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T-DNA direct repeat and 35S promoter methylation affect transgene expression but do not cause silencing in transgenic sweet orange

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Abstract

In transgenic plants, multiple T-DNA copies and cytosine methylation usually correlate with a loss of transgene expression while methylation of promoter is associated with transgene silencing. Here, six independent GFP transgenic ‘Bingtang’ sweet orange (Citrus sinensis (L.) Osb.) plants were analyzed with copy number, T-DNA repeat, cytosine methylation and transgene expression. The fluorescence of transgenic plants was normal and bright except that the transgenic B4 plant showed mottled fluorescence. Five of the six transgenic plants had multiple transgene copies and three transgenic plants had direct repeat sequences. The degrees of cytosine methylation within GFP and CaMV 35S promoter region were higher in multiple copies than in single copy transgenic plant B6. Cytosine methylation of nptII in multiple copies transgenic plants except B4 was slightly greater than single copy transgenic plant B6. Furthermore, transgenic B4 plant with mottled GFP expression had the highest degree of methylation of CG cytosine pattern. The transcript level of GFP and nptII was lower in three transgenic plants (B1, B2 and B3) with direct repeat, as revealed by real-time RT–PCR analysis. These results suggest that multiple T-DNA copies and methylation of CaMV 35S promoter affect transgene expression levels but do not cause gene silencing in transgenic sweet orange.

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Abbreviations

GFP:

Green fluorescent protein

nptII:

Neomycin phosphotransferase II

PCR:

Polymerase chain reaction

CaMV:

Cauliflower mosaic virus

RB:

Right border

LB:

Left border

References

  • Ahuja MR (2009) Transgene stability and dispersal in forest trees. Trees 23:1125–1135

    Article  CAS  Google Scholar 

  • Brodersen P, Voinnet O (2006) The diversity of RNA silencing pathways in plants. Trends Genet 22:269–278

    Article  Google Scholar 

  • Brunner AM, Li J, Di Fazio SP, Shevchenko O, Montgomery BE, Mohamed R, Wei H, Ma C, Elias AA, Van Wormer K, Strauss SH (2007) Genetic containment of forest plantations. Tree Genet Genome 3:75–100

    Article  Google Scholar 

  • Butaye KMJ, Cammue BPA, Delaure SL, De Bolle MFC (2005) Approaches to minimize variation of transgene expression in plants. Mol Breed 16:79–91

    Article  Google Scholar 

  • Carthew RW, Sontheimer EJ (2009) Origins and mechanisms of miRNAs and siRNAs. Cell 136:642–655

    Article  PubMed  CAS  Google Scholar 

  • Cervera M, Pina JA, Juarez J, Navarro L, Peña L (2000) A broad exploration of a transgenic population of citrus: stability of gene expression and phenotype. Theor Appl Genet 100:670–677

    Article  CAS  Google Scholar 

  • Chawla R, Ariza Nieto M, Wilson AJ, Moore SK, Srivastava V (2006) Transgene expression produced by biolistic-mediated, site-specific gene integration is consistently inherited by the subsequent generations. Plant Biotechnol J 4:209–218

    Article  PubMed  CAS  Google Scholar 

  • Cheng YJ, Guo WW, Yi HL, Pang XM, Deng XX (2003) An efficient protocol for genomic DNA extraction from Citrus species. Plant Mol Biol Rep 21:177a–177g

    Article  Google Scholar 

  • Diéguez MJ, Vaucheret H, Paszkowski J, Mittelsten SO (1998) Cytosine methylation at CG and CNG sites is not a prerequisite for the initiation of transcriptional gene silencing in plants, but it is required for its maintenance. Mol Gen Genet 259:207–215

    Article  PubMed  Google Scholar 

  • Domínguez A, Fagoaga C, Navarro L, Moreno P, Peña L (2002) Regeneration of transgenic citrus plants under non selective conditions results in high-frequency recovery of plants with silenced transgenes. Mol Gen Genet 267:544–556

    Google Scholar 

  • Domínguez A, Cervera M, Pérez RM, Romero J, Fagoaga C, Cubero J, López MM, Juárez JA, Navarro L, Peña L (2004) Characterisation of regenerants obtained under selective conditions after Agrobacterium-mediated transformation of citrus explants reveals production of silenced and chimeric plants at unexpected high frequencies. Mol Breed 14:171–183

    Article  Google Scholar 

  • Duan YX, Liu X, Fan J, Li DL, Wu RC, Guo WW (2007) Multiple shoot induction from seedling epicotyls and transgenic citrus plant regeneration containing the green fluorescent protein gene. Bot Stud 48:165–171

    Google Scholar 

  • Duan YX, Fan J, Guo WW (2010) Regeneration and characterization of transgenic kumquat plants containing the Arabidopsis APETALA1 gene. Plant Cell Tiss Organ Cult 100:273–281

    Article  CAS  Google Scholar 

  • Dutt M, Grosser JW (2009) Evaluation of parameters affecting Agrobacterium-mediated transformation of citrus. Plant Cell Tiss Organ Cult 98:331–340

    Article  CAS  Google Scholar 

  • Eamens A, Wang MB, Smith NA, Waterhouse PM (2008) RNA silencing in plants: yesterday, today, and tomorrow. Plant Physiol 147:456–468

    Article  PubMed  CAS  Google Scholar 

  • Fojtová M, Bleys A, Bedřichová J, Van Houdt H, Křížová K, Depicker A, Kovařík A (2006) The trans-silencing capacity of invertedly repeated transgenes depends on their epigenetic state in tobacco. Nucleic Acids Res 34:2280–2293

    Article  PubMed  Google Scholar 

  • Gambino G, Chitarra W, Maghuly F, Laimer M, Boccacci P, Marinoni DT, Gribaudo I (2009) Characterization of T-DNA insertions in transgenic grapevines obtained by Agrobacterium-mediated transformation. Mol Breed 24:305–320

    Article  CAS  Google Scholar 

  • Gambino G, Perrone I, Carra A, ChitarraW Boccacci P, Marinoni DT, Barberis M, Maghuly F, Laimer M, Gribaudo I (2010) Transgene silencing in grapevines transformed with GFLV resistance genes: analysis of variable expression of transgene, siRNAs production and cytosine methylation. Transgenic Res 19:17–27

    Article  PubMed  CAS  Google Scholar 

  • Goll MG, Anderson R, Stainier DYR, Spradling AC, Halpern ME (2009) Transcriptional silencing and reactivation in transgenic Zebrafish. Genetics 182:747–755

    Article  PubMed  CAS  Google Scholar 

  • Gruntman E, Qi YJ, Slotkin RK, Roeder T, Martienssen RA, Sachidanandam R (2008) Kismeth: Analyzer of plant methylation states through bisulfite sequencing. BMC Bioinformatics 9:371

    Article  PubMed  Google Scholar 

  • Hawkins S, Leple JC, Cornu D, Jouanin L, Pilate G (2003) Stability of transgene expression in poplar: a model forest tree species. Ann For Sci 60:427–438

    Article  Google Scholar 

  • Hsieh J, Fire A (2000) Recognition and silencing of repeated DNA. Annu Rev Genet 34:187–204

    Article  PubMed  CAS  Google Scholar 

  • Klimaszewska K, Lachance D, Bernier-Cardou M, Rutledge RG (2003) Transgene integration patterns and expression levels in transgenic tissue lines of Picea mariana, P. glauca and P. abies. Plant Cell Rep 21:1080–1087

    Article  PubMed  CAS  Google Scholar 

  • Kooter JM, Matzke MA, Meyer P (1999) Listening to the silent genes: transgene silencing, gene regulation and pathogen control. Trends Plant Sci 4:340–347

    Article  PubMed  Google Scholar 

  • Krayer von Krauss MP, Kaiser M, Almaas V, van der Sluijs J, Kloprogge P (2008) Diagnosing and prioritizing uncertainties according to their relevance for policy: the case of transgene silencing. Sci Total Environ 390:23–34

    Article  PubMed  CAS  Google Scholar 

  • Kumar S, Fladung M (2001) Gene stability in transgenic aspen (Populus) II. Molecular characterization of variable expression of transgene in wild and hybrid aspen. Planta 213:731–740

    Article  PubMed  CAS  Google Scholar 

  • Lechtenberg B, Schubert D, Forsbach A, Gils M, Schmidt R (2003) Neither inverted repeat T-DNA configurations nor arrangements of tandemly repeated transgenes are sufficient to trigger transgene silencing. Plant J 34:507–517

    Article  PubMed  CAS  Google Scholar 

  • Li JY, Brunner AM, Meilan R, Strauss SH (2009) Stability of transgenes in trees: expression of two reporter genes in poplar over three field seasons. Tree Physiol 29:299–312

    Article  PubMed  CAS  Google Scholar 

  • Liu Q, Xu J, Liu YZ, Zhao XL, Deng XX, Guo LL, Gu JQ (2007) A novel bud mutation that confers abnormal patterns of lycopene accumulation in sweet orange fruit (Citrus sinensis L. Osbeck). J Exp Bot 58:4161–4171

    Article  PubMed  CAS  Google Scholar 

  • Mette MF, Aufsatz W, van der Winden J, Matzke MA, Matzke AJ (2000) Transcriptional silencing and promoter methylation triggered by double-stranded RNA. EMBO J 19:5194–5201

    Article  PubMed  CAS  Google Scholar 

  • Mishiba K, Nishihara M, Nakatsuka T, Abe Y, Hirano H, Yokoi T, Kikuchi A, Yamamura S (2005) Consistent transcriptional silencing of 35S-driven transgenes in gentian. Plant J 44:541–556

    Article  PubMed  CAS  Google Scholar 

  • Morel JB, Mourrain P, Béclin C, Vaucheret H (2000) DNA methylation and chromatin structure affect transcriptional and post-transcriptional transgene silencing in Arabidopsis. Curr Biol 10:1591–1594

    Article  PubMed  CAS  Google Scholar 

  • Omar AA, Dekkers MGH, Graham JH, Grosser JW (2008) Estimation of transgene copy number in transformed citrus plants by quantitative multiplex real-time PCR. Biotechnol Prog 24:1241–1248

    Article  PubMed  CAS  Google Scholar 

  • Sijen T, Vijn I, Rebocho A, van Blokland R, Roelofs D, Mol JNM, Kooter JM (2001) Transcriptional and posttranscriptional gene silencing are mechanistically related. Curr Biol 11:436–440

    Article  PubMed  CAS  Google Scholar 

  • Skarn M, Eike MC, Meza TJ, Mercy IS, Jakobsen KS, Aalen RB (2006) An inverted repeat transgene with a structure that cannot generate double-stranded RNA, suffers silencing independent of DNA methylation. Transgenic Res 15:489–500

    Article  PubMed  CAS  Google Scholar 

  • Southern E (2006) Southern blotting. Nat Protoc 1:518–525

    Article  PubMed  CAS  Google Scholar 

  • Stam M, Mol JNM, Kooter JM (1997) The silence of genes in transgenic plants. Ann Bot 79:3–12

    Article  CAS  Google Scholar 

  • Tan B, Li DL, Xu SX, Fan GE, Fan J, Guo WW (2009) Highly efficient transformation of GFP and KNU genes into precocious trifoliate orange (Poncirus trifoliata [L.] Raf), a potential model genotype for functional genomics studies in Citrus. Tree Genet Genomes 5:529–537

    Article  Google Scholar 

  • Tang W, Newton RJ, Weidner DA (2007) Genetic transformation and gene silencing mediated by multiple copies of a transgene in eastern white pine. J Exp Bot 58:545–554

    Article  PubMed  CAS  Google Scholar 

  • Vaucheret H, Fagard M (2001) Transcriptional gene silencing in plants: targets, inducers and regulators. Trends Genet 17:29–35

    Article  PubMed  CAS  Google Scholar 

  • Wang MB, Waterhouse PM (2000) High-efficiency silencing of a β-glucuronidase gene in rice is correlated with repetitive transgene structure but is independent of DNA methylation. Plant Mol Biol 43:67–82

    Article  PubMed  CAS  Google Scholar 

  • Xu SX, Cai XD, Tan B, Guo WW (2011) Comparison of expression of three different sub-cellular targeted GFPs in transgenic Valencia sweet orange by confocal laser scanning microscopy. Plant Cell Tiss Organ Cult 104:199–207

    Article  Google Scholar 

Download references

Acknowledgments

This research was financially supported by the National 863 program of China (No. 2011AA100205), and the National NSF of China (No. 30921002).

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Authors and Affiliations

  1. Key Laboratory of Horticultural Plant Biology (Ministry of Education), National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, 430070, China

    Jing Fan, Xin Liu, Shi-Xiao Xu, Qiang Xu & Wen-Wu Guo

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  1. Jing Fan
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  2. Xin Liu
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  3. Shi-Xiao Xu
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  4. Qiang Xu
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  5. Wen-Wu Guo
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Correspondence to Wen-Wu Guo.

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Fan, J., Liu, X., Xu, SX. et al. T-DNA direct repeat and 35S promoter methylation affect transgene expression but do not cause silencing in transgenic sweet orange. Plant Cell Tiss Organ Cult 107, 225–232 (2011). https://doi.org/10.1007/s11240-011-9973-z

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  • DOI: https://doi.org/10.1007/s11240-011-9973-z

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