1.2.2 Dormant chlamydospore
The culture grown in a grain medium for 30 days was placed in a shallow
dish and treated at 28 ℃ and RH30% for 10 days. Then, the culture was
dried for 4 days under circulating air at 35 ℃, and the water content
was determined to be less than 3%. The spore germination rate was
calculated in accordance with the method described by Wang et al. (2019)
[3]. The germination rate should be below 5%, sealed, and stored in
a dry place for later use. The spores were purified and counted in the
same way as non-dormant spores.
1.3 Extraction and determination of cAMP from
chlamydospore
The purified chlamydospore-concentrated suspension was collected, added
with liquid nitrogen, and ground repeatedly in a mortar until
flocculation. Appropriate amount of PBS, 10 μL of 10 mmol/L EDTA, and
200 μL of 100 mmol/L phenylmethyl sulfonyl fluoride were added, and
ultrasonic crushing (power 400 w) was carried out in an ice water bath
for 10 min, with an interval of 2 s. After the spores broke the wall,
they were bathed in water at 80 ℃ for 2 h, cooled, and centrifuged at
8000 r/min for 10 min. The supernatant was absorbed, and 1 mL of
deionized water was added to the residue for centrifugation and
extraction two times. The obtained supernatant is the extraction liquid
of cAMP.
The protein was removed from the cAMP extract by using the Sevag method
[31], in which chloroform and n-butanol were first mixed at a volume
ratio of 4:1 to produce a mixture of chloroform and n-butanol. The
extract solution of cAMP was mixed with the mixed solution of chloroform
n-butanol at a volume ratio of 4:1, shaken for 30 min, and then
centrifuged at 12000 r/min for 5 min to separate the chloroform phase
from the water phase. The water phase was taken into a new centrifuge
tube to obtain a cAMP extract, which removes the protein.
The cAMP Direct Immunoassay Kit (Colorimetric, BioVion) was used to
detect cAMP in chlamydospore. The standard solution was prepared in
accordance with the kit instructions. The absorbance of the standard
solution was measured using the enzyme marker (RT-5000, Beijing
Yuanpinghao Biotechnology Co., LTD.), and the standard curve was drawn.
The OD value of the samples to be tested was measured using the enzyme
marker, with three parallel samples for each sample, and the average
value of each parallel sample was determined three times.
Determination of soluble proteins in chlamydospore
The spore wall was broken, centrifuged, and washed in accordance with
the abovementioned methods to remove the spore wall, and the resulting
supernatant is the spore protein extraction solution. Protein
concentration was determined using the Coomassie Brilliant Blue method
in accordance with the instruction of the Bradford Protein Concentration
Assay Kit (BioVion products). First, the protein standard stored at −20
℃ is melted and prepared into a series of standard solution. 200 μL of
G250 staining solution was added into each hole of the 96-well plate,
and various parameters of the enzyme marker were set. The 96-well plate
was placed into the enzyme-labeled instrument for testing, and the data
were recorded and saved. The OD value of the measured standard solution
was used to draw the standard curve. The volume of the added sample was
recorded during sample determination, and three parallels were repeated
per well. Then, the OD value of the sample to be tested was determined
using the enzyme marker by adding a dye solution in the same way as the
standard product, and the test was repeated three times for each
parallel sample. Furthermore, the protein concentration was determined
using the Coomassie Brilliant Blue method in accordance with the
instructions of the Bradford Protein Concentration Determination Kit
(BioVion product).
1.5 Data analysis
In this experiment, the content of spores in the original sample was
measured by the number of spores/mL, whereas the horizontal coordinate
of the standard curve was the concentration of W/V, that is, pmol/50 μL
(cAMP assay) or mg/mL (protein assay). First, the absorbance values
measured by different samples were substituted into the standard curve
to find the concentration of cAMP or protein in different samples
(pmol/50 μL or mg/mL), and then the protein or cAMP content in different
samples was determined (pmol or mg) in accordance with the volume of the
sample when determining the absorbance. Considering that the number of
spores in the sample is measured per milliliter, it is finally converted
to the protein or cAMP content (pmol or mg) per 107spores. The mean value was measured, and the difference in protein or
cAMP content between dormant and non-dormant spores was compared by
ANOVA in SPSS.