Binary vectors which allow the exchange of plant ...€¦ · selectable markers and reporter genes...
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Nucleic Acids Research, Vol. 18, No. 1 © Oxford University Press 1990 Binary vectors which allow the exchange of plant selectable markers and reporter genes Detlef Becker Institut fur Genetik der Universitat zu Koln, Weyertal 121, D-5000 Koln 41, FRG Submitted November 15, 1989 With the binary vectors pBIG and pBIB it is possible to: i) exchange the plant selectable marker/promoter fusion and the reporter gene, ii) use one vector and Agrobacterium strain system, thus reducing variables associated with using different vectors and strains, iii) allow for divergent transcription of the selectable marker gene and of promoter/reporter gene fusions. The two plasmids are derivatives of the binary vectors pBI 101 and pBIN 19 (1, 2). DNA fragments can be inserted into the T-DNA by the unique restriction sites of a pUC 19 polylinker. The RK2 functions of the plasmids allow the stable maintenance both in E. coli and A. tumefaciens. The two plasmids were constructed by removing the Bam HI site in pBI 101 (1) by filling in with Klenow-polymerase. The resulting plasmid was partially digested with Sst II, blunt ended and subsequently digested with Hind III and isolated on a low melting agarose gel. An Eco RI blunt ended/Bam HI fragment containing the polyadenylation site of gene 7 (3) and a Hind IWBam HI fragment which contains a Nos-promoter/kanamycin and a Nos-promoter/hygromycin gene fusion, respectively, were inserted into the vector, thus producing the vectors pBIG-KAN and pBIG-HYG. The Nos-promoter was taken from pPCV5013 Hyg (4), the neomycin-phosphotransferase (NPT II) gene from pBI 101 (1), and a modified hygromycin B phosphotransferase (HPT) gene was taken from pGDW 11 (5). Subsequently, the GUS-gene was exchanged by the pUC 19 polylinker {Hind WSst I) without a Bam HI site to create pBEB-KAN and pBIB-HYG, respectively. The vectors have been tested by leaf disk transformation of Nicotiana tabacum cv. Petit Havana SRI and were effective in producing resistant plants which produce shoots and roots on MS- medium containing the appropriate antibiotics (kanamycin sulfate 200 /tg/ml; hygromycin B 45 /ig/ml). REFERENCES 1. Jefferson,R.A. et al. (1987) EMBO J. 6, 3901-3907. 2. Bevan.M. (1984) Nucl. Acids Res. 12, 8711-8721. 3. Velten,J. and Schell,J. (1985) Nucl. Acids Res. 13, 6981-6998. 4. Koncz,C, Martini,N., Mayerhofer.R., Koncz-Kalman,Z., Korber.H., Redei.G.P. and SchellJ. (1989) PNAS 86, in press. 5. Wing,D., Koncz,C. and Schell,J. (1989) MGG 219, 9-16. * as CO en pBIG pfl g7 j HPT H HPT iP, •m Q (A IB a t icna. coxin ¥ m O u U GUS Pfir n ¥ W CO pBIB pfi g7 | m XI or HPT rnos •- Q w fl .o E a w XC0Q. COXtOXtQ o u 111 11 pfi nos *shows unique restriction enzyme sites for cloning and the grey box represents the resistance genes for selection of transgenic plants; only the T-DNA is shown, not drawn to scale. 203
Transcript of Binary vectors which allow the exchange of plant ...€¦ · selectable markers and reporter genes...
Nucleic Acids Research, Vol. 18, No. 1 © Oxford University Press 1990
Binary vectors which allow the exchange of plantselectable markers and reporter genes
Detlef BeckerInstitut fur Genetik der Universitat zu Koln, Weyertal 121, D-5000 Koln 41, FRG
Submitted November 15, 1989
With the binary vectors pBIG and pBIB it is possible to: i)exchange the plant selectable marker/promoter fusion and thereporter gene, ii) use one vector and Agrobacterium strain system,thus reducing variables associated with using different vectorsand strains, iii) allow for divergent transcription of the selectablemarker gene and of promoter/reporter gene fusions.
The two plasmids are derivatives of the binary vectors pBI 101and pBIN 19 (1, 2). DNA fragments can be inserted into theT-DNA by the unique restriction sites of a pUC 19 polylinker.The RK2 functions of the plasmids allow the stable maintenanceboth in E. coli and A. tumefaciens.
The two plasmids were constructed by removing the Bam HIsite in pBI 101 (1) by filling in with Klenow-polymerase. Theresulting plasmid was partially digested with Sst II, blunt endedand subsequently digested with Hind III and isolated on a lowmelting agarose gel. An Eco RI blunt ended/Bam HI fragmentcontaining the polyadenylation site of gene 7 (3) and a HindIWBam HI fragment which contains a Nos-promoter/kanamycinand a Nos-promoter/hygromycin gene fusion, respectively, wereinserted into the vector, thus producing the vectors pBIG-KAN
and pBIG-HYG. The Nos-promoter was taken from pPCV5013Hyg (4), the neomycin-phosphotransferase (NPT II) gene frompBI 101 (1), and a modified hygromycin B phosphotransferase(HPT) gene was taken from pGDW 11 (5). Subsequently, theGUS-gene was exchanged by the pUC 19 polylinker {Hind WSstI) without a Bam HI site to create pBEB-KAN and pBIB-HYG,respectively.
The vectors have been tested by leaf disk transformation ofNicotiana tabacum cv. Petit Havana SRI and were effective inproducing resistant plants which produce shoots and roots on MS-medium containing the appropriate antibiotics (kanamycin sulfate200 /tg/ml; hygromycin B 45 /ig/ml).
REFERENCES1. Jefferson,R.A. et al. (1987) EMBO J. 6, 3901-3907.2. Bevan.M. (1984) Nucl. Acids Res. 12, 8711-8721.3. Velten,J. and Schell,J. (1985) Nucl. Acids Res. 13, 6981-6998.4. Koncz,C, Martini,N., Mayerhofer.R., Koncz-Kalman,Z., Korber.H.,
Redei.G.P. and SchellJ. (1989) PNAS 86, in press.5. Wing,D., Koncz,C. and Schell,J. (1989) MGG 219, 9 -16 .
*
as
CO en
pBIG pflg 7j HPT H HPT iP,
•m Q (A IB a t
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GUS Pfir
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pBIB pfig7| m XI or HPT rnos
•- Q w fl .o E a wXC0Q. COXtOXtQ
ou111
11 pfin o s
*shows unique restriction enzyme sites for cloning and the grey box represents the resistance genes for selection of transgenic plants; only the T-DNA is shown,not drawn to scale.
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