Quantifiable downregulation of endogenous genes in Agaricus bisporus mediated by expression of RNA hairpins.
Functional gene studies in the cultivated white button mushroom Agaricus bisporus have been constrained by the absence of effective gene-silencing tools. Using two endogenous genes from A. bisporus, we have tested the utility of dsRNA hairpin constructs to mediate downregulation of specific genes. Hairpin constructs for genes encoding orotidine 5'- monophosphate decarboxylase (URA3) and carboxin resistance (CBX) were introduced into A. bisporus using Agrobacteriummediated transfection. Although predicted changes in phenotype were not observed in vitro, quantitative-PCR analyses indicated unambiguously that transcripts in several transformants were substantially reduced compared with the non-transformed controls. Interestingly, some hairpin transformants exhibited increased transcription of target genes. Our observations show that hairpin transgenic sequences can mediate downregulation of A. bisporus endogenous genes and that the technology has the potential to expedite functional genomics of the mushroom.
Warwick HRI, University of Warwick, Wellesbourne, Warwickshire, CV35 9EF, U.K.
Ana S M B
AS
Costa
D John I
DJ
Thomas
Daniel
D
Eastwood
Simon B
SB
Cutler
Andy M
AM
Bailey
Gary D
GD
Foster
Peter R
PR
Mills
Micheal P
MP
Challen
D000364:Agaricus / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D015536:Down-Regulation; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics; D055029:Inverted Repeat Sequences; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000096:biosynthesis* / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014162:Transfection; D015854:Up-Regulation
D005656:Fungal Proteins; D012330:RNA, Double-Stranded