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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

(A) Wild-type (left) and transgenic P. tricornutum cells (right) expressing the GUS gene, grown on agar plates and stained for GUS activity. Groups of cells are shown in blow up. GUS activity in the Pt/GUS strain used for the silencing analysis is indicated. (B) Schematic maps of the anti-sense and the inverted repeat constructs. Anti-sense constructs: GUS fragments of 240 or 390 bp cloned between the stop codon of the selectable Sh ble gene and the diatom FcpA terminator region. Inverted-repeat constructs: the GUS fragments were cloned in sense and anti-sense orientation. The region of self-complementarity is shown in blue, whereas the non-complementary region (corresponding to the spacer) is indicated by the diagonal lines. H4p (Histone 4 promoter), FcpBp (Fucoxanthin Chlorophyll a/c-binding Protein B promoter).
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Figure 1: (A) Wild-type (left) and transgenic P. tricornutum cells (right) expressing the GUS gene, grown on agar plates and stained for GUS activity. Groups of cells are shown in blow up. GUS activity in the Pt/GUS strain used for the silencing analysis is indicated. (B) Schematic maps of the anti-sense and the inverted repeat constructs. Anti-sense constructs: GUS fragments of 240 or 390 bp cloned between the stop codon of the selectable Sh ble gene and the diatom FcpA terminator region. Inverted-repeat constructs: the GUS fragments were cloned in sense and anti-sense orientation. The region of self-complementarity is shown in blue, whereas the non-complementary region (corresponding to the spacer) is indicated by the diagonal lines. H4p (Histone 4 promoter), FcpBp (Fucoxanthin Chlorophyll a/c-binding Protein B promoter).

Mentions: In order to establish a gene silencing methodology for diatoms we first targeted a reporter gene expressed in a transgenic line, as has been done in other organisms (31,32). We specifically used β-glucoronidase (GUS) because GUS activity can be easily detected and quantified in diatoms (Figure 1A) (11).Figure 1.


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)

(A) Wild-type (left) and transgenic P. tricornutum cells (right) expressing the GUS gene, grown on agar plates and stained for GUS activity. Groups of cells are shown in blow up. GUS activity in the Pt/GUS strain used for the silencing analysis is indicated. (B) Schematic maps of the anti-sense and the inverted repeat constructs. Anti-sense constructs: GUS fragments of 240 or 390 bp cloned between the stop codon of the selectable Sh ble gene and the diatom FcpA terminator region. Inverted-repeat constructs: the GUS fragments were cloned in sense and anti-sense orientation. The region of self-complementarity is shown in blue, whereas the non-complementary region (corresponding to the spacer) is indicated by the diagonal lines. H4p (Histone 4 promoter), FcpBp (Fucoxanthin Chlorophyll a/c-binding Protein B promoter).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: (A) Wild-type (left) and transgenic P. tricornutum cells (right) expressing the GUS gene, grown on agar plates and stained for GUS activity. Groups of cells are shown in blow up. GUS activity in the Pt/GUS strain used for the silencing analysis is indicated. (B) Schematic maps of the anti-sense and the inverted repeat constructs. Anti-sense constructs: GUS fragments of 240 or 390 bp cloned between the stop codon of the selectable Sh ble gene and the diatom FcpA terminator region. Inverted-repeat constructs: the GUS fragments were cloned in sense and anti-sense orientation. The region of self-complementarity is shown in blue, whereas the non-complementary region (corresponding to the spacer) is indicated by the diagonal lines. H4p (Histone 4 promoter), FcpBp (Fucoxanthin Chlorophyll a/c-binding Protein B promoter).
Mentions: In order to establish a gene silencing methodology for diatoms we first targeted a reporter gene expressed in a transgenic line, as has been done in other organisms (31,32). We specifically used β-glucoronidase (GUS) because GUS activity can be easily detected and quantified in diatoms (Figure 1A) (11).Figure 1.

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