Abstract
The structure and function of untranslated mRNA leader sequences and their role in controlling gene expression remains poorly understood. Previous research has suggested that the 5′ untranslated region (5′UTR) of the Vigna radiata aminocyclopropane-1-carboxylate synthase synthase (VR-ACS1) gene may function as a translational enhancer in plants. To test such hypothesis we compared the translation enhancing properties of three different 5′UTRs; those from the VR-ACS1, the chlorophyll a/b binding gene from petunia (Cab22L; a known translational enhancer) and the Vigna radiata pectinacetylesterase gene (PAE; used as control). Identical constructs in which the coding region of the β-glucuronidase (GUS) gene was fused to each of the three 5′UTRs and placed under the control of the cauliflower mosaic virus 35S promoter were prepared. Transient expression assays in tobacco cell cultures and mung bean leaves showed that the VR-ACS1 and Cab22L 5′UTRs directed higher levels of GUS activity than the PAE 5′UTR. Analysis of transgenic Arabidopsis thaliana seedlings, as well as different tissues from mature plants, confirmed that while transcript levels were equivalent for all constructs, the 5′UTRs from the VR-ACS1 and Cab22L genes can increase GUS activity twofold to fivefold compared to the PAE 5′UTR, therefore confirming the translational enhancing properties of the VR-ACS1 5′UTR.
Abbreviations
- Cab22L:
-
Chlorophyll a/b binding
- CaMV:
-
Cauliflower mosaic virus
- GUS:
-
β-Glucuronidase
- MUG:
-
4-Methylumbelliferyl-β-d-glucuronide
- PAE:
-
Pectinacetylesterase
- UTR:
-
Untranslated region
- VR-ACS1:
-
Vigna radiata aminocyclopropane-1-carboxylate synthase
References
Akama K, Shiraishi H, Ohta S, Nakamura K, Okada K, Shimura Y (1992) Efficient transformation of Arabidopsis thaliana: comparison of the efficiencies with various organs, plant ecotypes and Agrobacterium strains. Plant Cell Rep 12:7–11
Baulcombe D (2004) RNA silencing in plants. Nature 431:356–363
Benfey PN, Chua NH (1990) The cauliflower mosaic virus 35S promoter: combinatorial regulation of transcription in plants. Science 250:959–966
Breton C, Bordenave M, Richard L, Pernollet JC, Huet JC, Perez S, Goldberg R (1996) PCR cloning and expression analysis of a cDNA encoding a pectinacetylesterase from Vigna radiata L. FEBS Lett 388:139–142
Brummell DA, Balint-Kurti PJ, Harpster MH, Palys JM, Oeller PW, Gutterson N (2003) Inverted repeat of a heterologous 3′-untranslated region for high-efficiency, high-throughput gene silencing. Plant J 33:793–800
Burgess DG, Ralston EJ, Hanson WG, Heckert M, Ho M, Jenq T, Palys JM, Tang KL, Gutterson N (2002) A novel, two-component system for cell lethality and its use in engineering nuclear male-sterility in plants. Plant J 31:113–125
Cazzonelli CI, Velten J (2008) In vivo characterization of plant promoter element interaction using synthetic promoters. Transgenic Res 17:437–457
Cazzonelli CI, McCallum EJ, Lee R, Botella JR (2005) Characterization of a strong constitutive mung bean (Vigna radiata L.) promoter with a complex mode of regulation in planta. Transgenic Res 14:941–967
Chakravorty D, Botella JR (2007) Over-expression of a truncated Arabidopsis thaliana heterotrimeric G protein γ subunit results in a phenotype similar to α and β subunit knockouts. Gene 393:163–170
Danthinne X, van Emmelo J (1990) Studies on the translational properties of STNV RNA non-coding regions. 42nd International symposium on crop protection. Gent, Belgium
De Amicis F, Patti T, Marchetti S (2007) Improvement of the pBI121 plant expression vector by leader replacement with a sequence combining a poly(CAA) and a CT motif. Transgenic Res 16:731–738
De Loose M, Danthinne X, Van Bockstaele E, Van Montagu M, Depicker A (1995) Different 5′ leader sequences modulate β-glucuronidase accumulation levels in transgenic Nicotiana tabacum plants. Euphytica 85:209–216
Dunsmuir P (1985) The petunia chlorophyll a/b binding-protein genes - a comparison of Cab genes from different gene families. Nucleic Acids Res 13:2503–2518
Fornara F, Parenicova L, Falasca G, Pelucchi N, Masiero S, Ciannamea S, Lopez-Dee Z, Altamura MM, Colombo L, Kater MM (2004) Functional characterization of OsMADS18, a member of the AP1/SQUA subfamily of MADS box genes. Plant Physiol 135:2207–2219
Futterer J, Hohn T (1996) Translation in plants - rules and exceptions. Plant Mol Biol 32:159–189
Gallie DR (1993) Posttranscriptional regulation of gene expression in plants. Annu Rev Plant Physiol Plant Mol Biol 44:77–105
Gallie DR, Walbot V (1992) Identification of the motifs within the tobacco mosaic virus 5′-leader responsible for enhancing translation. Nucleic Acids Res 20:4631–4638
Harpster MH, Townsend JA, Jones JDG, Bedbrook J, Dunsmuir P (1988) Relative strengths of the 35S cauliflower mosaic virus, 1′, 2′, and nopaline synthase promoters in transformed tobacco sugarbeet and oilseed rape callus tissue. Mol Gen Genet 212:182–190
Joshi JP, Zhou H, Huang X, Chiang VL (1997) Context sequences of translation initiation codon in plants. Plant Mol Biol 35:993–1001
Kawaguchi R, Bailey-Serres J (2005) mRNA sequence features that contribute to translational regulation in Arabidopsis. Nucleic Acids Res 33:955–965
Klaff P, Riesner D, Steger G (1996) RNA structure and the regulation of gene expression. Plant Mol Biol 32:89–106
Kozak M (1989) The scanning model for translation: An update. J Cell Biol 108:229–241
Laurena AC, Magdalita PM, Hidalgo MSP, Villegas VN, Mendoza EMT, Botella JR (2002) Cloning and molecular characterization of ripening-related ACC synthase from papaya fruit (Carica papaya L.). In: Proceedings of the International Symposium on Tropical and Subtropical Fruits
Lodish HF (1976) Translational control of protein synthesis. Annu Rev Biochem 45:39–72
Matsui T, Hori M, Shizawa N, Nakayama I, Shinmyo A, Yoshida K (2006) High-efficiency secretory production of peroxidase C1a using vesicular transport engineering in transgenic tobacco. J Biosci Bioeng 102:102–109
Mylne J, Botella JR (1998) Binary vectors for sense and antisense expression of Arabidopsis ESTs. Plant Mol Biol Report 16:257–262
Narsai R, Howell KA, Millar AH, O’Toole N, Small I, Whelan J (2007) Genome-wide analysis of mRNA decay rates and their determinants in Arabidopsis thaliana. Plant Cell 19:3418–3436
O’Keefe DP, Tepperman JM, Dean C, Leto KJ, Erbes DL, Odell JT (1994) Plant expression of a bacterial cytochrome P450 that catalyzes activation of a sulfonylurea pro-herbicide. Plant Physiol 105:473–482
Odell JT, Nagy F, Chua N (1985) Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature 313:810–812
Ohta S, Mita S, Hattori T, Nakamura K (1990) Construction and expression in tobacco of a ß-glucoronidase (GUS) reporter gene containing an intron within the coding sequence. Plant Cell Physiol 31:805–813
Purnell MP, Botella JR (2007) Tobacco isoenzyme 1 of NAD(H)-dependent glutamate dehydrogenase catabolizes glutamate in vivo. Plant Physiol 143:530–539
Trusov Y, Zhang W, Assmann SM, Botella JR (2008) Heterotrimeric G protein Gγ-deficient mutants do not recapitulate all phenotypes of Gβ-deficient mutants. Plant Physiol 147:636–649
Trusov Y, Sewelam N, Rookes JE, Kunkel M, Nowak E, Schenk PM, Botella JR (2009) Heterotrimeric G proteins-mediated resistance to necrotrophic pathogens includes mechanisms independent of salicylic acid-, jasmonic acid/ethylene- and abscisic acid-mediated defense signaling. Plant J 58:69–81
Turner RL, Glynn M, Taylor SC, Cheung MK, Spurr C, Twell D, Foster GD (1999) Analysis of a translational enhancer present within the 5′-terminal sequence of the genomic RNA of potato virus S. Arch Virol 144:1451–1461
Venter M (2007) Synthetic promoters: genetic control through cis engineering. Trends Plant Sci 12:118–124
Wells DR, Tanguay RL, Le H, Gallie DR (1998) HSP101 functions as a specific translational regulatory protein whose activity is regulated by nutrient status. Gene Dev 12:3236–3251
Wu KQ, Hu M, Martin T, Wang CM, Li XQ, Tian LN, Brown D, Miki B (2003) The cryptic enhancer elements of the tCUP promoter. Plant Mol Biol 51:351–362
Zaccomer B, Haenni AL, Macaya G (1995) The remarkable variety of plant RNA virus genomes. J Gen Virol 76:231–247
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Wever, W., McCallum, E.J., Chakravorty, D. et al. The 5′ untranslated region of the VR-ACS1 mRNA acts as a strong translational enhancer in plants. Transgenic Res 19, 667–674 (2010). https://doi.org/10.1007/s11248-009-9332-6
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DOI: https://doi.org/10.1007/s11248-009-9332-6