Construction of Hypericin Gland-Specific cDNA Library via Suppression Subtractive Hybridization.
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.
Centre for the Research and Technology of Agro-Environment and Biological Sciences (CITAB), University of Minho, Campus de Gualtar, Braga, 4717-057, Portugal.
Rupesh Kumar
RK
Singh
Centre for the Research and Technology of Agro-Environment and Biological Sciences (CITAB), University of Minho, Campus de Gualtar, Braga, 4717-057, Portugal.
Weina
W
Hou
Centre for the Research and Technology of Agro-Environment and Biological Sciences (CITAB), University of Minho, Campus de Gualtar, Braga, 4717-057, Portugal. franklin@bio.uminho.pt.|Department of Integrative Plant Biology, Institute of Plant Genetics of the Polish Academy of Sciences, 34Strzeszynska street, Poznan, PL-60-479, Wielkopolska, Poland. franklin@bio.uminho.pt.
Gregory
G
Franklin
D000873:Anthracenes; D053898:Biosynthetic Pathways; D018076:DNA, Complementary / Q000235:genetics; D015723:Gene Library; D018525:Germination; D020902:Hypericum / Q000033:anatomy & histology / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D010569:Perylene / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000235:genetics; D018749:RNA, Plant / Q000235:genetics; D066251:Subtractive Hybridization Techniques / Q000379:methods*
D000873:Anthracenes; D018076:DNA, Complementary; D012333:RNA, Messenger; D018749:RNA, Plant; D010569:Perylene; C004965:hypericin
Dark glands; Gene sequencing; Hypericin biosynthesis; Hypericum perforatum; Polymerase chain reaction; Suppression subtractive hybridization