Advertisement

Planta

, Volume 235, Issue 6, pp 1397–1408 | Cite as

Analysis of the APX, PGD1 and R1G1B constitutive gene promoters in various organs over three homozygous generations of transgenic rice plants

  • Su-Hyun Park
  • Seung Woon Bang
  • Jin Seo Jeong
  • Harin Jung
  • Mark Christian Felipe Reveche Redillas
  • Hyung Il Kim
  • Kang Hyun Lee
  • Youn Shic Kim
  • Ju-Kon Kim
Original Article

Abstract

We have previously characterized the constitutively active promoters of the APX, PGD1 and R1G1B genes in rice (Park et al. 2010 in J Exp Bot 61:2459–2467). To have potential crop biotechnology applications, gene promoters must be stably active over many generations. In our current study, we report our further detailed analysis of the APX, PGD1 and R1G1B gene promoters in various organs and tissues of transgenic rice plants for three (T3–5) homozygous generations. The copy numbers in 37 transgenic lines that harbor promoter:gfp constructs were determined and promoter activities were measured by real-time qPCR. Analysis of the 37 lines revealed that 15 contained a single copy of one of the three promoter:gfp chimeric constructs. The promoter activity levels were generally higher in multi-copy lines, whereas variations in these levels over the T3–5 generations studied were observed to be smaller in single-copy than in multi-copy lines. The three promoters were further found to be highly active in the whole plant body at both the vegetative and reproductive stages of plant growth, with the exception of the APX in the ovary and R1G1B in the pistil and filaments where zero or very low levels of activity were detected. Of note, the spatial activities of the PGD1 promoter were found to be strikingly similar to those of the ZmUbi1, a widely used constitutive promoter. Our comparison of promoter activities between T3, T4 and T5 plants revealed that the APX, PGD1 and R1G1B promoters maintained their activities at comparable levels in leaves and roots over three homozygous generations and are therefore potentially viable alternative promoters for crop biotechnology applications.

Keywords

APX (ascorbate peroxidase gene) Constitutive promoter Homozygous generations PGD1 (phosphogluconate dehydrogenase gene) R1G1B Single copy 

Abbreviations

APX

Ascorbate peroxidase

PGD1

Phosphogluconate dehydrogenase

R1G1B

R1G1 domain containing protein B

CaMV 35S

Cauliflower mosaic virus 35S

OsCc1

Rice cytochrome c

ZmUbi

Maize ubiquitin1

Act1

Rice actin1

Notes

Acknowledgments

This study was supported by the Rural Development Administration under the “Cooperative Research Program for Agriculture Science and Technology Development” (Project No. PJ906910), the Next-Generation BioGreen 21 Program (Project No. PJ007971 to J.-K.K.), and by the Ministry of Education, Science and Technology under “Mid-career Researcher Program” (Project No. 20100026168 to J.-K.K.).

Supplementary material

425_2011_1582_MOESM1_ESM.pdf (341 kb)
Supplementary material 1 (PDF 345 kb)

References

  1. An YQ, McDowell JM, Huang S, McKinney EC, Chambliss S, Meagher RB (1996) Strong, constitutive expression of the Arabidopsis ACT2/ACT8 actin subclass in vegetative tissues. Plant J 10:107–121PubMedCrossRefGoogle Scholar
  2. Anand A, Trick HN, Gill BS, Muthukrishnan S (2003) Stable transgene expression and random gene silencing in wheat. Plant Biotechnol J 1:241–251PubMedCrossRefGoogle Scholar
  3. Atkinson RG, Bieleski LRF, Gleave AP, Janssen B-J, Morris BAM (1998) Post-transcriptional silencing of chalcone synthase in petunia using a geminivirus-based episomal vector. Plant J 15:593–604CrossRefGoogle Scholar
  4. Callis J, Raasch JA, Vierstra RD (1990) Ubiquitin extension proteins of Arabidopsis thaliana. J Biol Chem 265:12486–12493PubMedGoogle Scholar
  5. Chareonpornwattana S, Thara KV, Wang L, Datta SK, Panbangred W, Muthukrishnan N (1999) Inheritance, expression, and silencing of a chitinase transgene in rice. Theor Appl Genet 98:371–378CrossRefGoogle Scholar
  6. Cho M-J, Choi HW, Buchanan BB, Lemaux PG (1999) Inheritance of tissue-specific expression of hordein promoter-uidA fusions in transgenic barley plants. Theor Appl Genet 98:1253–1262CrossRefGoogle Scholar
  7. Choi HW, Lemaux PG, Cho MJ (2003) Long-term stability of transgene expression driven by barley endosperm-specific hordein promoters in transgenic barley. Plant Cell Rep 21:1108–1120PubMedCrossRefGoogle Scholar
  8. Cornejo MJ, Luth D, Blankenship KM, Anderson OD, Blechl AE (1993) Activity of a maize ubiquitin promoter in transgenic rice. Plant Mol Biol 23:567–581PubMedCrossRefGoogle Scholar
  9. De Pater BS, van der Mark F, Rueb S, Katagiri F, Chua N-H, Schilperoort RA, Hensgens LAM (1992) The promoter of the rice gene GOS2 is active in various different monocot tissues and binds rice nuclear factor ASF-1. Plant J 2:837–844PubMedCrossRefGoogle Scholar
  10. Demeke T, Hucl P, Baga M, Caswell K, Leung N, Chibbar RN (1999) Transgene inheritance and silencing in hexaploid spring wheat. Theor Appl Genet 99:947–953CrossRefGoogle Scholar
  11. Dietz-Pfeilstetter A (2010) Stability of transgene expression as a challenge for genetic engineering. Plant Sci 179:164–167CrossRefGoogle Scholar
  12. He C, Lin Z, McElroy D, Wu R (2009) Identification of a rice Actin2 gene regulatory region for high-level expression of transgenes in monocots. Plant Biotechnol J 7:227–239PubMedCrossRefGoogle Scholar
  13. Hernandez-Garcia CM, Adriana P, Martinelli AP, Bouchard RA, Finer JJ (2009) A soybean (Glycine max) polyubiquitin promoter gives strong constitutive expression in transgenic soybean. Plant Cell Rep 28:837–849PubMedCrossRefGoogle Scholar
  14. Hensgens LAM, de Bakker EPHM, van Os-Ruygrok EP, Rueb S, van de Mark F, van der Maas HM, van der Veen S, Kooman-Gersmann M, Hart L, Schilperoort RA (1993) Transient and stable expression of gusA fusions with rice genes in rice, barley and perennial ryegrass. Plant Mol Biol 23:643–669Google Scholar
  15. Horvath H, Jensen LG, Wong OT, Kohl E, Ullrich SE, Cochran J, Kannangara CG, Wettstein D (2001) Stability of transgene expression, field performance and recombination breeding of transformed barley lines. Theor Appl Genet 102:1–11CrossRefGoogle Scholar
  16. James VA, Worland B, Snape JW, Vain P (2004) Strategies for precise quantification of transgene expression levels over several generations in rice. J Exp Bot 5:1307–1313CrossRefGoogle Scholar
  17. Jang IC, Choi WB, Lee KH, Song SI, Nahm BH, Kim JK (2002) High-level and ubiquitous expression of the rice cytochrome c gene OsCc1 and its promoter activity in transgenic plants provides a useful promoter for transgenesis of monocots. Plant Physiol 129:1473–1481PubMedCrossRefGoogle Scholar
  18. Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusion: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907PubMedGoogle Scholar
  19. Kwak MS, Oh MJ, Lee SW, Shin JS, Paek KH, Bae JM (2007) A strong constitutive gene expression system derived from ibAGP1 promoter and its transit peptide. Plant Cell Rep 26:1253–1262PubMedCrossRefGoogle Scholar
  20. 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 34:507–517PubMedCrossRefGoogle Scholar
  21. Lessard PA, Kulaveerasingam H, York GM, Strong A, Sinskey AJ (2002) Manipulating gene expression for the metabolic engineering of plants. Metab Eng 4:67–79PubMedCrossRefGoogle Scholar
  22. Liang YS, Bae HJ, Kang SH, Lee T, Kim MG, Kim YM, Ha SH (2009) The Arabidopsis beta-carotene hydroxylase gene promoter for a strong constitutive expression of transgene. Plant Biotechnol Rep 3:325–331CrossRefGoogle Scholar
  23. Lu J, Sivamani E, Li X, Qu R (2008) Activity of the 5′ regulatory regions of the rice polyubiquitin rubi3 gene in transgenic rice plants as analyzed by both GUS and GFP reporter genes. Plant Cell Rep 27:1587–1600PubMedCrossRefGoogle Scholar
  24. Mandel T, Fleming AJ, Krahenbuhl R, Kuhlemeier C (1995) Definition of constitutive gene expression in plants: the translation initiation factor 4A gene as a model. Plant Mol Biol 29:995–1004PubMedCrossRefGoogle Scholar
  25. Mascarenhas JP, Hamilton DA (1992) Artifacts in the localization of GUS activity in anthers of petunia transformed with a CaMV 35S-GUS construct. Plant J 2:405–408CrossRefGoogle Scholar
  26. McElroy D, Zhang W, Cao J, Wu R (1990) Isolation of an efficient actin promoter for use in rice transformation. Plant Cell 2:163–171Google Scholar
  27. Meyer P, Saedler H (1996) Homology-dependent gene silencing in plants. Annu Rev Plant Mol Biol 47:23–48CrossRefGoogle Scholar
  28. Muller AE (2010) Gene silencing in plants: transgenes as targets and effectors. In: Kempken F, Jung C (eds) Genetic modification of plants agriculture horticulture and forestry. Springer, Berlin, pp 79–101Google Scholar
  29. Odell JT, Nagy F, Chua NH (1985) Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature 313:810–812PubMedCrossRefGoogle Scholar
  30. Park S-H, Yi N, Kim YS, Jeong M-H, Bang S-W, Choi YD, Kim J-K (2010) Analysis of five novel putative constitutive gene promoters in transgenic rice plants. J Exp Bot 61:2459–2467PubMedCrossRefGoogle Scholar
  31. Plesse B, Criqui MC, Durr A, Parmentier Y, Fleck J, Genschik P (2001) Effects of the polyubiquitin gene Ubi.U4 leader intron and first ubiquitin monomer on reporter gene expression in Nicotiana tabacum. Plant Mol Biol 45:655–667PubMedCrossRefGoogle Scholar
  32. Potenza C, Aleman L, Sengupta-Gopalan C (2004) Targeting transgene expression in research, agricultural, and environmental applications: promoters used in plant transformation. In Vitro Cell Dev Biol Plant 40:1–22Google Scholar
  33. Rasco-Gaunt S, Liu D, Li CP, Doherty A, Hagemann K, Riley A, Thompson T, Brunkan C, Mitchell M, Lowe K, Krebbers E, Lazzeri P, Jayne S, Rice D (2003) Characterisation of the expression of a novel constitutive maize promoter in transgenic wheat and maize. Plant Cell Rep 21:569–576PubMedGoogle Scholar
  34. 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 58:545–554PubMedCrossRefGoogle Scholar
  35. Van der Leede-Plegt LM, van de Ven BCE, Bino RJ, van der Salm TPM, van Tunen AJ (1992) Introduction and differential use of various promoters in pollen grains of Nicotiana glutinosa and Lilium longiflorum. Plant Cell Rep 11:20–24CrossRefGoogle Scholar
  36. Wei H, Wang ML, Moore PH, Albert HH (2003) Comparative expression analysis of two sugarcane polyubiquitin promoters and flanking sequences in transgenic plants. J Plant Physiol 160:1241–1251PubMedCrossRefGoogle Scholar
  37. Wilkinson JE, Twell D, Lindsey K (1997) Activities of CaMV 35S and nos promoters in pollen: implications for field release of transgenic plants. J Exp Bot 48:265–275CrossRefGoogle Scholar
  38. Xiao K, Zhang C, Harrison M, Wang ZY (2005) Isolation and characterization of a novel plant promoter that directs strong constitutive expression of transgenes in plants. Mol Breed 15:221–231CrossRefGoogle Scholar
  39. Yang G, Lee Y-H, Jiang Y, Kumpatla SP, Hall TC (2005) Organization, not duplication, triggers silencing in a complex transgene locus in rice. Plant Mol Biol 58:351–366PubMedCrossRefGoogle Scholar
  40. Yang Y, Costa A, Leonhardt N, Siegel RS, Schroeder JI (2008) Isolation of a strong Arabidopsis guard cell promoter and its potential as a research tool. Plant Methods 4:6PubMedCrossRefGoogle Scholar
  41. Zhang W, McElroy D, Wu R (1991) Analysis of rice Act1 5′ region activity in transgenic rice plants. Plant Cell 3:1155–1165PubMedCrossRefGoogle Scholar
  42. Zhou X, Carranco R, Vitha S, Hall TC (2005) The dark side of green fluorescent protein. New Phytol 168:313–322PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Su-Hyun Park
    • 1
  • Seung Woon Bang
    • 1
  • Jin Seo Jeong
    • 1
  • Harin Jung
    • 1
  • Mark Christian Felipe Reveche Redillas
    • 1
  • Hyung Il Kim
    • 1
  • Kang Hyun Lee
    • 1
  • Youn Shic Kim
    • 1
  • Ju-Kon Kim
    • 1
  1. 1.School of Biotechnology and Environmental EngineeringMyongji UniversityYonginKorea

Personalised recommendations