International Journal of Marine Science 2016, Vol.6, No.4, 1-12
4
for 1 min. Equal quantities of total RNA were
examined in triplicate for each condition. The relative
expression levels of each group were normalized to
ef1-α and expressed as the mean ± S.E. Student’s
t-test was used to statistically analyse and compare
two groups. Multiple-group comparisons were
analyzed for significant differences between group
using one-way ANOVA with a Tukey test (Statistica
version 5.1; StatSoft. Inc., USA). The differences
were defined as significant at
p
< 0.05.
Table 1 Primer sequences and gene names.
Gene name
Primer sequence
(5’→3’)
PCR
size
(bp)
Accession
number
Estrogen receptor (
ER
)
F:CCGGCCCTACACAGAGATCA
R:AGCCAAGAGCTCTCCAACAACT
150 bp
NM_152959
Estrogen receptor 1 (
ER1
)
F: CTGTGCCGTCTGCAGTGATT
R: CGGCGGTTCTTGTCGATAGT
150 bp
AF516874
Estrogen receptor 2 (
ER 2
)
F: TCCGACACCTCAGCAACAAA
R: TTTCTGGGCTCTGTTGTCTGTCT
150 bp
AF349413
NK2 homeobox 5 (
Nkx2.5
)
F: CGGGATGGTAAACCGTGTCT
R: GCTCGACGGATAGTTGCATGA
150 bp
NM_131421
NK2 homeobox 7 (
Nkx2.7
)
F: AGCTCACATCCACACAGGTCAA
R: GAGCTCCGTGACAGGGTTTG
150 bp
NM_131419
Heart and neural crest derivatives
expressed 2 (
Hand2
)
F: TGTCATGAAGAACCCCCCTAT
R: CCCCGGTACTCCTCCGTAGT
150 bp
NM_131626
GATA-binding protein 4
(
GATA-4
)
F: CCAGTCTGCAACGCATGTG
R: GATCGCCGACTGACCTTCAG
150 bp
NM_131236
GATA-binding protein 5
(
GATA-5
)
F: GGGACGCCAGGGAACTCTA
R: CACGCGTTGCACAGGTAGTG
150 bp
NM_131235
GATA-binding protein 6
(
GATA-6
)
F:AGTCGCGACCAGTACCTTTCAA
R: CCTTCGGGATTGCAGTGAGT
150 bp
NM_131557
Fibroblast growth factor 1a
(
FGF1a
)
F: ATGGCAAGCTGTACGCTTCA
R: GGCCCCGTTTCATTTTCC
150 bp
NM_200760
T-box 2a (
Tbx2a
)
F:ACGTTTTCCCTGAGACCGATT
R:ATGGAAGGGTCAGCTGTTTCC
150 bp
AF179405
T-box 2b (
Tbx2b
)
F: ACGTTTTCCCTGAGACCGATT
R:ATGGAAGGGTCAGCTGTTTCC
150 bp
NM_131051
T-box 5a (
Tbx5a
)
F: CGGATGTTTCCCAGCTTCAA
R: CATCGCAGGCTCAGCTTTC
150 bp
NM_130915
Elongation factor 1 (
ef-1
)
F: TGGTGGTGTCGGTGAGTTTG
R: AAACGAGCCTGGCTGTAAGG
150 bp
AY422992
3 Results
3.1 Developmental toxicity of 4-tert-octylphenol
To evaluate the toxic effects of 4-t-OP on zebrafish
embryogenesis, embryos were exposed to 0.1
M, 0.5
M, 1
M, 2.5
M and 5
M 4-t-OP and compared
with their corresponding control group (embryo
medium contain PTU only). The survival rate,
hatching rate and malformation rate were used as
criteria to evaluate the toxic ity of 4-t-OP to
zebrafish embryos. As a result showed in Table 2,
the survival rate, hatching rate and malformat ion
rate exhibit dose effects to 4-t-OP concentration.
Embryos treated with 0.1 M 4-t-OP as well as control
group developed normally, and the survival rate and
hatching rate at 3 dpf were more than 95%. However,
the survival rate and hatching rate at 3 dpf were
declined accompanied by the increasing of 4-t-OP
concentration. A 12% of survival rate and 23% of
hatching rate at 3 dpf were showed in the presence of
5
M 4-t-OP, and all the embryos were seen to be
deformed. Concentration higher than 5
M resulted in
100% mortality at 2 dpf and 3 dpf. Around 50% of
survival rate and 60% of malformation rate at 3 dpf
were shown in the embryos treated with 1
M of
4-t-OP, and a high proportion of cardiovascular defect
was revealed in malformation samples. Thus, 1
M of
4-t-OP concentration was used to characterize the
