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.
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References
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 45:1150–1154
Mayer AM (2006) Polyphenol oxidases in plants and fungi: going places? A review. Phytochemistry 67:2318–2331
Tran LT, Taylor JS, Constabel CP (2012) The polyphenol oxidase gene family in land plants: lineage-specific duplication and expansion. BMC Genomics 13:395
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 14:62
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 12:1101–1105
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 49:652–657
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 65:383–388
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 25:200–210
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 40:261–270
Ossowski S, Schwab R, Weigel D (2008) Gene silencing in plants using artificial microRNAs and other small RNAs. Plant J 53:674–690
Schwab R, Ossowski S, Riester M, Warthmann N, Weigel D (2006) Highly specific gene silencing by artificial microRNAs in Arabidopsis. Plant Cell 18:1121–1133
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 49:1589–1595
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
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 56:2907–2914
Millam S (2006) Potato (Solanum tuberosum L.). Methods Mol Biol 344:25–36
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Chi, M., Bhagwat, B., Tang, G., Xiang, Y. (2016). Knockdown of Polyphenol Oxidase Gene Expression in Potato (Solanum tuberosum L.) with Artificial MicroRNAs. In: Fett-Neto, A. (eds) Biotechnology of Plant Secondary Metabolism. Methods in Molecular Biology, vol 1405. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3393-8_15
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DOI: https://doi.org/10.1007/978-1-4939-3393-8_15
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