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The functionality of α-kafirin promoter and α-kafirin signal peptide

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Abstract

Cereal grains offer great potential as a storage system for production of highly valuable proteins using biotechnological approaches, but such applications require tight temporal and spatial control of transgene expression. Towards this aim, we have undertaken a detailed analysis of α-kafirin (α-kaf) promoter and α-kaf signal peptide (sp) in transgenic sorghum plants, using green fluorescent protein gene (gfp) as a reporter. Constructs containing either the α-kaf promoter or the constitutive maize ubiquitin-1 (ubi) promoter driving either gfp or sp-gfp translational fusion were introduced into Sorghum bicolor inbred line Tx430 by particle bombardment. We show for the first time that the α-kaf promoter directs endosperm-specific transgene expression, with activity first detected at 10 days post-anthesis (dpa), peaking at 20 dpa, and remaining active through to physiological maturity. Furthermore, we demonstrate for the first time that the α-kafirin sp is sufficient to direct foreign protein to protein bodies in the endosperm. The evidence is also provided for possible mis-targeting by α-kaf sp in vegetative tissues of transgenic lines with ubi-sp-gfp, resulting in loss of reporter gene translational activity that no GFP signal was observed. These results demonstrate that α-kaf promoter and α-kaf sp are well suited for seed bioengineering to produce recombinant proteins in sorghum endosperm or deposit foreign proteins into sorghum protein bodies.

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References

  • Ahmad N, Sant R, Bokan M, Steadman KJ, Godwin ID (2012) Expression pattern of the alpha-kafirin promoter coupled with a signal peptide from Sorghum bicolor L. Moench. J Biomed Biotechnol 2012:1–8

    Article  Google Scholar 

  • Belton PS, Delgadillo I, Halford NG, Shewry PR (2006) Kafirin structure and functionality. J Cereal Sci 44(3):272–286

    Article  CAS  Google Scholar 

  • Boothe JG, Saponja JA & Parmenter DL (1997) Molecular farming in plants: oilseeds as vehicles for the production of pharmaceutical proteins. Drug Dev Res 42(3–4):172–181

    Article  CAS  Google Scholar 

  • Christensen AH, Quail PH (1996) Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants. Transgenic Res 5(3):213–218

    Article  CAS  PubMed  Google Scholar 

  • Coleman CE, Lopes MA, Gillikin JW, Boston RS, Larkins BA (1995) A defective signal peptide in the maize high-lysine mutant floury 2. Proc Natl Acad Sci USA 92(15):6828–6831

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • DeRose RT, Begum D, Hall TC (1996) Analysis of kafirin promoter activity in transgenic tobacco seeds. Plant Mol Biol 32(6):1029–1035

    Article  CAS  PubMed  Google Scholar 

  • Frisch DA, Vandergeest AHM, Dias K, Hall TC (1995) Chormosomal integration is required for spatial regulation of expression from the beta-phaseolin promoter. Plant J 7(3):503–512

    Article  CAS  Google Scholar 

  • Giddings G, Allison G, Brooks D, Carter A (2000) Transgenic plants as factories for biopharmaceuticals. Nat Biotechnol 18(11):1151–1155

    Article  CAS  PubMed  Google Scholar 

  • Gnanasambandam A, Birch RG (2004) Efficient developmental mis-targeting by the sporamin NTPP vacuolar signal to plastids in young leaves of sugarcane and Arabidopsis. Plant Cell Rep 23(7):435–447

    Article  CAS  PubMed  Google Scholar 

  • Hamaker BR, Mohamed AA, Habben JE, Huang CP & Larkins BA (1995) Efficient procedure for extracting maize and sorghum kernel proteins reveals higher prolamin contents than the conventional method. Cereal Chem 72(6):583–588

    CAS  Google Scholar 

  • Heim R, Cubitt AB, Tsien RY (1995) Improved green fluorescence. Nature 373(6516):663–664

    Article  CAS  PubMed  Google Scholar 

  • Lamphear BJ, Streatfield SJ, Jilka JA, Brooks CA, Barker DK, Turner DD, Delaney DE, Garcia M, Wiggins B, Woodard SL, Hood EE, Tizard IR, Lawhorn B, Howard JA (2002) Delivery of subunit vaccines in maize seed. J Control Release 85(1–3):169–180

    Article  CAS  PubMed  Google Scholar 

  • Larkins BA, Hurkman WJ (1978) Synthesis and deposition of zein in protein bodies of maize endosperm. Plant Physiol 62(2):256–263

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Liu GQ, Godwin ID (2012) Highly efficient sorghum transformation. Plant Cell Rep 31(6):999–1007

    Article  PubMed  PubMed Central  Google Scholar 

  • Liu G, Gilding EK, Godwin ID (2015) A robust tissue culture system for sorghum [Sorghum bicolor (L.) Moench]. S Afr J Bot 98:157–160

    Article  CAS  Google Scholar 

  • Mace ES, Tai SS, Gilding EK, Li YH, Prentis PJ, Bian LL, Campbell BC, Hu WS, Innes DJ, Han XL, Cruickshank A, Dai CM, Frere C, Zhang HK, Hunt CH, Wang XY, Shatte T, Wang M, Su Z, Li J, Lin XZ, Godwin ID, Jordan DR, Wang J (2013) Whole-genome sequencing reveals untapped genetic potential in Africa’s indigenous cereal crop sorghum. Nat Commun 4:1–9

    Google Scholar 

  • Matsubayashi Y (2012) Recent progress in research on small post-translationally modified peptide signals in plants. Genes Cells 17(1):1–10

    Article  CAS  PubMed  Google Scholar 

  • Melnik S, Stoger E (2013) Green factories for biopharmaceuticals. Curr Med Chem 20(8):1038–1046

    CAS  PubMed  Google Scholar 

  • Muntz K (1998) Deposition of storage proteins. Plant Mol Biol 38(1–2):77–99

    Article  CAS  PubMed  Google Scholar 

  • Obembe OO, Popoola JO, Leelavathi S, Reddy SV (2011) Advances in plant molecular farming. Biotechnol Adv 29(2):210–222

    Article  PubMed  Google Scholar 

  • Qin KZ, Qiu P, Wen JY, Zhu YG, Li NW, Li SQ (2016) High throughput transformation of a Sorghum cDNA library for rice improvement. Plant Cell Tissue Org Cult 125(3):471–478

    Article  CAS  Google Scholar 

  • Ramakers C, Ruijter JM, Deprez RHL, Moorman AFM (2003) Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci Lett 339(1):62–66

    Article  CAS  PubMed  Google Scholar 

  • Shewry PR, Napier JA, Tatham AS (1995) Seed storage proteins: structures and biosynthesis. Plant Cell 7(7):945–956

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Stoger E, Ma JKC, Fischer R, Christou P (2005) Sowing the seeds of success: pharmaceutical proteins from plants. Curr Opin Biotechnol 16(2):167–173

    Article  CAS  PubMed  Google Scholar 

  • Sunilkumar G, Campbell LM, Puckhaber L, Stipanovic RD, Rathore KS (2006) Engineering cottonseed for use in human nutrition by tissue-specific reduction of toxic gossypol. Proc Natl Acad Sci USA 103(48):18054–18059

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Twyman RM, Stoger E, Schillberg S, Christou P, Fischer R (2003) Molecular farming in plants: host systems and expression technology. Trends Biotechnol 21(12):570–578

    Article  CAS  PubMed  Google Scholar 

  • Urriola J, Rathore KS (2014) Temporal and spatial activities of a rice glutelin promoter in transgenic sorghum. Plant Cell Tissue Org Cult 116(2):227–234

    Article  CAS  Google Scholar 

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Acknowledgments

We are thankful to ARC (Australian Research Council) and Pacific Seeds Pty. Ltd. for their financial support on the Linkage Project LP0883808. We are also grateful to GRDC (Grains Research & Development Corporation) for providing a Grains Research Top-up scholarship (GRS10405). We are indebted to SRA (Sugar Research Australia) for using their microscope. We deeply appreciate Dr. Yue Sun for her effort during the editing process.

Author contributions

GL and AT performed sorghum transformation. GL, SM, and KCL carried out microscopic observation of GFP. NA designed and cloned all constructs. IDG and EKG contributed experimental design, supervised all experiments and provided valuable discussions. GL, KCL, SRM and IDG contributed to the final manuscript.

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Authors and Affiliations

  1. School of Agriculture and Food Sciences, The University of Queensland, Level 3, John Hines Building 62#, Brisbane, QLD, 4072, Australia

    Guoquan Liu, Kyle C. Lamont, Alison Tomkins, Stephen R. Mudge & Ian D. Godwin

  2. Faculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam Campus, 42300, Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia

    Norazlina Ahmad

  3. The Institute of Molecular Biosciences, The University of Queensland, Brisbane, QLD, 4072, Australia

    Edward K. Gilding

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  1. Guoquan Liu
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  2. Kyle C. Lamont
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  3. Norazlina Ahmad
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  4. Alison Tomkins
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  5. Stephen R. Mudge
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Correspondence to Guoquan Liu.

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Liu, G., Lamont, K.C., Ahmad, N. et al. The functionality of α-kafirin promoter and α-kafirin signal peptide. Plant Cell Tiss Organ Cult 128, 133–143 (2017). https://doi.org/10.1007/s11240-016-1093-3

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  • DOI: https://doi.org/10.1007/s11240-016-1093-3

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