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Gene silencing in the marine diatom Phaeodactylum tricornutum.

De Riso V, Raniello R, Maumus F, Rogato A, Bowler C, Falciatore A - Nucleic Acids Res. (2009)

Bottom Line: We report the successful silencing of a GUS reporter gene expressed in transgenic lines, as well as the knockdown of endogenous phytochrome (DPH1) and cryptochrome (CPF1) genes.Initial molecular analyses reveal that targeted downregulation likely occurs through transcriptional and post-transcriptional gene silencing mechanisms.Interestingly, molecular players involved in RNA silencing in other eukaryotes are only poorly conserved in diatoms.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Ecology and Evolution of Plankton, Stazione Zoologica Anton Dohrn, Villa Comunale, 80121, Napoli, Italy.

ABSTRACT
Diatoms are a major but poorly understood phytoplankton group. The recent completion of two whole genome sequences has revealed that they contain unique combinations of genes, likely recruited during their history as secondary endosymbionts, as well as by horizontal gene transfer from bacteria. A major limitation for the study of diatom biology and gene function is the lack of tools to generate targeted gene knockout or knockdown mutants. In this work, we have assessed the possibility of triggering gene silencing in Phaeodactylum tricornutum using constructs containing either anti-sense or inverted repeat sequences of selected target genes. We report the successful silencing of a GUS reporter gene expressed in transgenic lines, as well as the knockdown of endogenous phytochrome (DPH1) and cryptochrome (CPF1) genes. To highlight the utility of the approach we also report the first phenotypic characterization of a diatom mutant (cpf1). Our data open the way for reverse genetics in diatoms and represent a major advance for understanding their biology and ecology. Initial molecular analyses reveal that targeted downregulation likely occurs through transcriptional and post-transcriptional gene silencing mechanisms. Interestingly, molecular players involved in RNA silencing in other eukaryotes are only poorly conserved in diatoms.

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Related in: MedlinePlus

Molecular analysis of silenced clones. (A) PCR analysis of the full length GUS gene in the untransformed wild-type strain (wt), in the transgenic GUS expressing cells (Pt/GUS), and in selected silenced clones. M, 1 Kb DNA size marker. (B) GUS activity of the clones shown in (A). Values are normalized to the mean GUS activity of the Pt/GUS strains (100% activity). Pt/GUS data are the mean of 10 independent sub-clones from the GUS parental transgenic strain. (C) Relative GUS mRNA levels analysed by qRT-PCR in the same strains, and normalized with respect to the expression of an internal standard RPS gene.
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Figure 2: Molecular analysis of silenced clones. (A) PCR analysis of the full length GUS gene in the untransformed wild-type strain (wt), in the transgenic GUS expressing cells (Pt/GUS), and in selected silenced clones. M, 1 Kb DNA size marker. (B) GUS activity of the clones shown in (A). Values are normalized to the mean GUS activity of the Pt/GUS strains (100% activity). Pt/GUS data are the mean of 10 independent sub-clones from the GUS parental transgenic strain. (C) Relative GUS mRNA levels analysed by qRT-PCR in the same strains, and normalized with respect to the expression of an internal standard RPS gene.

Mentions: The different constructs were introduced in a transgenic P. tricornutum line expressing the GUS reporter gene (Pt/GUS) and putative transformants expressing the silencing construct were first selected on phleomycin. A preliminary screening of GUS expression levels performed by histochemical assays indeed indicated that around 50% of clones displayed a reduced GUS staining (data not shown). We therefore initiated a more detailed analysis by randomly selecting 10 independent antibiotic resistant clones as well as 10 sub-clones from the parental Pt/GUS strain. In each case, the presence of the full-length GUS gene was verified by PCR, and the level of GUS activity quantified (Figure 2 and Supplementary Table 1).Figure 2.


Gene silencing in the marine diatom Phaeodactylum tricornutum.

De Riso V, Raniello R, Maumus F, Rogato A, Bowler C, Falciatore A - Nucleic Acids Res. (2009)

Molecular analysis of silenced clones. (A) PCR analysis of the full length GUS gene in the untransformed wild-type strain (wt), in the transgenic GUS expressing cells (Pt/GUS), and in selected silenced clones. M, 1 Kb DNA size marker. (B) GUS activity of the clones shown in (A). Values are normalized to the mean GUS activity of the Pt/GUS strains (100% activity). Pt/GUS data are the mean of 10 independent sub-clones from the GUS parental transgenic strain. (C) Relative GUS mRNA levels analysed by qRT-PCR in the same strains, and normalized with respect to the expression of an internal standard RPS gene.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2724275&req=5

Figure 2: Molecular analysis of silenced clones. (A) PCR analysis of the full length GUS gene in the untransformed wild-type strain (wt), in the transgenic GUS expressing cells (Pt/GUS), and in selected silenced clones. M, 1 Kb DNA size marker. (B) GUS activity of the clones shown in (A). Values are normalized to the mean GUS activity of the Pt/GUS strains (100% activity). Pt/GUS data are the mean of 10 independent sub-clones from the GUS parental transgenic strain. (C) Relative GUS mRNA levels analysed by qRT-PCR in the same strains, and normalized with respect to the expression of an internal standard RPS gene.
Mentions: The different constructs were introduced in a transgenic P. tricornutum line expressing the GUS reporter gene (Pt/GUS) and putative transformants expressing the silencing construct were first selected on phleomycin. A preliminary screening of GUS expression levels performed by histochemical assays indeed indicated that around 50% of clones displayed a reduced GUS staining (data not shown). We therefore initiated a more detailed analysis by randomly selecting 10 independent antibiotic resistant clones as well as 10 sub-clones from the parental Pt/GUS strain. In each case, the presence of the full-length GUS gene was verified by PCR, and the level of GUS activity quantified (Figure 2 and Supplementary Table 1).Figure 2.

Bottom Line: We report the successful silencing of a GUS reporter gene expressed in transgenic lines, as well as the knockdown of endogenous phytochrome (DPH1) and cryptochrome (CPF1) genes.Initial molecular analyses reveal that targeted downregulation likely occurs through transcriptional and post-transcriptional gene silencing mechanisms.Interestingly, molecular players involved in RNA silencing in other eukaryotes are only poorly conserved in diatoms.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Ecology and Evolution of Plankton, Stazione Zoologica Anton Dohrn, Villa Comunale, 80121, Napoli, Italy.

ABSTRACT
Diatoms are a major but poorly understood phytoplankton group. The recent completion of two whole genome sequences has revealed that they contain unique combinations of genes, likely recruited during their history as secondary endosymbionts, as well as by horizontal gene transfer from bacteria. A major limitation for the study of diatom biology and gene function is the lack of tools to generate targeted gene knockout or knockdown mutants. In this work, we have assessed the possibility of triggering gene silencing in Phaeodactylum tricornutum using constructs containing either anti-sense or inverted repeat sequences of selected target genes. We report the successful silencing of a GUS reporter gene expressed in transgenic lines, as well as the knockdown of endogenous phytochrome (DPH1) and cryptochrome (CPF1) genes. To highlight the utility of the approach we also report the first phenotypic characterization of a diatom mutant (cpf1). Our data open the way for reverse genetics in diatoms and represent a major advance for understanding their biology and ecology. Initial molecular analyses reveal that targeted downregulation likely occurs through transcriptional and post-transcriptional gene silencing mechanisms. Interestingly, molecular players involved in RNA silencing in other eukaryotes are only poorly conserved in diatoms.

Show MeSH
Related in: MedlinePlus