Ma, R.Q.
(Fisheries College, Ocean University of China)
,
He, F.
(Fisheries College, Ocean University of China)
,
Wen, H.S.
(Fisheries College, Ocean University of China)
,
Li, J.F.
(Fisheries College, Ocean University of China)
,
Mu, W.J.
(Fisheries College, Ocean University of China)
,
Liu, M.
(Fisheries College, Ocean University of China)
,
Zhang, Y.Q.
(Fisheries College, Ocean University of China)
,
Hu, J.
(Fisheries College, Ocean University of China)
,
Qun, L.
(Fisheries College, Ocean University of China)
The cytochrome P450c17-I (CYP17-I) is one of the enzymes critical to gonadal development and the synthesis of androgens. Two single nucleotide polymorphisms (SNPs) were detected within the coding region of the CYP17-I gene in a population of 75 male Japanese flounder (Paralichthys olivaceus). They w...
The cytochrome P450c17-I (CYP17-I) is one of the enzymes critical to gonadal development and the synthesis of androgens. Two single nucleotide polymorphisms (SNPs) were detected within the coding region of the CYP17-I gene in a population of 75 male Japanese flounder (Paralichthys olivaceus). They were SNP1 (c.C445T) located in exon2 and SNP2 (c.T980C (p.Phe307Leu)) located in exon5. Four physiological indices, which were serum testosterone (T), serum $17{\beta}$-estradiol ($E_2$), Hepatosomatic index (HSI), and Gonadosomatic index (GSI), were studied to examine the effect of the two SNPs on the reproductive endocrines of Japanese flounder. Multiple comparisons revealed that CT genotype of SNP1 had a much lower T level than CC genotype (p<0.05) and the GSI of individuals with CC genotype of SNP2 was higher than those with TT genotype (p<0.05). Four diplotypes were constructed based on the two SNPs and the diplotype D3 had a significantly lower T level and GSI. In conclusion, the two SNPs were significantly associated with reproductive traits of Japanese flounder.
The cytochrome P450c17-I (CYP17-I) is one of the enzymes critical to gonadal development and the synthesis of androgens. Two single nucleotide polymorphisms (SNPs) were detected within the coding region of the CYP17-I gene in a population of 75 male Japanese flounder (Paralichthys olivaceus). They were SNP1 (c.C445T) located in exon2 and SNP2 (c.T980C (p.Phe307Leu)) located in exon5. Four physiological indices, which were serum testosterone (T), serum $17{\beta}$-estradiol ($E_2$), Hepatosomatic index (HSI), and Gonadosomatic index (GSI), were studied to examine the effect of the two SNPs on the reproductive endocrines of Japanese flounder. Multiple comparisons revealed that CT genotype of SNP1 had a much lower T level than CC genotype (p<0.05) and the GSI of individuals with CC genotype of SNP2 was higher than those with TT genotype (p<0.05). Four diplotypes were constructed based on the two SNPs and the diplotype D3 had a significantly lower T level and GSI. In conclusion, the two SNPs were significantly associated with reproductive traits of Japanese flounder.
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문제 정의
However, there has been no report about SNPs of CYP17-I gene in fish until now. The purpose of this study was to investigate the SNPs of Japanese flounder CYP17-I gene which had been obtained (Genbank ID: FJ815204) and then analyze the effects of these SNPs on the reproductive endocrines. Four physiological indices were used to measure these effects.
05). To the best of our knowledge, this is the first report on the CYP17-I gene polymorphisms in the teleosts. SNPs identified in the present study have potential applications in breeding programs.
제안 방법
The PCR reaction was performed in a volume of 25 μl containing of 2.5 μl of 10×PCR Buffer, 80 ng of genomic DNA, 0.20 mM each dNTP, 0.50 mM each primer and 0.25 U Taq DNA polymerase (rTaq TaKaRa).
The SNP genotype of an individual at each SNP site was determined based on SSCP patterns and sequencing chromatograms. The genotype frequencies and allele frequencies for each polymorphism were calculated by Microsoft Excel.
GLM estimation was performed to analyze the associations of SNPs in CYP17-I gene with reproductive traits (Table 3). The results showed that SNP1 was significantly associated with T level (p<0.
대상 데이터
5℃; ≥4 mg/L O2; 14:10 h light:dark cycle). One hundred male Japanese flounder were sampled randomly and then anesthetized before the experiment. Blood was extracted from the fish tail with disposable syringes, and then stored at 4℃.
0 software. Seven primer sets were designed for the CYP17-I gene of Japanese flounder based on the 8 exons (Table 1). The primers of exon 4 were not designed because its sequence was less than 100 base pairs (bp).
데이터처리
The SNP genotype of an individual at each SNP site was determined based on SSCP patterns and sequencing chromatograms. The genotype frequencies and allele frequencies for each polymorphism were calculated by Microsoft Excel. ANOVA tests for associations of SNP genotypes with reproductive traits (T, E2 , HSI and GSI) were performed using Stat View software version 9.
The genotype frequencies and allele frequencies for each polymorphism were calculated by Microsoft Excel. ANOVA tests for associations of SNP genotypes with reproductive traits (T, E2 , HSI and GSI) were performed using Stat View software version 9.0 (SAS Institute Inc., Cary, NC). Significant differences among means of different genotypes were calculated using Duncan’s multiple-range test and p values no more than 0.
이론/모형
Total DNA was extracted from the frozen muscle samples using the phenolchloroform method. The DNA purity and concentration were measured using an UV spectrotometer (Amersham, American).
성능/효과
Significant differences among means of different genotypes were calculated using Duncan’s multiple-range test and p values no more than 0.05 were considered statistically significant.
The CT genotype of SNP1 and the CC genotype of SNP2 were respectively 10.67% and 13.33% which were obviously lower than the other genotypes. In addition, the two SNPs were both bi-allelic, each SNP site varied between only two nucleotides.
The results showed that SNP1 was significantly associated with T level (p<0.05) while SNP2 had significantly associations with GSI (p<0.05).
The results indicated that there was a significant association between diplotypes and T level and GSI (p<0.05).
The most important result we found was that individuals with CT genotype of SNP1 had a much lower T level than those with CC genotype, although the SNP1 was a synonymous mutation. Many studies on both humans and animals have reported that a genetic polymorphism existing in the coding region or promoter region is expected to be associated with a small or moderate affect on the gene function (Weber and Nathanson, 2000).
In conclusion, two SNPs were detected and they were both associated with the reproductive endocrines of Japanese flounder. The SNP1 located in exon2 was significantly associated with serum T level (p<0.
후속연구
SNPs identified in the present study have potential applications in breeding programs. Therefore, further studies are needed at the transcriptional regulation level and protein level for deeper insights into the SNPs effects on the physiological functions of P450c17-I. In general, SNPs have high potential for applications in aquaculture.
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