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) Sm/Sp(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-region::aph, 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) recA::bla, 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).
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pCAMBIA vectors offer:
high copy number in E.coli for high DNA yields
pVS1 replicon for high stability in Agrobacterium
small size, 7-12kb depending on which plasmid
restriction sites designed for modular plasmid modifications and
small but adequate poly-linkers for introducing your DNA of interest
• bacterial selection with chloramphenicol or kanamycin
• plant selection with hygromycin B or kanamycin (phosphinothricin
selection was discontinued at the request of the IP owner, Bayer,
after the initial distribution in 1997)
• simple means to construct translational fusions to gusA reporter
genes
Nomenclature of pCAMBIA vectors
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The four digit numbering system works as follows:
First digit - indicates plant selection: 0 for absence; 1 for hygromycin resistance; 2 for kanamycin; and 3 for
phosphinothricin (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).
Second digit - indicates bacterial selection: 1 for spectinomycin/streptomycin resistance; 2 for chloramphenicol; 3
for kanamycin; 4 for spec/strep and kanamycin.
Third digit - indicates polylinker used: 0 for pUC18 polylinker; 8 for pUC8 polylinker; 9 for pUC9 polylinker.
Fourth digit - indicates reporter gene(s) present: 0 for no reporter gene; 1 for E.coli gusA; 2 for mgfp5; 3 for
gusA:mgfp5 fusion; 4 for mgfp5:gusA fusion; 5 for Staphylococcus sp. gusA (GUSPlus).
Fifth digit - notes some other special feature. So far this has been used only with: pCAMBIA1305.1 and plasmids
derived from it, where the .1 denotes the absence of a signal peptide from the GUSPlus™ protein; and
pCAMBIA1305.2 where the .2 denotes the presence of the GRP signal peptide for in planta secretion of the
GUSPlus™ protein.
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; a/b/c indicates the reading
frame for fusions with the Fuse and Use vectors.
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 don't have, for example, a pCAMBIA2205.2. 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.
Types
Ach5 .......... agrocinopine, octopine type
B6S3, A6 .... octopine type
Bo542 ........ leucinopine, succinamopine, agropine type, vir weaker than A281
C58, T37 .... nopaline types
A281 ......... succinamopine, leucinopine, agrocinopine
Antibiotics
Chloramphenicol, 100 µg/mL for strain AGL1, 10 µg/mL for LBA4404, 25 µg/mL for EHA105 and for E. coli.
Kanamycin, 50 µg/mL for both Agrobacterium and E. coli.
For selection of transformed rice plants we use hygromycin at 50 µg/mL and 25 µg/mL 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 18:25-31
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 9:957-962
Christou P (1997) Rice transformation: bombardment. Plant Mol Biol 35:197-203.
Deblaere R, Reynaerts A, Hofte H, Hernalsteens JP, Leemans J, and Van Montagu M (1987) Vectors for cloning in plant cells. Meth
Enzymol 153:277-292
Hajdukiewicz,P, Svab, Z, Maliga, P., (1994) The small versatile pPZP family of Agrobacterium binary vectors for plant transformation.
Plant Mol Biol 25:989-994
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 6:271-282
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 303:179-180
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 168:1291-1301
Hood EE, Gelvin SB, Melchers S, Hoekema A (1993) New Agrobacterium helper plasmids for gene transfer to plants (EHA105). Trans
Res 2:208-218
Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: Beta-glucuronidase as a sensitive and versatile gene fusion marker in higher
plants. EMBO J 6:3901-3907
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 141:129-136
Lazo GR, Stein PA, Ludwig RA (1991) A DNA transformation-competent Ara bidopsis genomic library in Agrobacterium. BioTechnology
9:963-967
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 31:805-814
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 5:1137-1142
Porath, J. (1992). Immobilized metal ion affinity chromatography. Protein Expre Purif 3:263-281
Siemering KR, Golbik R, Sever R, Haseloff J (1996) Mutations that suppress the thermosensitivity of green fluorescent protein. Curr Biol
6:1653-1663
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 18:6767-6770
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GROWTH MEDIA FOR AGROBACTERIUM
YEP Per liter:
Bacto peptone 10 g
NaCl
5g
Yeast extract
10 g
(Agar)
15 g
No pH adjustment
AB
AB Buffer Per liter (20x stock solution):
K2HPO4
60 g
(or K2HPO4.3H2O
78.6 g)
NaH2PO4
20 g
(or NaH2PO4.H20
23 g)
AB Salts Per liter (20x stock solution):
NH4Cl
20 g
MgSO4.7H20
6g
(or MgSO4
2.9 g)
KCl
3g
CaCl2
0.2 g
(or CaCl2.2H20
0.26 g)
FeSO4.7H20
50 mg
Sterilize the AB Buffer and the AB Salts separately. Shake the salts before use to disperse the FeSO4.
Add sucrose to a final concentration of 0.5%.
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)
• Cloning vectors
Schematic representation of the modular binary destination vectors generated
Himmelbach A. et.al. Plant Physiol. 2010:145:1192-1200
Copyright © 2007. American Society of Plant Biologists. All rights reserved.
HindIII
EcoRI
AHLG
(12~13kb)
SmaI
HindIII
ApaI
HindIII
XbaI
插入的所克隆基因
Actin promotor
1.3kb
GFP
HA tag
1.2kb
0.7kb
启动子
一个融合的TAG,用于纯化蛋白质
待插入的克隆片段
一个融合的GFP片段,用于定位
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.
Schematic representation of the modular binary destination vectors generated
Himmelbach, A., et al. Plant Physiol. 2007;145:1192-1200
Copyright ©2007 American Society of Plant Biologists
bar, gene for phosphinothricin acetyltransferase; nptIII, gene for neomycin phosphotransferase
for 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; RB,right 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 TMV.
The numbers under each DNA region indicate the approximate size of that region in base pairs and the arrow
indicates the orientation.
The superscript number on each restriction site indicates how many times that restriction site occurs in the
indicated plasmid.
Plant Molecular Biology 40: 711–717, 1999.
ags: agropine synthase,
A, ApaI; B,BamHI; Bc, BclI; Bg, BglII; Bx, BstXI;H, HindIII; N, NotI; Nc, NcoI; P,
PstI;RI, EcoRI; SI, SacI; SII, SacII; S, SalI; Sm, SmaI; Sp, SpeI; Xb, XbaI; X, XhoI.
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Aocs,
ocs transcriptional activating element;
AmasPmas, mas2#-activating and promoter
elements; ags-ter, poly(A) addition
signal from the agropine synthase
gene; ADHin, intron from the maize
alcohol dehydrogenase I gene.
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 phosphinothricin/
Basta/Bialophos. Other symbols
and restriction endonuclease sites
are as in the legend to Figure 1. The bar
gene contains KpnI and SalI sites; therefore,
these sites are not unique to
T-DNA binary vectors containing the
bar gene (indicated by [K] and [Sl]). A,
T-DNA binary vectors based on the
pMSP-1 series of plasmids. B, T-DNA
binary vectors based upon the pMSP-2
series of plasmids. C, T-DNA binary
vectors based on the pMSP-3 series of
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
phosphinothricin/Basta/Bialophos
Lee L. et.al. Plant Physiol. 2010:145:1294-1300
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; ubi:
maize Ubiquitin promoter; nptII: neomycin phosphotransferase gene; CaMVter:
CaMV terminator; GUS: -glucuronidase gene; Actin1: rice Actin1 promoter;
ubi 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.