Genomics and Applied Biology 2015, Vol. 6, No. 7, 1-8
4
Bacto agar) plate and incubated at 30
C for 2 days.
Percent survival was obtained by dividing the number
of viable colonies on plates subjected to heat
treatment by the number of viable colonies on plates
held at 30
C (control) and multiplying by 100.
Carotenoid accumulation
To measure carotenoid accumulation, ~1 X 10
6
cells/ml of each
N. crassa
strains were inoculated into
Vogel’s sucrose (2%) medium supplemented with
0.2% Tween 80 used as a wetting agent to prevent
conidiation (Zalokar, 1954) and kept for growth at
C
Figure 2 Growth phenotypes. (A) Rate of apical growth of the
wild-type,
trm-9
,
nca-2
, and
trm-9
nca-2
strains were
measured using race tubes. Growth rate of
trm-9
nca-2
double mutant strain was lesser as compared to parental single
mutants and wild-type strain. (B) Colony
morphology of
wild-type,
trm-9,
nca-2,
and
trm-9
nca-2
double mutant
strains. The
trm-9
nca-2
double mutant strain showed
matty-like growth and reduced pigmentation. (C) Dry weight of
trm-9,
nca-2,
trm-9
nca-2
and wild-type strains. Dry
weight yield of
trm-9
nca-2
double mutant strain was less
than parental single mutants and the wild-type
30
C for two days in dark, and at room temperature
for one day under light. After that mycelia were
filtered, lyophilized and powdered. Carotenoids were
extracted from 50 mg lyophilized powder by using
acetone and hexane. Total carotenoids content were
calculated by measuring the maximum absorbance
value at 470 nm and using formula: Total carotenoid
content (µg/g) = [Total absorbance x Total volume of
extract (ml) x 10
4
]/ [Absorbance coefficient (2500) x
Sample weight (g)] (Rodriguez-Amaya and Kimura
2004).
Results and Discussion
NCU04120, NCU04898, and NCU04736 genes
encode CaM, TRM-9, and NCA-2, respectively,
and contain conserved domains
The
N. crassa
calmodulin
gene (
cmd
) NCU04120
encodes a highly conserved Ca
2+
signaling protein
CaM that possesses four conserved EF hand motifs
(Supplementary Figure 3A). Similarly, the NCU04898
encodes a Ca
2+
/cation ATPases annotated as TRM-9
