pcambia vectors. the pcambia vector backbone is derived from the ppzp vectors. the sphi site outside...

pCambia Vectors

The pCAMBIA vector backbone is derived from the pPZP vectors the SphI site outside the right T-DNA Border or the SacII site outside the left T-DNA Border The smaller polylinkers also eliminate potential conflicts from sites such as SphI (which has an ATG) or XbaI (which has a TAG) Plant selection genes in the pCAMBIA vectors are driven by a double-enhancer version of the CaMV35S promoter and terminated by the CaMV35S polyA signal

1 LBA4404 (Ach5 pTiAch5) SmSp(R) in the virulence plasmid (from Tn904) all T-DNA of pTiAch5 eliminated in pAL4404 (Hoekema et al 1983)

2 EHA101 genotype C58 pTiBo542 T-regionaph Km(R) A281 derivative harboring pEHA101 T-DNA replaced with nptII elimination of T-DNA boundaries uncofirmed super-virulent (Hood et al 1986)

3 EHA105 is a Km(S) derivative of EHA101 (Hood et al 1993)

4 AGL1 genotype is AGL0 (C58 pTiBo542) recAbla T-region deleted Mop(+) Cb(R) [AGL0 is an EHA101 with the T-region deleted which also deletes the aph gene] (Lazo et al 1991)

5 A281 reconstructed strain derivative of A136 (cured C58) harboring pTiBo542 super-virulent (Hood et al 1986)

bull pCAMBIA vectors offerbull high copy number in Ecoli for high DNA yields bull pVS1 replicon for high stability in Agrobacterium bull small size 7-12kb depending on which plasmid bull restriction sites designed for modular plasmid modifications and sm

all but adequate poly-linkers for introducing your DNA of interest bull bacterial selection with chloramphenicol or kanamycin bull plant selection with hygromycin B or kanamycin (phosphinothricin se

lection was discontinued at the request of the IP owner Bayer after the initial distribution in 1997)

bull simple means to construct translational fusions to gusA reporter genes

Nomenclature of pCAMBIA vectors

bull The four digit numbering system works as followsbull First digit - indicates plant selection 0 for absence 1 for hygromycin resistance 2 for kanamycin and 3 for phos

phinothricin (the vectors containing the phosphinothricin resistance gene are no longer available from CAMBIA at the request of Bayer which owns patents restricting its use in some countries)

bull Second digit - indicates bacterial selection 1 for spectinomycinstreptomycin resistance 2 for chloramphenicol 3 for kanamycin 4 for specstrep and kanamycin

bull Third digit - indicates polylinker used 0 for pUC18 polylinker 8 for pUC8 polylinker 9 for pUC9 polylinkerbull Fourth digit - indicates reporter gene(s) present 0 for no reporter gene 1 for Ecoli gusA 2 for mgfp5 3 for gusA

mgfp5 fusion 4 for mgfp5gusA fusion 5 for Staphylococcus sp gusA (GUSPlus)bull Fifth digit - notes some other special feature So far this has been used only with pCAMBIA13051 and plasmids

derived from it where the 1 denotes the absence of a signal peptide from the GUSPlustrade protein and pCAMBIA13052 where the 2 denotes the presence of the GRP signal peptide for in planta secretion of the GUSPlustrade protein

bull Lagging letter - X indicates that the reporter gene lacks its own start codon and the vector is for creating fusions to the reporter Z indicates presence of a functional lacZa for blue-white screening abc indicates the reading frame for fusions with the Fuse and Use vectors

bull Important note Due to resource limitations not all possible vector feature combinations have been created at CAMBIA You may initially be disappointed to find that we dont have for example a pCAMBIA22052 The vectors were designed however such that it should be a relatively simple matter for a researcher needing such a vector to construct it from the components in other vectors If you have created a pCAMBIA vector derivative that other researchers will find useful and you want to share with other researchers email us

TypesAch5 agrocinopine octopine type B6S3 A6 octopine type Bo542 leucinopine succinamopine agropine type vir weaker than A281 C58 T37 nopaline types A281 succinamopine leucinopine agrocinopine AntibioticsChloramphenicol 100 microgmL for strain AGL1 10 microgmL for LBA4404 25 microgmL for EHA105 and for E coli Kanamycin 50 microgmL for both Agrobacterium and E coli For selection of transformed rice plants we use hygromycin at 50 microgmL and 25 microgmL for tobacco

Minimal Selection Vectors pCAMBIA1200 pCAMBIA1300 pCAMBIA1380 pCAMBIA1390 pCAMBIA2200 pCAMBIA2300

Selected References Chen L Zhang S Beachy RN Fauquet CM (1998) A protocol for consistent large-scale production of fertile transgenic rice plants Plant Cell Reports 1825-31 Christou P (1991) Production of transgenic rice (Oryza sativa L) plants from agronomically important indica and japonica varieties via electric discharge particle acceleration of exogenous DNA into immature zygotic embryos Biotechnology 9957-962 Christou P (1997) Rice transformation bombardment Plant Mol Biol 35197-203 Deblaere R Reynaerts A Hofte H Hernalsteens JP Leemans J and Van Montagu M (1987) Vectors for cloning in plant cells Meth Enzymol 153277-292 HajdukiewiczP Svab Z Maliga P (1994) The small versatile pPZP family of Agrobacterium binary vectors for plant transformation Plant Mol Biol 25989-994 Hiei Y Ohta S Komari T Kumashiro T (1994) Efficient transformation of rice (Oryza sativa L) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA Plant J 6271-282 Hoekema A Hirsch PR Hooykaas PJJ Schilperoort RA (1983) Binary vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid Nature 303179-180 Hood EE Helmer GL Fraley RT Chilton MD (1986) The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA J Bac 1681291-1301 Hood EE Gelvin SB Melchers S Hoekema A (1993) New Agrobacterium helper plasmids for gene transfer to plants (EHA105) Trans Res 2208-218 Jefferson RA Kavanagh TA Bevan MW (1987) GUS fusions Beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants EMBO J 63901-3907 Klapwijk PM van Breukelen J Korevaar K Ooms G Schilperoort RA (1980) T ransposition of Tn904 encoding streptomycin resistance into octopine Ti plasmid of Agrobacterium tumefaciens J Bac 141129-136 Lazo GR Stein PA Ludwig RA (1991) A DNA transformation-competent Ara bidopsis genomic library in Agrobacterium BioTechnology 9963-967 Ohta S Mita S Hattori T Nakamura K (1990) Construction and expression in tobacco of a beta-glucuronidase (GUS) reporter gene containing an intron within the coding sequence Plant Cell Physiol 31805-814 Ooms G Hooykaas PJJ Van Veen RJM Van Beelen P Regensburg-Tunk JG Schilperoort RA (1982) Octopine Ti-plasmid deletion mutants of Agrobacterium tumefaciens with emphasis on the right side of the T-region Plasmid 7 15-29 Peralta EG Hellmiss R Ream W (1986) Overdrive a T-DNA transmission enhancer on the A tumefaciens tumour-inducing plasmid EMBO J 51137-1142 Porath J (1992) Immobilized metal ion affinity chromatography Protein Expre Purif 3263-281 Siemering KR Golbik R Sever R Haseloff J (1996) Mutations that suppress the thermosensitivity of green fluorescent protein Curr Biol 61653-1663 Tanaka A Mita S Ohta S Kyozuka J Shimamoto K Nakamura K (1990) Enhancement of foreign gene expression by a dicot intron in rice but not in tobacco is correlated with an increased level of mRNA and an efficient splicing of the intron Nucl Acids Res 186767-6770

bull GROWTH MEDIA FOR AGROBACTERIUMbull YEP Per liter bull Bacto peptone 10 g

NaCl 5 gYeast extract 10 g(Agar) 15 g

bull No pH adjustmentbull AB bull AB Buffer Per liter (20x stock solution) bull K2HPO4 60 g

(or K2HPO43H2O 786 g)NaH2PO4 20 g(or NaH2PO4H20 23 g)

bull AB Salts Per liter (20x stock solution) bull NH4Cl 20 g

MgSO47H20 6 g(or MgSO4 29 g)KCl 3 gCaCl2 02 g(or CaCl22H20 026 g)FeSO47H20 50 mg

bull Sterilize the AB Buffer and the AB Salts separately Shake the salts before use to disperse the FeSO4 bull Add sucrose to a final concentration of 05

MCS EcoRI(10578) SacI(10584) KpnI(10590) SmaI(10594) BamHI(10599) XbaI(10605) SalI(10611) Plant kanamycin selection replaces hygromycin in pcambia1300双 T载体 pCDMAR-Hyg(HDF)

bull Cloning vectors

Schematic representation of the modular binary destination vectors generated

Himmelbach A etal Plant Physiol 20101451192-1200

Copyright copy 2007 American Society of Plant Biologists All rights reserved

HindIII EcoRI

Actin promotor HA tag

SmaI

HindIII HindIII

插入的所克隆基因

启动子

一个融合的TAG用于纯化蛋白质

待插入的克隆片段

XbaI

GFP

一个融合的GFP片段用于定位

07kb12kb13kb

构建时用ApaI和XbaI组合酶切AHLG或用SmaI和XbaI组合但是如用SmaI须在起始密码子ATG前smaI后加一个碱基才能正确读码

AHLG

(12~13kb)

ApaI


SmaI

HindIII HindIII


启动子



XbaI

GFP


07kb12kb13kb


AHLG

(12~13kb)

ApaI

Copyright copy2007 American Society of Plant Biologists

Himmelbach A et al Plant Physiol 20071451192-1200


Plant Molecular Biology 40 711ndash717 1999

bar gene for phosphinothricin acetyltransferase nptIII gene for neomycin phosphotransferasefor kanamycin resistance (from pBIN19) oriV part of RK2 origin of replication (from pBIN19) P35S 35S promoter of cauliflower mosaic virus P35S2 35S promoter with double enhancers Pnos promoter of nos (nopaline synthase) gene Pubi maize ubiquitin-1 promoter RBright border of T-DNA Tnos terminator of nos (nopaline synthase) gene TP plastid targeting sequence of Rubisco small subunit trfA part of RK2 origin of replication ubi intron intron-1 from maize ubiquitin-1 gene uidA gene for -glucuronidase (GUS) the translational enhancer of TMVThe numbers under each DNA region indicate the approximate size of that region in base pairs and the arrow indicates the orientationThe superscript number on each restriction site indicates how many times that restriction site occurs in the indicated plasmid

ags agropine synthaseA ApaI BBamHI Bc BclI Bg BglII Bx BstXIH HindIII N NotI Nc NcoI P PstIRI EcoRI SI SacI SII SacII S SalI Sm SmaI Sp SpeI Xb XbaI X XhoI

bull Aocsbull ocs transcriptional activating elementbull AmasPmas mas2-activating and promoterbull elements ags-ter poly(A) additionbull signal from the agropine synthasebull gene ADHin intron from the maizebull alcohol dehydrogenase I genebull Pnos nos promoterbull tAg7 poly(A) addition signal forbull T-DNA gene 7 hptII gene conferringbull resistance to hygromycin nptII genebull conferring resistance to kanamycin barbull gene conferring resistance to phosphinothricinbull BastaBialophos Other symbolsbull and restriction endonuclease sitesbull are as in the legend to Figure 1 The barbull gene contains KpnI and SalI sites thereforebull these sites are not unique tobull T-DNA binary vectors containing thebull bar gene (indicated by [K] and [Sl]) Abull T-DNA binary vectors based on thebull pMSP-1 series of plasmids B T-DNAbull binary vectors based upon the pMSP-2bull series of plasmids C T-DNA binarybull vectors based on the pMSP-3 series ofbull plasmids

Maps of the T-DNA binary vectors based upon the pMSP series of plasmids pExxxx numbers at the right of each plasmid map indicate Gelvin laboratory stock numbers kanr Plasmid confers resistance to kanamycin upon host bacteria Pnos nos promoter tAg7 poly(A)

addition signal for T-DNA gene 7 hptII gene conferring resistance to hygromycin nptII gene conferring resistance to kanamycin bar gene conferring resistance to

phosphinothricinBastaBialophos

Lee L etal Plant Physiol 20101451294-1300

羧苄青霉素红霉素庆大霉素卡那霉素利福平

A schematic diagram of the T-DNA region from the binary vector pCAS04 RB the right border of T-DNA LB the left border of T-DNA ubimaize Ubiquitin promoter nptII neomycin phosphotransferase gene CaMVter CaMV terminator GUS 1049109-glucuronidase gene Actin1 rice Actin1 promoterubi Ist intron the first intron of ubiquitin promoter is derived from the binary vector pEP200

Chu C J Genet Genomics 36 (2009) 267-276

The OsAct2-based expression vectors for use in monocot transformation Cyt108 a truncated cytochrome c gene of rice (Li et al 2003) Hpt the coding sequence of the hygromycin phosphotransferase gene LB T-DNA left border MCS multiple cloning sites P35S the 35S promoter from cauliflower mosaic virus (CaMV) RB T-DNA right border T35S the 35S terminator of CaMV

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The pCAMBIA vector backbone is derived from the pPZP vectors the SphI site outside the right T-DNA Border or the SacII site outside the left T-DNA Border The smaller polylinkers also eliminate potential conflicts from sites such as SphI (which has an ATG) or XbaI (which has a TAG) Plant selection genes in the pCAMBIA vectors are driven by a double-enhancer version of the CaMV35S promoter and terminated by the CaMV35S polyA signal


































HindIII EcoRI


SmaI

HindIII HindIII


启动子



XbaI

GFP


07kb12kb13kb


AHLG

(12~13kb)

ApaI


SmaI

HindIII HindIII


启动子



XbaI

GFP


07kb12kb13kb


AHLG

(12~13kb)

ApaI
















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HindIII EcoRI


SmaI

HindIII HindIII


启动子



XbaI

GFP


07kb12kb13kb


AHLG

(12~13kb)

ApaI


SmaI

HindIII HindIII


启动子



XbaI

GFP


07kb12kb13kb


AHLG

(12~13kb)

ApaI
















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SmaI

HindIII HindIII


启动子



XbaI

GFP


07kb12kb13kb


AHLG

(12~13kb)

ApaI


SmaI

HindIII HindIII


启动子



XbaI

GFP


07kb12kb13kb


AHLG

(12~13kb)

ApaI
















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pcambia vectors. the pcambia vector backbone is derived from the ppzp vectors. the sphi site outside...

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