p Cambia Vectors The p CAMBIA vector backbone
p. Cambia Vectors
The p. CAMBIA vector backbone is derived from the p. PZP vectors. the Sph. I site outside the right T-DNA Border, or the Sac. II site outside the left T-DNA Border. The smaller polylinkers also eliminate potential conflicts from sites such as Sph. I (which has an ATG) or Xba. I (which has a TAG). Plant selection genes in the p. CAMBIA vectors are driven by a double-enhancer version of the Ca. MV 35 S promoter and terminated by the Ca. MV 35 S poly. A signal.
1. LBA 4404 (Ach 5 p. Ti. Ach 5) Sm/Sp(R) in the virulence plasmid (from Tn 904); all T-DNA of p. Ti. Ach 5 eliminated in p. AL 4404 (Hoekema et al. , 1983). 2. EHA 101, genotype C 58 p. Ti. Bo 542; T-region: : aph, Km(R); A 281 derivative harboring p. EHA 101, T-DNA replaced with npt. II, elimination of T-DNA boundaries uncofirmed, super-virulent (Hood et al. , 1986). 3. EHA 105 is a Km(S) derivative of EHA 101 (Hood et al. , 1993). 4. AGL 1, genotype is AGL 0 (C 58 p. Ti. Bo 542) rec. A: : bla, T-region deleted Mop(+) Cb(R) [AGL 0 is an EHA 101 with the T-region deleted, which also deletes the aph gene] (Lazo et al. , 1991). 5. A 281, reconstructed strain, derivative of A 136 (cured C 58) harboring p. Ti. Bo 542, super-virulent (Hood et al. , 1986).
• • • p. CAMBIA vectors offer: high copy number in E. coli for high DNA yields p. VS 1 replicon for high stability in Agrobacterium small size, 7 -12 kb 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 gus. A reporter genes
Nomenclature of p. CAMBIA 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 p. UC 18 polylinker; 8 for p. UC 8 polylinker; 9 for p. UC 9 polylinker. Fourth digit - indicates reporter gene(s) present: 0 for no reporter gene; 1 for E. coli gus. A; 2 for mgfp 5; 3 for gus. A: mgfp 5 fusion; 4 for mgfp 5: gus. A fusion; 5 for Staphylococcus sp. gus. A (GUSPlus). Fifth digit - notes some other special feature. So far this has been used only with: p. CAMBIA 1305. 1 and plasmids derived from it, where the. 1 denotes the absence of a signal peptide from the GUSPlus™ protein; and p. CAMBIA 1305. 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 lac. Za 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 p. CAMBIA 2205. 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 p. CAMBIA vector derivative that other researchers will find useful and you want to share with other researchers, email us.
Types Ach 5. . agrocinopine, octopine type B 6 S 3, A 6. . octopine type Bo 542. . . . leucinopine, succinamopine, agropine type, vir weaker than A 281 C 58, T 37. . nopaline types A 281. . succinamopine, leucinopine, agrocinopine Antibiotics Chloramphenicol, 100 µg/m. L for strain AGL 1, 10 µg/m. L for LBA 4404, 25 µg/m. L for EHA 105 and for E. coli. Kanamycin, 50 µg/m. L for both Agrobacterium and E. coli. For selection of transformed rice plants we use hygromycin at 50 µg/m. L and 25 µg/m. L for tobacco. Minimal Selection Vectors p. CAMBIA 1200; p. CAMBIA 1380; p. CAMBIA 1390; p. CAMBIA 2200; p. CAMBIA 2300
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 p. PZP 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 A 281 is encoded in a region of p. Ti. Bo 542 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 (EHA 105). 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 Tn 904 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. Bio. Technology 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 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 m. RNA and an efficient splicing of the intron. Nucl Acids Res 18: 6767 -6770
• • • GROWTH MEDIA FOR AGROBACTERIUM YEP Per liter: Bacto peptone 10 g Na. Cl 5 g Yeast extract 10 g (Agar) 15 g No p. H adjustment AB AB Buffer Per liter (20 x stock solution): K 2 HPO 4 60 g (or K 2 HPO 4. 3 H 2 O 78. 6 g) Na. H 2 PO 4 20 g (or Na. H 2 PO 4. H 20 23 g) AB Salts Per liter (20 x stock solution): NH 4 Cl 20 g Mg. SO 4. 7 H 20 6 g (or Mg. SO 4 2. 9 g) KCl 3 g Ca. Cl 2 0. 2 g (or Ca. Cl 2. 2 H 20 0. 26 g) Fe. SO 4. 7 H 20 50 mg Sterilize the AB Buffer and the AB Salts separately. Shake the salts before use to disperse the Fe. SO 4. Add sucrose to a final concentration of 0. 5%.
MCS: Eco. RI(10578), Sac. I(10584), Kpn. I(10590), , Sma. I(10594), Bam. HI(10599), Xba. I(10605), Sal. I(10611) Plant kanamycin selection replaces hygromycin in pcambia 1300 双T载体p. CDMAR-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.
Hind. III Eco. RI
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; npt. III, gene for neomycin phosphotransferase for kanamycin resistance (from p. BIN 19); ori. V, part of RK 2 origin of replication (from p. BIN 19); P 35 S , 35 S promoter of cauliflower mosaic virus; P 35 S 2, 35 S 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; trf. A, part of RK 2 origin of replication; ubi intron, intron-1 from maize ubiquitin-1 gene; uid. A, 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, Apa. I; B, Bam. HI; Bc, Bcl. I; Bg, Bgl. II; Bx, Bst. XI; H, Hind. III; N, Not. I; Nc, Nco. I; P, Pst. I; RI, Eco. RI; SI, Sac. I; SII, Sac. II; S, Sal. I; Sm, Sma. I; Sp, Spe. I; Xb, Xba. I; X, Xho. I.
• • • • • • • Aocs, ocs transcriptional activating element; Amas. Pmas, mas 2#-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; t. Ag 7, poly(A) addition signal for T-DNA gene 7; hpt. II, gene conferring resistance to hygromycin; npt. II, 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 Kpn. I and Sal. I 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 p. MSP-1 series of plasmids. B, T-DNA binary vectors based upon the p. MSP-2 series of plasmids. C, T-DNA binary vectors based on the p. MSP-3 series of plasmids.
Maps of the T-DNA binary vectors based upon the p. MSP series of plasmids. p. Exxxx 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; t. Ag 7, poly(A) addition signal for T-DNA gene 7; hpt. II, gene conferring resistance to hygromycin; npt. II, gene conferring resistance to kanamycin; bar, gene conferring resistance to phosphinothricin/Basta/Bialophos Lee L. et. al. Plant Physiol. 2010: 145: 1294 -1300
A schematic diagram of the T-DNA region from the binary vector p. CAS 04. RB: the right border of T-DNA; LB: the left border of T-DNA; ubi: maize Ubiquitin promoter; npt. II: neomycin phosphotransferase gene; Ca. MVter: Ca. MV terminator; GUS: �� -glucuronidase gene; Actin 1: rice Actin 1 promoter; ubi Ist intron: the first intron of ubiquitin promoter. is derived from the binary vector p. EP 200. Chu C J. Genet. Genomics 36 (2009) 267 -276
The Os. Act 2 -based expression vectors for use in monocot transformation. Cyt 108, 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; P 35 S, the 35 S promoter from cauliflower mosaic virus (Ca. MV); RB, T-DNA right border; T 35 S, the 35 S terminator of Ca. MV.
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