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).

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 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: pCAMBIA and plasmids derived from it, where the.1 denotes the absence of a signal peptide from the GUSPlus™ protein; and pCAMBIA 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 pCAMBIA 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, us.

Types Ach agrocinopine, octopine type B6S3, A6.... octopine type Bo leucinopine, succinamopine, agropine type, vir weaker than A281 C58, T nopaline types A 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: Christou P (1997) Rice transformation: bombardment. Plant Mol Biol 35: Deblaere R, Reynaerts A, Hofte H, Hernalsteens JP, Leemans J, and Van Montagu M (1987) Vectors for cloning in plant cells. Meth Enzymol 153: Hajdukiewicz,P, Svab, Z, Maliga, P., (1994) The small versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol Biol 25: 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: 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: 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: Hood EE, Gelvin SB, Melchers S, Hoekema A (1993) New Agrobacterium helper plasmids for gene transfer to plants (EHA105). Trans Res 2: 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: 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: Lazo GR, Stein PA, Ludwig RA (1991) A DNA transformation-competent Ara bidopsis genomic library in Agrobacterium. BioTechnology 9: 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: 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: Porath, J. (1992). Immobilized metal ion affinity chromatography. Protein Expre Purif 3: Siemering KR, Golbik R, Sever R, Haseloff J (1996) Mutations that suppress the thermosensitivity of green fluorescent protein. Curr Biol 6: 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:

GROWTH MEDIA FOR AGROBACTERIUM YEP Per liter: Bacto peptone 10 g NaCl 5 g 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 6 g (or MgSO4 2.9 g) KCl 3 g CaCl2 0.2 g (or CaCl2.2H 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: Copyright © American Society of Plant Biologists. All rights reserved.

HindIII EcoRI

Copyright ©2007 American Society of Plant Biologists Himmelbach, A., et al. Plant Physiol. 2007;145: Schematic representation of the modular binary destination vectors generated

Plant Molecular Biology 40: 711–717, 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.

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.

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:

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)

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.