Mafia e corruzione: differenze concettuali, connessioni, strumenti di contrasto

Normalized ACL, AACT and HMGR mRNA levels in transgenic and wild-type T. brevicorniculatum plants quantified by qRT-PCR. The corresponding mRNA levels were normalized against the constitutive gene elongation factor 1 α (TbEF1α) from T. brevicorniculatum. Bars represent standard errors (n = three biological replicates). A: AtACLA1, AtAACT2 and AtHMGR1 transgene mRNA levels in transgenic lines. No significant differences at p < 0.05 were detected among the transgenic lines using the Mann-Whitney U test. B: Endogenous TbACLA, TbAACT and TbHMGR1 mRNA levels in all transgenic lines and wild-type (Wt) plants. No significant differences at p < 0.05 were detected between the wild-type plants and transgenic lines using the Mann-Whitney U test. (TIFF 1510 kb

Effect of DNA concentration on transformation efficiency.

<p>Protoplasts were injected with 100/µl (A and B), 500 ng/µl (C and D) and 1000 ng/µl (E and F) DNA solutions and monitored for evidence of GFP fluorescence after culture for one month. The transformation efficiencies represent the mean of three replicates. Arrows indicate the surviving injected protoplasts and small dots indicate dead cells. Scale bar  = 100 µm. All cells were injected in the visial field shown in this figure, but uninjected cells also developed into (non-fluorescent) microcalli.</p

Transfection efficiency is affected by different concentrations of MgCl₂ and the DNA incubation time.

<p>Oil palm protoplasts were transfected with 10 µg CFDV-hrGFP plasmid using 40% (w/v) PEG solution with MgCl₂ at concentrations of 10 mM (A), 25 mM (B), 50 mM (C) and 100 mM (D). Oil palm protoplasts were incubated with 10 µg CFDV-hrGFP plasmid DNA for 15 min (E) or 30 min (F), and then mixed with PEG-MgCl₂ solution. Transfection efficiency was calculated as the number of GFP-fluorescent protoplasts divided by the total number of protoplasts in one representative microscope field. The transfection efficiencies represent the mean of three replicates. Scale bar  = 10 µm in (A)–(E), 75 µm in (F).</p

Effects of DNA and PEG concentrations on transfection efficiency.

<p>Oil palm protoplasts were transfected with 25 µg (A) or 50 µg (B) of CFDV-hrGFP plasmid DNA, and with 50 µg CFDV-hrGFP plasmid DNA in the presence of 25% (C), 40% (D) or 50% PEG (E). Black arrows indicate damaged protoplasts caused by PEG toxicity. The transfection efficiencies represent the mean of three replicates. Scale bar  = 10 µm in (A) and (B), 25 µm in (C)–(F).</p

Oil palm protoplasts showing GFP fluorescence.

<p>A 3-month-old oil palm cell suspension culture in Y35N5D2iP liquid medium (A) was collected and cultured on Y35N5D2iP solid medium (B) for protoplast isolation (C). Transient GFP fluorescence was observed in protoplasts isolated from the 3-month-old cell suspension culture after subculture for 7 days (D) and 14 days (E), and protoplasts isolated from the 4-month-old cell suspension culture (F). CLSM images are shown representing GFP fluorescence (GFP), autofluorescence (Auto) and bright field (Bright) as well as three-layer images (Merged) of the protoplasts. Red arrows indicate autofluorescence. Scale bar  = 1 cm in (A) and (B), 100 µm in (C), 10 µm in (D), 25 µm in (E) and (F).</p

Development of microcalli expressing GFP.

<p>Five days after DNA microinjection, the alginate layer was transferred to Y3A liquid medium comprising 5.5% (w/v) sucrose and 8.2% (w/v) glucose supplemented with 10 µM NAA, 2 µM 2,4–D, 2 µM IBA, 2 µM GA₃, 2 µM 2iP and 200 mg/l ascorbic acid and cultured at 28°C for 2 weeks. The medium was then replaced with similar Y3A liquid medium comprising 4% (w/v) sucrose and 7.2% (w/v) glucose to allow the development of microcolonies (A and B, after 2 months). The medium was then replaced with Y3A liquid medium comprising 4% (w/v) sucrose to promote the conversion of microcolonies (C and D, after 4 months) into microcalli (E and F, after 6 months). Finally, the alginate layer containing microcalli (G) was transferred onto Y31N0.1BA solid medium (H) for the regeneration of oil palm plants. Arrows indicate the injected protoplasts. The transformation efficiencies represent the mean of three replicates. Scale bar  = 100 µm in (A)–(F), 1 cm in (G) and (H).</p

Microinjection of DNA into oil palm protoplasts.

<p>Oil palm protoplasts were isolated from a 3-month-old cell suspension culture after subculture for 7 days, mixed with 1% alginate solution in Y3A medium and distributed as a thin layer onto supporting medium (A). The embedded protoplasts were arranged in a single planar layer as confirmed by using the 10× objective (B). The protoplasts were incubated at 28°C in the dark for 3 days (C), and then placed on the microscope stage for DNA microinjection (D). The DNA solution was injected into the protoplast (E) and confirmed by Lucifer yellow fluorescence (F). GFP fluorescence was detected in the cytoplasm after 3 days (G and H). The injected protoplast is indicated by an arrow. Scale bar  = 1 cm in (A), (C) and (D), 100 µm in (B), 25 µm in (E)–(H).</p

Down-Regulation of Small Rubber Particle Protein Expression Affects Integrity of Rubber Particles and Rubber Content in <em>Taraxacum brevicorniculatum</em>

<div><p>The biosynthesis of rubber is thought to take place on the surface of rubber particles in laticifers, highly specialized cells that are present in more than 40 plant families. The small rubber particle protein (SRPP) has been supposed to be involved in rubber biosynthesis, and recently five SRPPs (TbSRPP1–5) were identified in the rubber-producing dandelion species <em>Taraxacum brevicorniculatum</em>. Here, we demonstrate by immunogold labeling that TbSRPPs are localized to rubber particles, and that rubber particles mainly consist of TbSRPP3, 4 and 5 as shown by high-resolution two-dimensional gel electrophoresis and mass spectrometric analysis. We also carried out an RNA-interference approach in transgenic plants to address the function of TbSRPPs in rubber biosynthesis as well as rubber particle morphology and stability. TbSRPP-RNAi transgenic <em>T. brevicorniculatum</em> plants showed a 40–50% reduction in the dry rubber content, but neither the rubber weight average molecular mass nor the polydispersity of the rubber were affected. Although no phenotypical differences to wild-type particles could be observed <em>in vivo</em>, rubber particles from the TbSRPP-RNAi transgenic lines were less stable and tend to rapidly aggregate in expelling latex after wounding of laticifers. Our results prove that TbSRPPs are very crucial for rubber production in <em>T. brevicorniculatum</em>, probably by contributing to a most favourable and stable rubber particle architecture for efficient rubber biosynthesis and eventually storage.</p> </div

Association of TbSRPPs with rubber particles. A.

<p>Fifty micrograms of purified rubber particle proteins were separated by isoelectric focussing (pH 3–10 IPG strips) and subsequent SDS-PAGE. The gel was stained with colloidal Coomassie Brilliant Blue. <b>B.</b> Backscattered electron imaging of rubber particles labeled with 10 nm gold particles by immunodetection using the TbSRPP-antibody. <b>C.</b> Gold labeling of rubber particles was not appreciably detected in backscattered electron images, when the corresponding pre-immune serum was used as primary antibody. Micrographs are shown as inverted images. Scale bars  = 400 nm.</p

<i>In vitro</i> analysis of the colloidal stability of rubber particles from TbSRPP-RNAi transgenic plants and wild-type plants.

<p><b>A.</b> Measurement of the zeta (ζ)-potential of rubber particles from wild-type plants (filled circles) and TbSRPP-RNAi transgenic plants (open circles) suspended in modified rubber extraction buffer (REB) at given pH values. Values represent mean ± SD of three independent plant lines (S3, S76 and S85) respectively of six wild-type plants (wt). No significant differences were detected among the plants using Students t-test (P, 0.05). <b>B.</b> Measurement of the z-average diameter of rubber particles from wild-type plants (filled circles) and TbSRPP-RNAi transgenic plants (open circles) suspended in modified REB with the indicated pH values. Values represent mean ± SD of three independent plant lines (S3, S76 and S85) respectively of six wild-type plants (wt). Significant differences have been detected among TbSRPP-RNAi lines and wild-type plants using Students t-test (P, 0.01). <b>C, F.</b> Confocal laser scanning microscopic analysis of isolated rubber particles from TbSRPP-RNAi transgenic plants (C) and wild-type plants (F) that were stained with nile red. <b>D, E </b><b>and </b><b>G, H.</b> Transmission electron microscopic analysis of isolated rubber particles from TbSRPP-RNAi transgenic and wild-type plants. <b>D.</b> Micrograph of isolated rubber particles from TbSRPP-RNAi transgenic plant line S3 suspended in modified rubber extraction buffer (REB) pH 7.2 at low and <b>E.</b> higher magnification. <b>G.</b> Micrograph of rubber particles from wild-type suspended in modified REB pH 7.2 at low and <b>H.</b> higher magnification. Enlarged areas are indicated with dashed boxes. Scale bars = 1 µm.</p