Current address: Department of Applied Biology, Gyeongsang National University, Jinju 660-701, Korea.
A novel light-dependent selection marker system in plants
Version of Record online: 23 AUG 2010
© 2010 The Authors. Plant Biotechnology Journal © 2010 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd
Plant Biotechnology Journal
Volume 9, Issue 3, pages 348–358, April 2011
How to Cite
Koh, S., Kim, H., Kim, J., Goo, E., Kim, Y.-J., Choi, O., Jwa, N.-S., Ma, J., Nagamatsu, T., Moon, J. S. and Hwang, I. (2011), A novel light-dependent selection marker system in plants. Plant Biotechnology Journal, 9: 348–358. doi: 10.1111/j.1467-7652.2010.00557.x
tflA (GenBank accession number GQ921834).
- Issue online: 6 MAR 2011
- Version of Record online: 23 AUG 2010
- Received 14 February 2010; revised 21 June 2010; accepted 24 June 2010.
Figure S1 Identification of tflA. A cosmid clone, pJ9, was isolated from a genomic library of P. polymyxa JH2. The 1.2- kb HindIII fragment of pJ90 contained one possible open reading frame encoding tflA.
Figure S2 Alignment of amino acid sequences of TflA with other dioxygenases. Amino acid sequences of TflA were aligned with glyoxalase/bleomycin resistance protein/dioxygenase from Exiguobacterium sp. 255-15 (gi172057369), hypothetical protein from Bacillus halodurans C-125 (gi15614705), NahC from Bacillus sp. JF8 (gi41349767), NahH from Bacillus sp. JF8 (gi41349762), ThnC from Sphingopyxis macrogoltabida TFA (gi5817852) and BphC from Pseudomonas sp. LB400 (giABE37053.1). Three conserved residues of all 7 sequences are boxed and indicated by asterisks. The sequence alignment was performed using DNAMAN (Lynnon Co., Quebec, Canada).
Figure S3 Homology tree of TflA with other dioxygenases. The tree shows glyoxalase/bleomycin resistance protein/dioxygenase from Exiguobacterium sp. 255-15 (gi172057369) and hypothetical protein from Bacillus halodurans C-125 (gi15614705) that are closely related to TflA from P. polymixa JH2.
Figure S4 Purification of His-TflA protein. Overexpression of His-tagged TflA protein in E.coli BL21 (DE3) was induced by 1 mm IPTG and purified by Ni-NTA column by washing 20 mm imidazole. Purified His-TflA was shown in a Coomassie blue-stained SDS-PAGE. Lane 1, molecular mass 2 standards; lane 2, total cell extracts of BL21 (DE3) (pLysS/pH904) before IPTG induction; lane 3, total cell extracts of BL21 (DE3) (pLysS/pH904) after 1 mm IPTG induction; lane 4, soluble fraction; lane 5, flow through; lane 6, wash with 20 mm imidazole; lane 7, final elution containing His-TflA through a Ni-NTA column. Separation was on a 12% SDS-polyacrylamide gel. Bands were visualized after staining with Coomassie blue. The band corresponding to His-TflA is indicated.
Figure S5 Toxoflavin degradation activity at different concentration of purified His-TflA. All lanes contained 100 μm toxoflavin. Lane 1, no purified His-TflA; lane 2, 1 μm purified His-TflA; lane 3, 2 μm purified His-TflA; lane 4, 5 μm purified His-TflA; lane 5, 10 μm purified His-TflA.
Figure S6 Substrate specificity of purified His-TflA to toxoflavin and its derivatives. 100 μm of each substrate was tested either with or without 5 μm of purified toxoflavin. Degradation activity of purified toxoflavin was determined as − (0–20%) to +++ (80–100%).
Figure S7 Southern blot analysis of transgenic rice (T2) lines. Genomic DNA (15 μg) of all Dongjinbyeo transgenic rice plants was digested with EcoRI and separated in 0.8% agarose gel. The 0.7- kb PCR product containing tflA was used as a probe. M: DNA marker. 1, Dt1; 2, Dt4; 3, Dt5; 4, Dt7; 5, Dt19; 6, Dt40; 7, Dt18; 8, Dt19; 9, Dt16; 10, Dt15; 11, Dt9; 12, pJ904.
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