2. Prepare a similar mix for reference β-tubulin gene with primers
tub-2F and tub-2R.
3. Run the qPCR protocol in a LightCycler 480 II real-time
instrument. Analyze transcription levels with LightCycler
480. Normalize carS (the gene of interest) expression with
β-tubulin as a reference gene (see Note 14). Addition of Dox
resulted in an induction of carS gene (Fig. 3).
3.4 Quantification of
Carotenoids in
Transformants with
Modulated Expression
3.4.1 Cultivation,
Harvesting and Preparation
of Mycelia
1. Prepare DG agar and DG-Dox agar (DG with 20 μg/ml Dox)
media and lay them in 8.9-cm Petri dishes. Inoculate strains
SG39 and SG262 at 7 symmetrical points on both media (see
Note 15).
2. Incubate the Petri dishes at 30
C, for 7 days under $6 W/m
2
white light, $360 lx. In our case, the cultures were illuminated
with four fluorescent tubes Philips TL-D 18 W/840 at a distance of ca. 60 cm (see Note 16).
3. Cut the agar around the mycelial colonies with a clean scalpel
blade. Scrap carefully the rest of the agar from the mycelia with
the blade. Press the mycelial samples on filter papers to remove
excess of water. Freeze in liquid nitrogen, and lyophilize the
samples overnight.
3.4.2 Extraction and
Quantification of
Carotenoids
1. Take a 2-ml screw-cap microtube (see Note 17). Add washed
sea sand (see Note 18), approximately 20–50 mg of dry mycelia
(see Note 19), and 1 ml acetone (see Notes 20 and 21).
2. Shake the microtubes containing the samples in a FASTPREP24 with 2 pulses of 30 s at 6 m/s. This allows extraction
of carotenoids.
3. Centrifuge the extraction tubes at maximal speed
(>12,000 Â g) for 2 min and transfer the acetone supernatant
to a clean 2-ml microcentrifuge tube (see Note 22). Dry the
samples in a speed vacuum concentrator (see Note 23).
4. Repeat the extraction procedures (steps 2–3) up to total
bleaching of the sample, and add the supernatant to the tube
of the same sample already dried in the speed vacuum
concentrator.
5. Dissolve the dry extract in 1 ml of acetone for carotenoid-rich
samples or in 100 μl of acetone for low-content carotenoid
samples (see Note 24). Centrifuge the sample at maximal
speed (>12,000 Â g) for 2 min (see Note 25).
6. Transfer the supernatant to a clean 1.5-ml microfuge tube.
Take the sample to a spectrophotometer UV cuvette and measure the absorbance in the complete spectrum from 350 nm to
650 nm in a spectrophotometer (Fig. 4b) (see Note 26).
Modulated Expression of a Key Regulatory Gene in Carotenoid Biosynthesis
355
tub-2F and tub-2R.
3. Run the qPCR protocol in a LightCycler 480 II real-time
instrument. Analyze transcription levels with LightCycler
480. Normalize carS (the gene of interest) expression with
β-tubulin as a reference gene (see Note 14). Addition of Dox
resulted in an induction of carS gene (Fig. 3).
3.4 Quantification of
Carotenoids in
Transformants with
Modulated Expression
3.4.1 Cultivation,
Harvesting and Preparation
of Mycelia
1. Prepare DG agar and DG-Dox agar (DG with 20 μg/ml Dox)
media and lay them in 8.9-cm Petri dishes. Inoculate strains
SG39 and SG262 at 7 symmetrical points on both media (see
Note 15).
2. Incubate the Petri dishes at 30
C, for 7 days under $6 W/m
2
white light, $360 lx. In our case, the cultures were illuminated
with four fluorescent tubes Philips TL-D 18 W/840 at a distance of ca. 60 cm (see Note 16).
3. Cut the agar around the mycelial colonies with a clean scalpel
blade. Scrap carefully the rest of the agar from the mycelia with
the blade. Press the mycelial samples on filter papers to remove
excess of water. Freeze in liquid nitrogen, and lyophilize the
samples overnight.
3.4.2 Extraction and
Quantification of
Carotenoids
1. Take a 2-ml screw-cap microtube (see Note 17). Add washed
sea sand (see Note 18), approximately 20–50 mg of dry mycelia
(see Note 19), and 1 ml acetone (see Notes 20 and 21).
2. Shake the microtubes containing the samples in a FASTPREP24 with 2 pulses of 30 s at 6 m/s. This allows extraction
of carotenoids.
3. Centrifuge the extraction tubes at maximal speed
(>12,000 Â g) for 2 min and transfer the acetone supernatant
to a clean 2-ml microcentrifuge tube (see Note 22). Dry the
samples in a speed vacuum concentrator (see Note 23).
4. Repeat the extraction procedures (steps 2–3) up to total
bleaching of the sample, and add the supernatant to the tube
of the same sample already dried in the speed vacuum
concentrator.
5. Dissolve the dry extract in 1 ml of acetone for carotenoid-rich
samples or in 100 μl of acetone for low-content carotenoid
samples (see Note 24). Centrifuge the sample at maximal
speed (>12,000 Â g) for 2 min (see Note 25).
6. Transfer the supernatant to a clean 1.5-ml microfuge tube.
Take the sample to a spectrophotometer UV cuvette and measure the absorbance in the complete spectrum from 350 nm to
650 nm in a spectrophotometer (Fig. 4b) (see Note 26).
Modulated Expression of a Key Regulatory Gene in Carotenoid Biosynthesis
355