Transient RNA Silencing of Scoulerine 9-O-Methyltransferase Expression by Double Stranded RNA in Coptis japonica Protoplasts

  title={Transient RNA Silencing of Scoulerine 9-O-Methyltransferase Expression by Double Stranded RNA in Coptis japonica Protoplasts},
  author={Joseph Gogo Dubouzet and Takashi Morishige and Nanae Fujii and Chung-Il An and Eiichiro Fukusaki and Kentaro Ifuku and Fumihiko Sato},
  journal={Bioscience, Biotechnology, and Biochemistry},
  pages={63 - 70}
RNAi (RNA interference, RNA silencing) is a powerful tool for functional genomics, but the construction of an RNAi vector(s) and the establishment of stable transformants are time-consuming and laborious. Here we report the transient RNAi of endogenous biosynthetic genes involved in isoquinoline alkaloid biosynthesis in Coptis japonica protoplasts. Double stranded (ds) RNA fragments of various lengths prepared from several different positions of the coding sequence of scoulerine 9-O… 
Identification of regulatory protein genes involved in alkaloid biosynthesis using a transient RNAi system.
The use of transient RNAi is reported here to isolate regulatory factor genes involved in isoquinoline alkaloid biosynthesis in Coptis japonica protoplasts using double-stranded RNAs prepared against candidate regulatory factors predicted from an EST library.
Identification of a WRKY protein as a transcriptional regulator of benzylisoquinoline alkaloid biosynthesis in Coptis japonica.
A regulator of transcription in berberine biosynthesis is identified using functional genomics with a transient RNA interference (RNAi) and overexpression of the candidate gene, which is named CjWRKY1.
Isoquinoline alkaloid biosynthesis is regulated by a unique bHLH-type transcription factor in Coptis japonica.
The isolation of a novel basic helix-loop-helix protein, CjbHLH1, from IQA-producing Coptis japonica is reported here, and the unique role of Cj b HLH1 in IQA biosynthesis is discussed.
Functional Analysis of Norcoclaurine Synthase in Coptis japonica*
Results suggested that CjNCS1 is the major NCS in C. japonica, whereas native NCS extracted from cultured C.Japonica cells was more active and formed a larger complex compared with recombinant Cj NCS1.
RNAi and functional genomics
The further development of a gene-substitution method based on dRNAi could provide a new tool for the characterization of plant gene functions using a more rational approach.
Transcription factors in alkaloid biosynthesis.
Structures of the three homoeologous loci of wheat benzoxazinone biosynthetic genes TaBx3 and TaBx4 and characterization of their promoter sequences
The results imply that stage-specific transcription ofTaBx3 and TaBx4 is not controlled by global sequence similarity of their promoters but by some essential cis-elements.
Molecular Characterization of LjSWEET3, a Sugar Transporter in Nodules of Lotus japonicus
Analysis of transporters functioning in nodules of Lotus japonicus shows consistent with a role for LjSWEET3 in sugar translocation towards nodules and also suggest the possible existence of multiple routes of carbon supply into nodules.


Specific RNA Interference in psbP Genes Encoded by a Multigene Family in Nicotiana tabacum with a Short 3′-Untranslated Sequence
It is suggested that double-stranded RNA having a 3′-untranslated sequence could be useful for an isogene-specific RNA interference of the family genes in Nicotiana tabacum.
A biochemical framework for RNA silencing in plants.
This finding supports the view that plant miRNAs direct RNAi and that miRNA-specified mRNA destruction is important for proper plant development and endonuclease complexes guided by small RNAs are a common feature of RNA silencing in both animals and plants.
Suppression of gene expression by RNA interference in cultured plant cells.
A quantitative study of the effects of RNAi in cultured plant cells that are uniform and divide synchronously for functional analysis of genes of interest and constructed dsRNA expression plasmids for a luciferase gene under the control of the cauliflower mosaic virus 35S promoter by simply connecting sense and antisense sequences in a head-to-head manner.
Technical advance. Double-stranded RNA interferes with gene function at the single-cell level in cereals.
The results suggest that direct delivery of dsRNA to cereals leads to a rapid and sequence-specific interference with gene function at the single-cell level.
A Transient RNA Interference Assay System Using Arabidopsis Protoplasts
This work reports a dsRNA-mediated transient RNAi assay system using protoplasts from Arabidopsis mesophyll cells and suspension-cultured cells (cell line T87), which led to marked silencing of target transgenes.
Molecular cloning and functional expression of O-methyltransferases common to isoquinoline alkaloid and phenylpropanoid biosynthesis.
  • S. Frick, T. Kutchan
  • Biology, Chemistry
    The Plant journal : for cell and molecular biology
  • 1999
Ten isoforms produced displayed distinct substrate specificities and in some cases co-infection with two different recombinant baculoviruses led to the O-methylation of new substrates, suggesting that some biosynthetic enzymes may be common to both phenylpropanoid and alkaloid anabolism.
A simple and rapid system for the quantitation of RNA interference in plant cultured cells.
The phenomenon known as RNA interference (RNAi) by double-stranded RNA (dsRNA) that was reported recently in the nematode Caenorhabditis elegans has been shown to operate by a mechanism that is
Molecular Cloning and Characterization of CoclaurineN-Methyltransferase from Cultured Cells of Coptis japonica *
Recombinant CNMT was purified to homogeneity, and enzymological characterization confirmed that CoptisCNMT has quite broad substrate specificity, i.e. not only for 6-O-methylnorlaudanosoline and norreticuline but also for 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline.
Antisense RNA-Mediated Suppression of Benzophenanthridine Alkaloid Biosynthesis in Transgenic Cell Cultures of California Poppy1
Results show that alterations in the metabolic flux through benzophenanthridine alkaloid biosynthesis can affect the regulation of amino acid pools in California poppy cell cultures.
Molecular cloning of columbamine O-methyltransferase from cultured Coptis japonica cells.
The result clearly indicated that EST analysis was useful for isolating the candidate gene in a relatively well-characterized biosynthetic pathway and the relationship between the structure and substrate recognition of the O-methyltransferases involved in isoquinoline alkaloid biosynthesis, and a reconsideration of the biosynthesis pathway to palmatine are discussed.