快来呀,大家来呀， 我们静听你的歌，咕咕!咕咕!歌声使我们快乐 14、谁会飞 谁会飞呀，鸟会飞，鸟儿鸟儿怎样飞？拍拍翅膀飞呀飞 谁会游呀，鱼会游，鱼儿鱼儿怎样游？摇摇尾巴点点头 谁会跑呀，马会跑，马儿马儿怎样跑？四脚离地身不摇。 15、好朋友 你帮我来梳梳头，我帮你来扣纽扣 团结友爱手拉手，我们都是好朋友 嘿嘿！ 16、数鸭子 门前大桥下，游过一群鸭， 快来快来数一数，二四六七八。 17、小雪花 小雪花，小雪花，飘在空中像朵花，小雪花，小雪花 飘在窗上变窗花，小雪花，小雪花，飘在手上不见了

幼儿英语歌词及翻译：The finger family

幼儿英语歌词及翻译：The finger family Daddy Finger, Daddy Finger. Here I am! Here I am! How are you, today? Very well, I thank you. Run away. Run away. Mommy Finger, Mommy Finger, Here I am! Here I am! How are you, today? Very well, I thank you. Run away. Run away. Brother Finger, Brother Finger, Here I am! Here I am! How are you, today? Very well, I thank you. Run away. Run away. Sister Finger, Sister Finger, Here I am! Here I am! How are you, today? Very well, I thank you.

Run away. Run away. Baby Finger, Baby Finger, Here I am! Here I am! How are you, today? Very well, I thank you. Run away. Run away. 手指家庭 手指爸爸，手指爸爸，我在这里！我在这里！你今天怎么样？ 很好，谢谢你。 跑开。跑开。 手指妈妈，手指妈妈，我在这里！我在这里！你今天怎么样？ 很好，谢谢你。 跑开。跑开。 手指哥哥，手指哥哥，我在这里！我在这里！你今天怎么样？ 很好，谢谢你。 跑开。跑开。 手指妹妹，手指妹妹，我在这里！我在这里！你今天怎么样？

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五年级教学设计thespringfestivaliscoming完整版

五年级教学设计 t h e s p r i n g f e s t i v a l i s c o m i n g Document serial number【NL89WT-NY98YT-NC8CB-NNUUT-NUT108】

教学设计 基本信息 姓名石玉平工作单位廊坊市第十小学 所用教科书冀教版小学英语三年级起点 所教年级五年级所教册次、单元上册Unit4 设计主题 Lesson20TheSpringFestivalisComing!（Part1）（课题名 称） 1.整体设计思路、指导依据说明 本节课是冀教版小学英语五年级上册第四单元 Lesson20TheSpringFestivalisComing!本单元主要的语言功能为：介绍春 节的习俗及人们在春节时干的活动。本课主要是让学生学会描述人们在春 节的时候干的活动。本课堂为随后的教学知识做铺垫，是本单元的基础性 教学内容。根据《英语新课程标准》，本人将采用引导型，任务型以及情 景交际法，采用多媒体课件进行教学。课堂的任务设计贴近实际生活的内 容，提供符合真实生活的学习情景。通过自主，合作，探究培养学生主动 学习英语的兴趣，自学能力及合作能力。 2.教学背景分析 教学内容分析： 本节课为Unit4，Lesson20第一部分，教材以春节习俗为中心话题，在春 节期间人们干的活动为主要内容。本堂课的教学内容与实际生活息息相 关，学生很熟悉，易于引导学生运用英语来进行交流。因此在本课教学过 程中，我将引导学生自主学习发现并总结字母发音规律，从而达到能自然 拼读出包含这个字母组合的单词的目的。同时采用多种活动形式，引导学 生学会用英语来表达春节的习俗。 学生情况分析： 本课的授课对象是五年级学生。他们已经掌握了简单的字母组合的发音规 则，因此也为本节课的字母组合发音以及其所涉及到的单词的整体拼读打 下基础，让学生结合单词来辨别字母发音并鼓励学生大胆积极地去探索发 现其发音特点。 3.教学目标分析 知识目标：能够听懂、会说、认读单词：room,door,window,kid,make；能 够认读、理解并运用句型及回答句型WhatdoyoudofortheSpringFestival? 情感目标：能用英语简单的描述中国的传统节日——春节；进一步培养学