Abstract
Hypericin, an important determinant of the pharmacological properties of the genus Hypericum, is considered as a major molecule for drug development. However, biosynthesis and accumulation of hypericin is not well understood. Identification of genes differentially expressed in tissues with and without hypericin accumulation is a useful strategy to elucidate the mechanisms underlying the development of the dark glands and hypericin biosynthesis. Suppression Subtractive Hybridization (SSH) is a unique method for PCR-based amplification of specific cDNA fragments that differ between a control (driver) and experimental (tester) transcriptome. This technique relies on the removal of dsDNA formed by hybridization between a control and test sample, thus eliminating cDNAs of similar abundance, and retaining differentially expressed or variable in sequence cDNAs. In our laboratory we applied this method to identify the genes involved in the development of dark glands and accumulation of hypericin in Hypericum perforatum. Here we describe the complete procedure for the construction of hypericin gland-specific subtracted cDNA library.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Guedes AP, Franklin G, Ferreira MF (2012) Hypericum sp.: essential oil composition and biological activities. Phytochem Rev 11:127–152
Karioti A, Bilia AR (2010) Hypericins as potential leads for new therapeutics. Int J Mol Sci 11:562–594
Nafee N, Youssef A, Hanan EG, Heba A, Sherif K (2013) Pharmaceutical nanotechnology antibiotic-free nanotherapeutics: hypericin nanoparticles thereof for improved in vitro and in vivo antimicrobial photodynamic therapy and wound healing. Int J Pharm 454:249–258
Cole WR, Mostofsky SH, Larson JC, Denckla MB, Mahone EM (2008) Age-related changes in motor subtle signs among girls and boys with ADHD. Neurology 71:1514–1520
Bruni R, Sacchetti G (2009) Factors affecting polyphenol biosynthesis in wild and field grown St. John’s Wort (Hypericum perforatum L. Hypericaceae/Guttiferae). Molecules 14:682–725
Conceição LFR, Ferreres F, Tavares RM, Dias ACP (2006) Induction of phenolic compounds in Hypericum perforatum L. cells by Colletotrichum gloeosporioides elicitation. Phytochemistry 67:149–155
Franklin G, Conceição LFR, Kombrink E, Dias ACP (2009) Xanthone biosynthesis in Hypericum perforatum cells provides antioxidant and antimicrobial protection upon biotic stress. Phytochemistry 70:60–68
Zobayed SMA, Afreen F, Goto E, Kozai T (2006) Plant–environment interactions: accumulation of hypericin in dark glands of Hypericum perforatum. Ann Bot 98:793–804
Kornfeld A, Kaufman PB, Lu CR, Gibson DM, Bolling SF, Warber SL, Chang SC, Kirakosyan A (2007) The production of hypericins in two selected Hypericum perforatum shoot cultures is related to differences in black gland structure. Plant Physiol Biochem 45:24–32
Franklin G, Oliveira M, Dias ACP (2007) Production of transgenic Hypericum perforatum plants via particle bombardment-mediated transformation of novel organogenic cell suspension cultures. Plant Sci 172:1193–1203
Franklin G, Dias ACP (2011) Chlorogenic acid participates in the regulation of shoot, root and root hair development in Hypericum perforatum. Plant Physiol Biochem 49:835–842
Ayan AK, Cirak C, Kevseroglu K, Ozen T (2004) Hypericin in some Hypericum species from Turkey. Asian J Plant Sci 3:200–202
Karppinen K, Hokkanen J, Mattila S, Neubauer P, Hohtola A (2008) Octaketide producing type III polyketide synthase from Hypericum perforatum is expressed in dark glands accumulating hypericins. FEBS J 275:4329–4342
Acknowledgment
This work was supported by Fundação para a Ciência e a Tecnologia project (PTDC/AGR-GPL/119211/2010). We acknowledge Dr Caroline J Sheeba, ICVS, University of Minho for critically reading and commenting on the manuscript.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Singh, R.K., Hou, W., Franklin, G. (2016). Construction of Hypericin Gland-Specific cDNA Library via Suppression Subtractive Hybridization. In: Jain, S. (eds) Protocols for In Vitro Cultures and Secondary Metabolite Analysis of Aromatic and Medicinal Plants, Second Edition. Methods in Molecular Biology, vol 1391. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3332-7_22
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3332-7_22
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3330-3
Online ISBN: 978-1-4939-3332-7
eBook Packages: Springer Protocols