Page 33 - 《水产学报》2026年第2期

2 期 李 双，等：跨组织/性别系统评估曼氏无针乌贼内参基因的稳定性 50 卷

Pad Prism 9 software (GraphPad Software, Inc., Tab. 4 Average C t values and standard deviation of
USA). Values are shown as means ± SD. Differences candidate genes in S. japonica
in C values between males and females were assessed gene male/female average C t value standard deviation
t
using unpaired two-tailed t-test. P < 0.05 was con- 18S M 16.29 1.39
sidered statistically significant. F 15.47 1.76
ef-1α M 18.81 1.91
2 RESULTS F 17.91 1.32
ef-1γ M 20.83 1.72
2.1 Evaluation of amplification efficiency and F 19.63 1.50
primer specificity β-actin M 19.97 3.53
F 19.66 3.23
Total RNA was successfully extracted from each
gapdh M 19.75 2.31
sample and subsequently reverse transcribed into com-
F 19.73 1.83
plementary DNA (cDNA). Standard curves were gen-
erated by qPCR amplification using serial dilutions of
instability. ef-1α and ef-1γ were expressed at moderate
cDNA as templates. The amplification efficiencies for
levels with intermediate C value variability (Fig. 1-g-
t
the five candidate reference genes were between 93%
h, i-j; Tab. 4).
and 105% (Tab. 1), and the regression coefficients
This study compared the expression levels of
were above 0.97, showing strong linear relationships.
individual reference genes between sexes within the
All candidate reference gene primers met the criteria
same tissues. Three of the five genes (18S, β-actin, and
for reliable qPCR expression analysis. Melting curve
ef-1γ) exhibited significant sex-specific differences
analysis further confirmed the presence of a single
(P < 0.05) in expression in certain tissues (Fig. 2). For
peak for each primer pair, thus verifying their high
instance, 18S showed sex bias in the brain, optic lobe,
specificity.
heart, branchial heart, intestine, stomach, and white
2.2 Expression profiling of candidate reference body; β-actin displayed sex bias in the brain, heart,
genes gill, branchial heart, stomach, pancreas, and skin; and
ef-1γ was sex-biased in the optic lobe, stomach, pan-
qPCR assays were performed on diverse tissues
creas, and white body. Conversely, ef-1α and gapdh
of male and female S. japonica. Expression profiles of
displayed no significant sex-related expression differ-
the candidate reference genes were evaluated using
ences (P > 0.05) across any of the tissues analyzed.
raw C values, where lower C values correspond to
t
t
Interestingly, a comprehensive analysis integrating
higher transcript abundance. The overall C values
t
data from all tissues indicated that β-actin and gapdh
ranged from 16.29 ± 1.39 to 20.83 ± 1.72 in males (M)
expression levels did not significantly differ between
and 15.47 ± 1.76 to 19.73 ± 1.83 in females (F) (Tab. 4).
sexes (Fig. 2-s).
All candidate reference genes exhibited some
degree of tissue-dependent variability (Fig.1), with the 2.3 geNorm analysis
broadest C ranges observed in gonad-associated tis- Gene stability was evaluated using the geNorm
t
sues studied (testis, spermatophore, ovary, nidamental
algorithm, yielding average stability values (M values)
gland, and accessory nidamental gland). In both sexes, where an M value < 1.5 is indicative of stable expres-
among the candidates, 18S was the most abundantly sion, with lower M values correlating with greater sta-
expressed gene in all tissues except the male heart, bility. The results revealed tissue-specific stability
showing the smallest C variation (Fig. 1-a-b; Tab. 4). rankings for these genes in both male and female cut-
t
In contrast, β-actin and gapdh exhibited the widest tlefish. In males (Fig. 3), all candidate genes had M <
fluctuations in expression across tissues (Fig. 1-c-d, e- 1.5 detected in all tissues examined, with the excep-
f; Tab. 4), indicating significant transcriptional tion of gapdh in the stomach (Fig. 3-g). This generally

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