Feasibility of the Seed Specific Cruciferin C Promoter in the Self Excision Cre/loxP Strategy Focused on Generation of Marker-free Transgenic Plants

Overview

Journal Theor Appl Genet

Publisher Springer

Specialty Genetics

Date 2008 Sep 10

PMID 18779945

Citations 14

Authors

Jana Moravcikova

Eva Vaculkova

Miroslav Bauer

Jana Libantova

Affiliations

Soon will be listed here.

Abstract

This work is focused on the generation of selectable marker-free transgenic tobacco plants using the self excision Cre/loxP system that is controlled by a strong seed specific Arabidopsis cruciferin C (CRUC) promoter. It involves Agrobacterium-mediated transformation using a binary vector containing the gus reporter gene and one pair of the loxP sites flanking the cre recombinase and selectable nptII marker genes (floxed DNA). Surprisingly, an ectopic activation of CRUC resulting in partial excision of floxed DNA was observed during regeneration of transformed cells already in calli. The regenerated T(0) plants were chimeric, but no ongoing ectopic expression was observed in these one-year-long invitro maintained plants. The process of the nptII removal was expected in the seeds; however, none of the analysed T(0) transgenic lines generated whole progeny sensitive to kanamycin. Detailed analyses of progeny of selected T(0)-30 line showed that 10.2% GUS positive plants had completely removed nptII gene while the remaining 86.4% were still chimeras. Repeated activation of the cre gene in T(2) seeds resulted in increased rate of marker-free plants, whereas four out of ten analysed chimeric T(1) plants generated completely marker-free progenies. This work points out the feasibility as well as limits of the CRUC promoter in the Cre/loxP strategy.

Citing Articles

Engineered Expression of Vip3A in Green Tissues as a Feasible Approach for the Control of Insect Pests in Maize.

Yuan G, Zeng C, Shi H, Yang Y, Du J, Zou C Insects. 2023; 14(10).

PMID: 37887815 PMC: 10607264. DOI: 10.3390/insects14100803.

Effect of marker-free transgenic Chlamydomonas on the control of Aedes mosquito population and on plankton.

Fei X, Huang X, Li Z, Li X, He C, Xiao S Parasit Vectors. 2023; 16(1):18.

PMID: 36653886 PMC: 9847121. DOI: 10.1186/s13071-022-05647-3.

An Efficient Gene Excision System in Maize.

Wang N, Arling M, Hoerster G, Ryan L, Wu E, Lowe K Front Plant Sci. 2020; 11:1298.

PMID: 32983193 PMC: 7492568. DOI: 10.3389/fpls.2020.01298.

Recent Advances of In Vitro Culture for the Application of New Breeding Techniques in Citrus.

Poles L, Licciardello C, Distefano G, Nicolosi E, Gentile A, La Malfa S Plants (Basel). 2020; 9(8).

PMID: 32722179 PMC: 7465985. DOI: 10.3390/plants9080938.

Heat-Shock-Induced Removal of Transgenes Using the Gene-Deletor System in Hybrid Aspen ( × ).

Wang B, Zhang Y, Zhao J, Dong M, Zhang J Genes (Basel). 2018; 9(10).

PMID: 30297683 PMC: 6210648. DOI: 10.3390/genes9100484.

References

van Engelen F, Molthoff J, Conner A, Nap J, Pereira A, Stiekema W . pBINPLUS: an improved plant transformation vector based on pBIN19. Transgenic Res. 1995; 4(4):288-90. DOI: 10.1007/BF01969123. View

Trindade L, Horvath B, Bachem C, Jacobsen E, Visser R . Isolation and functional characterization of a stolon specific promoter from potato (Solanum tuberosum L.). Gene. 2003; 303:77-87. DOI: 10.1016/s0378-1119(02)01147-2. View

Mlynarova L, Nap J . A self-excising Cre recombinase allows efficient recombination of multiple ectopic heterospecific lox sites in transgenic tobacco. Transgenic Res. 2003; 12(1):45-57. DOI: 10.1023/a:1022112221427. View

Zheng X, Deng W, Luo K, Duan H, Chen Y, McAvoy R . The cauliflower mosaic virus (CaMV) 35S promoter sequence alters the level and patterns of activity of adjacent tissue- and organ-specific gene promoters. Plant Cell Rep. 2007; 26(8):1195-203. DOI: 10.1007/s00299-007-0307-x. View

Mlynarova L, Conner A, Nap J . Directed microspore-specific recombination of transgenic alleles to prevent pollen-mediated transmission of transgenes. Plant Biotechnol J. 2006; 4(4):445-52. DOI: 10.1111/j.1467-7652.2006.00194.x. View

Becerra C, Puigdomenech P, Vicient C . Computational and experimental analysis identifies Arabidopsis genes specifically expressed during early seed development. BMC Genomics. 2006; 7:38. PMC: 1420293. DOI: 10.1186/1471-2164-7-38. View

Zimmermann P, Hirsch-Hoffmann M, Hennig L, Gruissem W . GENEVESTIGATOR. Arabidopsis microarray database and analysis toolbox. Plant Physiol. 2004; 136(1):2621-32. PMC: 523327. DOI: 10.1104/pp.104.046367. View

Hoff T, Schnorr K, Mundy J . A recombinase-mediated transcriptional induction system in transgenic plants. Plant Mol Biol. 2001; 45(1):41-9. DOI: 10.1023/a:1006402308365. View

Jefferson R, Kavanagh T, Bevan M . GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 1987; 6(13):3901-7. PMC: 553867. DOI: 10.1002/j.1460-2075.1987.tb02730.x. View

10.

Suzuki M, Kao C, Cocciolone S, McCarty D . Maize VP1 complements Arabidopsis abi3 and confers a novel ABA/auxin interaction in roots. Plant J. 2001; 28(4):409-18. DOI: 10.1046/j.1365-313x.2001.01165.x. View

11.

Coppoolse E, de Vroomen M, Roelofs D, Smit J, van Gennip F, Hersmus B . Cre recombinase expression can result in phenotypic aberrations in plants. Plant Mol Biol. 2003; 51(2):263-79. DOI: 10.1023/a:1021174726070. View

12.

Belostotsky D, Meagher R . A pollen-, ovule-, and early embryo-specific poly(A) binding protein from Arabidopsis complements essential functions in yeast. Plant Cell. 1996; 8(8):1261-75. PMC: 161239. DOI: 10.1105/tpc.8.8.1261. View

13.

Li Z, Xing A, Moon B, Burgoyne S, Guida A, Liang H . A Cre/loxP-mediated self-activating gene excision system to produce marker gene free transgenic soybean plants. Plant Mol Biol. 2007; 65(3):329-41. DOI: 10.1007/s11103-007-9223-2. View

14.

Marjanac G, De Paepe A, Peck I, Jacobs A, De Buck S, Depicker A . Evaluation of CRE-mediated excision approaches in Arabidopsis thaliana. Transgenic Res. 2007; 17(2):239-50. DOI: 10.1007/s11248-007-9096-9. View

15.

Stuitje A, Verbree E, van der Linden K, Mietkiewska E, Nap J, Kneppers T . Seed-expressed fluorescent proteins as versatile tools for easy (co)transformation and high-throughput functional genomics in Arabidopsis. Plant Biotechnol J. 2006; 1(4):301-9. DOI: 10.1046/j.1467-7652.2003.00028.x. View

16.

Luo K, Duan H, Zhao D, Zheng X, Deng W, Chen Y . 'GM-gene-deletor': fused loxP-FRT recognition sequences dramatically improve the efficiency of FLP or CRE recombinase on transgene excision from pollen and seed of tobacco plants. Plant Biotechnol J. 2007; 5(2):263-274. DOI: 10.1111/j.1467-7652.2006.00237.x. View

17.

Bradford M . A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976; 72:248-54. DOI: 10.1016/0003-2697(76)90527-3. View

18.

Schmid M, Davison T, Henz S, Pape U, Demar M, Vingron M . A gene expression map of Arabidopsis thaliana development. Nat Genet. 2005; 37(5):501-6. DOI: 10.1038/ng1543. View

19.

Hoglund A, Rodin J, Larsson E, Rask L . Distribution of napin and cruciferin in developing rape seed embryos. Plant Physiol. 1992; 98(2):509-15. PMC: 1080218. DOI: 10.1104/pp.98.2.509. View

20.

Verweire D, Verleyen K, De Buck S, Claeys M, Angenon G . Marker-free transgenic plants through genetically programmed auto-excision. Plant Physiol. 2007; 145(4):1220-31. PMC: 2151720. DOI: 10.1104/pp.107.106526. View