Advertisement

Knockdown of Polyphenol Oxidase Gene Expression in Potato (Solanum tuberosum L.) with Artificial MicroRNAs

  • Ming Chi
  • Basdeo Bhagwat
  • Guiliang Tang
  • Yu XiangEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1405)

Abstract

It is of great importance and interest to develop crop varieties with low polyphenol oxidase (PPO) activity for the food industry because PPO-mediated oxidative browning is a main cause of post-harvest deterioration and quality loss of fresh produce and processed foods. We recently demonstrated that potato tubers with reduced browning phenotypes can be produced by inhibition of the expression of several PPO gene isoforms using artificial microRNA (amiRNA) technology. The approach introduces a single type of 21-nucleotide RNA population to guide silencing of the PPO gene transcripts in potato tissues. Some advantages of the technology are: small RNA molecules are genetically transformed, off-target gene silencing can be avoided or minimized at the stage of amiRNA designs, and accuracy and efficiency of the processes can be detected at every step using molecular biological techniques. Here we describe the methods for transformation and regeneration of potatoes with amiRNA vectors, detection of the expression of amiRNAs, identification of the cleaved product of the target gene transcripts, and assay of the expression level of PPO gene isoforms in potatoes.

Key words

Artificial microRNA (amiRNA) Gene knockdown Polyphenol oxidase (PPO) Potato genetic transformation RNA silencing Solanum tuberosum StuPPO 

References

  1. 1.
    Holderbaum DF, Kon T, Kudo T, Guerra MP (2010) Enzymatic browning, polyphenol oxidase activity, and polyphenols in four apple cultivars: dynamics during fruit development. HortScience 45:1150–1154Google Scholar
  2. 2.
    Mayer AM (2006) Polyphenol oxidases in plants and fungi: going places? A review. Phytochemistry 67:2318–2331CrossRefPubMedGoogle Scholar
  3. 3.
    Tran LT, Taylor JS, Constabel CP (2012) The polyphenol oxidase gene family in land plants: lineage-specific duplication and expansion. BMC Genomics 13:395PubMedCentralCrossRefPubMedGoogle Scholar
  4. 4.
    Chi M, Bhagwat B, Lane WD, Tang G, Su Y, Sun R, Oomah BD, Wiersma PA, Xiang Y (2014) Reduced polyphenol oxidase gene expression and enzymatic browning in potato (Solanum tuberosum L.) with artificial microRNAs. BMC Plant Biol 14:62PubMedCentralCrossRefPubMedGoogle Scholar
  5. 5.
    Bachem CW, Speckmann G-J, van der Linde PC, Verheggen FT, Hunt MD, Steffens JC, Zabeau M (1994) Antisense expression of polyphenol oxidase genes inhibits enzymatic browning in potato tubers. Nat Biotechnol 12:1101–1105CrossRefGoogle Scholar
  6. 6.
    Coetzer C, Corsini D, Love S, Pavek J, Tumer N (2001) Control of enzymatic browning in potato (Solanum tuberosum L.) by sense and antisense RNA from tomato polyphenol oxidase. J Agric Food Chem 49:652–657CrossRefPubMedGoogle Scholar
  7. 7.
    Murata M, Nishimura M, Murai N, Haruta M, Homma S, Itoh Y (2001) A transgenic apple callus showing reduced polyphenol oxidase activity and lower browning potential. Biosci Biotechnol Biochem 65:383–388CrossRefPubMedGoogle Scholar
  8. 8.
    Richter C, Dirks ME, Gronover CS, Prüfer D, Moerschbacher BM (2012) Silencing and heterologous expression of ppo-2 indicate a specific function of a single polyphenol oxidase isoform in resistance of dandelion (Taraxacum officinale) against Pseudomonas syringae pv. tomato. Mol Plant Microbe Interact 25:200–210CrossRefPubMedGoogle Scholar
  9. 9.
    Bhagwat B, Chi M, Su L, Tang H, Tang G, Xiang Y (2013) An in vivo transient expression system can be applied for rapid and effective selection of artificial microRNA constructs for plant stable genetic transformation. J Genet Genomics 40:261–270CrossRefPubMedGoogle Scholar
  10. 10.
    Ossowski S, Schwab R, Weigel D (2008) Gene silencing in plants using artificial microRNAs and other small RNAs. Plant J 53:674–690CrossRefPubMedGoogle Scholar
  11. 11.
    Schwab R, Ossowski S, Riester M, Warthmann N, Weigel D (2006) Highly specific gene silencing by artificial microRNAs in Arabidopsis. Plant Cell 18:1121–1133PubMedCentralCrossRefPubMedGoogle Scholar
  12. 12.
    Beaujean A, Sangwan RS, Lecardonnel A, Sangwan-Norreel BS (1998) Agrobacterium-mediated transformation of three economically important potato cultivars using sliced internodal explants: an efficient protocol of transformation. J Exp Bot 49:1589–1595CrossRefGoogle Scholar
  13. 13.
    Bhagwat B, Chi M, Han D, Tang H, Tang G, Xiang Y (2015) Design, construction and validation of artificial microRNA vectors using Agrobacterium-mediated transient expression system. Methods Mol Biol Google Scholar
  14. 14.
    Nicot N, Hausman JF, Hoffmann L, Evers D (2005) Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress. J Exp Bot 56:2907–2914Google Scholar
  15. 15.
    Millam S (2006) Potato (Solanum tuberosum L.). Methods Mol Biol 344:25–36PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Ming Chi
    • 1
    • 2
  • Basdeo Bhagwat
    • 1
  • Guiliang Tang
    • 3
    • 4
  • Yu Xiang
    • 1
    Email author
  1. 1.Pacific Agri-Food Research CenterAgriculture and Agri-Food CanadaSummerlandCanada
  2. 2.College of ForestryNorthwest A & F UniversityYanglingChina
  3. 3.Department of Biological SciencesMichigan Technological UniversityHoughtonUSA
  4. 4.Provincial State Key Laboratory of Wheat and Maize Crop ScienceHenan Agricultural UniversityZhengzhouChina

Personalised recommendations