Abstract
Despite the advances in transgenesis, transformation technologies still rely on the introduction of a selectable marker gene to identify cells and tissues that have integrated the gene of interest in their genome. The continuous presence of the marker genes in the transgenics is often controversial as it can potentially have multiple undesirable impacts. The present study employed the self-excising Cre-loxP system to generate marker-free Arabidopsis thaliana expressing the agronomically important glyoxalase I (glyI) gene from Brassica juncea to confer salt stress tolerance. A binary vector was constructed wherein the salt-inducible rd29A promoter was used to drive the expression of the glyI gene and the transformants of A. thaliana were recovered using kanamycin resistance as the selectable marker. The neomycin phosphotransferase II (nptII) gene was flanked by the loxP sites followed by the introduction of a heat-inducible Cre-recombinase in between the loxP sites. The kanamycin-resistant transgenic lines of A. thaliana using this vector showed an ability to withstand stress imposed by 150 mM NaCl. The exposure of the T2 transgenic lines to a mild heat shock (37°C) resulted in the recovery of salt-tolerant, kanamycin-sensitive T3 progeny. Molecular analyses of the T3 transgenic lines following the heat shock treatment confirmed the excision of the nptII gene and the completion of their life cycle in the presence of 150 mM NaCl-induced stress.
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Abbreviations
- ABA:
-
Abscisic acid
- ABRE:
-
ABA- responsive element
- Cre:
-
Cre-recombinase
- DRE:
-
Drought responsive element
- gly I:
-
Glyoxalase I
- hsp:
-
Heat shock promoter
- MS:
-
Murashige and Skoog
- SPB:
-
Sodium Phosphate Buffer
- GSH:
-
Reduced glutathione
- PMSF:
-
Phenylmethylsulfonylfluoride
- PVPP:
-
Polyvinyl polypyrrolidone
References
Alscher RG (1989) Biosynthesis and antioxidant function of glutathione in plants. Physiol Plant 77:457–464. doi:10.1111/j.1399-3054.1989.tb05667.x
Arumugam N, Gupta V, Jagannath A, Mukhopadhyay, Pradhan AK, Burma PK and Pental D (2006) A passage through in vitro culture leads to efficient production of marker-free transgenic plants in Brassica juncea using the Cre-loxP system. Trans Res 16:703–712. doi:10.1007/s11248-006-9058-7
Bechtold N, Ellis J, Pelletier G (1993) In planta Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. CR Acad Sci Paris Life Sci 316:1194–1199
Blumwald E, Grover A, Good AG (2004) Breeding for abiotic stress tolerance: challenges and opportunities. Proc. 4th Int. Crop Science Congress
Chen S, Li X, Liu X, Xu H, Meng K, Xiao G et al (2005) Green fluorescent protein as a vital elimination marker to easily screen marker-free transgenic progeny derived from plants co-transformed with double T-DNA binary vector system. Plant Cell Rep 23:625–631. doi:10.1007/s00299-004-0853-4
Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159. doi:10.1016/0003-2697(87)90021-2
Coppoolse ER, de Vroomen MJ, Roelofs D, Smit J, van Gennip F, Hersmus BJ et al (2003) Cre recombinase expression can result in phenotypic aberrations in plants. Plant Mol Biol 51:263–279. doi:10.1023/A:1021174726070
Cuellar W, Gaudin A, Solorzano D, Casas A, Nopo L, Chudalayandi P et al (2006) Self-excision of the antibiotic resistance gene nptII using a heat inducible Cre-loxP system for transgenic potato. Plant Mol Biol 62:71–82. doi:10.1007/s11103-006-9004-3
Dale EC, Ow DW (1991) Gene transfer with subsequent removal of the selection gene from the host genome. Proc Natl Acad Sci USA 88:10558–10562. doi:10.1073/pnas.88.23.10558
Esparteo J, Sanchez-Aguayo I, Pardo JM (1995) Molecular characterization of glyoxalase I from a higher plant; upregulated by stress. Plant Mol Biol 29:1223–1233. doi:10.1007/BF00020464
Goldsbrough AP, Lastrella CN, Yoder JI (1993) Transposition mediated re-positioning and subsequent elimination of marker genes from transgenic tomato. Bio/technology 11:1286–1292
Hill R, Sendashonga C (2006) Conservation biology, genetically modified organism, and the biosafety protocol. Conserv Biol 20(6):1620–1625. doi:10.1111/j.1523-1739.2006.00534.x
Hoa TT, Bong BB, Huq E, Hodges TK (2002) Cre-lox site specific recombination controls the excision of a transgene from the rice genome. Theor Appl Genet 104:518–525. doi:10.1007/s001220100748
Hoff T, Schnorr KM, Mundy J (2001) A recombinase–mediated transcriptional induction system in transgenic plants. Plant Mol Biol 45:41–49. doi:10.1023/A:1006402308365
Iamtham S, Day A (2000) Removal of antibiotic resistance genes from transgenic tobacco plastids. Nat Biotechnol 18:1172–1176
Li Z, Xing A, Moon BP, Burgoyne SA, Guida AD, Liang H et al (2007) A Cre/loxP- mediated self-activating gene excision system to produce marker gene free transgenic soyabean plants. Plant Mol Biol 65:329–341. doi:10.1007/s11103-007-9223-2
Marjanac G, De Paepe A, Peck I, Jacobs A, De Buck S, Depicker A (2008) Evaluation of CRE-mediated excision approaches in Arabidopsis thaliana. Transgenic Res 17:239–250
Matthews PR, Wang MB, Waterhouse PM, Thornton S, Fieg SJ, Gubler F et al (2001) Marker gene elimination from transgenic barley, using co-transformation with adjacent ‘twin T-DNA’ on a standard Agrobacterium transformation vector. Mol Breed 7:195–202. doi:10.1023/A:1011333321893
Murray GC, Thompson WF (1980) Rapid isolation of high molecular weight DNA. Nucl Acid Res 8:4321–4325
Park J, Lee YK, Kang BK, Chung WH (2004) Co-transformation using a negative selectable marker gene for the production of selectable marker gene-free transgenic plants. Theor Appl Genet 109:1562–1567. doi:10.1007/s00122-004-1790-x
Pooggin M, Shivaprasad PV, Veluthambi K, Hohn T (2003) RNAi targeting of DNA virus in plants. Nat Biotechnol 21:131–132. doi:10.1038/nbt0203-131b
Ramaswamy O, Guha-Mukherjee S, Sopory SK (1983) Presence of glyoxalase I in pea. Biochem Int 7:307–318
Russell SH, Hoopes JL, Odell JT (1992) Directed excision of a transgene from the plant genome. Mol Gen Genet 234:49–59
Sambrook J, Fritsch EF, Maniatis T (1989) In Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
Singla-Pareek SL, Reddy M, Sopory SK (2003) Genetic engineering of the glyoxalase pathway in tobacco leads to enhanced salinity tolerance. Proc Natl Acad Sci USA 100(25):14672–14677. doi:10.1073/pnas.2034667100
Singla-Pareek SL, Yadav SK, Pareek A, Reddy MK, Sopory SK (2006) Transgenic tobacco overexpressing glyoxalase pathway enzymes grow and set viable seeds in zinc spiked soils. Plant Physiol 140:613–623. doi:10.1104/pp.105.073734
Singla-Pareek SL, Yadav SK, Pareek A, Reddy MK, Sopory SK (2008) Enhancing salt tolerance in a crop plant by overexpression of glyoxalase II. Trans. Res. 17:171–180. doi:10.1007/s11248-007-9082-2
Sreekala C, Wu L, Gu K, Wang D, Tian D, Yin Z (2005) Excision of selectable marker in transgenic rice (Oryza sativa L.) using a chemically regulated Cre/loxP system. Plant Cell Rep 24:86–94. doi:10.1007/s00299-004-0909-5
Stemberg N, Sauer B, Hoess R, Abremski K (1986) Bacteriophage P1 cre gene and its regulatory region. Evidence for multiple promoters and regulation by DNA methylation. J Mol Biol 187:197–212. doi:10.1016/0022-2836(86)90228-7
Sternberg N, Hamilton D (1981) Bacteriophage P1 site-specific recombination between lox P sites. J Mol Biol 150:467–486. doi:10.1016/0022-2836(81)90375-2
Thornalley PJ (1990) Glyoxalase system: new developments towards functional characterization of metabolic pathways fundamental to biological life. Biochem J Kasu 269:1–11
Uotila L (1989) Glutathione thiol esterases. In: Dolphin D, Poulson R, Avramovic O (eds) Glutahione: Chemical, Biochemical and Medical Aspects, Coenzymes and Cofactors; Vol III. Part A. Wiley-Interscience, New York, pp 767–804
Veena, Reddy VS, Sopory SK (1999) Glyoxalase I from Brassica juncea: molecular cloning, regulation and its overexpression confer tolerance in transgenic tobacco under stress. Plant J 17:385–395
Wang Y, Chen B, Hu Y, Li J, Lin Z (2005) Inducible excision of selectable marker gene from transgenic plants by the cre/lox site-specific recombination system. Transgenic Res 14:605–614
Yamaguchi-Shinozaki K, Shinozaki K (1993a) Characterization of the expression of a desiccation-responsive rd29 gene of Arabidopsis thaliana and analysis of its promoter in transgenic plants. Mol Gen Genet 236:331–340. doi:10.1007/BF00277130
Yamaguchi-Shinozaki K, Shinozaki K (1993b) Arabidopsis DNA encoding two dessication–responsive rd29 genes. Plant Physiol 101:1119–1120. doi:10.1104/pp.101.3.1119
Yamaguchi-Shinozaki K, Shinozaki K (1994) A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, low temperature or high salt stress. Plant Cell 6:251–254
Zhang W, Subbarao S, Addae P, Shen A, Armstrong C, Peschke V et al (2003) Cre/lox-mediated marker gene excision in transgenic maize (Zea mays L.) plants. Theor Appl Genet 107:1157–1168. doi:10.1007/s00122-003-1368-z
Zuo J, Niu QW, Moller SG, Chua NH (2001) Chemical-regulated, site specific DNA excision in transgenic plants. Nat Biotechnol 19:157–161. doi:10.1038/84428
Acknowledgements
We thank Prof. S. K. Sopory I.C.G.E.B., New Delhi, for the gift of the glyI cDNA, Prof. John Mundy, University of Copenhagen, Denmark, for the pCrox vector, Prof. Barbara Hohn, F.M.I., Switzerland for discussions and suggestions and Dr. Mohd. Aslam Yusuf and Mr. Ravi Rajwanshi, Jawaharlal Nehru University, New Delhi for their valuable inputs. Thanks are due to Prof. Anna Depicker for her help with advice and vectors. This research project has been implemented with financial contributions from the Swiss Agency for Development and Cooperation, Government of Switzerland and the Department Biotechnology, Government of India under the Indo-Swiss Collaboration of Biotechnology.
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Roy, S.D., Saxena, M., Bhomkar, P.S. et al. Generation of marker free salt tolerant transgenic plants of Arabidopsis thaliana using the gly I gene and cre gene under inducible promoters. Plant Cell Tiss Organ Cult 95, 1–11 (2008). https://doi.org/10.1007/s11240-008-9402-0
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DOI: https://doi.org/10.1007/s11240-008-9402-0