研究论文 郑尧 1, 陈家长 1, 邴旭文 1 2 1B(NR5A1B), 2(FOXL2), DMRT1 [2 3] 摘要 : F 1 (Carassius auratus var. pengze) (Pcc) CYP19A1A.

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1 2015 9, 22(5): Journal of Fishery Sciences of China 研究论文 DOI: /SP.J 郑尧 1, 陈家长 1, 邴旭文 1 2, 王在照 1.,,, ; 2.,, 摘要 : F 1 (Carassius auratus var. pengze) (Pcc),,, PccF 1 (43.6±3.0)%, PccF 1 (4.7±1.2)%, Pcc-vasa Pcc-esr1 Pcc-esr2b, PccF 1 (P<0.01) (P<0.05) PccF 1, Pcc-amh Pcc-dmrt1b Pcc-dmrt1c Pcc-foxl2 Pcc-vasa Pcc-esr2b, (P<0.01) (P<0.05) ; (P<0.01) PccF 1,, : ; ; ; ; ; 中图分类号 : S917 文献标志码 : A 文章编号 : (2015) EDCs [1],, (Carassius auratus var. pengze, Pcc),, CYP19A1A,, (17 -estradiol, E2), AMH, DAX1, 1B(NR5A1B), 2(FOXL2), DMRT1 [2 3] [4], /, E2 [2], 3β (3βHSD), 11βHSD (11-KT), 1(cyp11a1), 17 /17, 20 收稿日期 : ; 修订日期 : 基金项目 : ( ); (2015BAD13B03); (CARS-49). 作者简介 : (1986 ),,,,. zhengy@ffrc.cn 通信作者 :,,,. Tel: , zzwang@nwsuaf.edu.cn

2 5 : 987 a1(cyp17a1), (star) F 1,,, V ;, I~II,, Pcc-amh Pcc-dax1 Pcc- cyp19a1a [5], 3, : (1) (Pcc-dmrt1a Pcc-dmrt1b Pcc-dmrt1c Pcc-foxl2 Pcc-nr5a1b SRY HMG 9a Pcc-sox9a DEAD-box RNA Pcc-vasa); (2) ( α1 Pcc-esr1 α2 Pcc-er alpha2 β2 Pcc-esr2a β1 Pcc-esr2b Pcc-ar); (3) (Pcc-3bhsd 11β 2 Pcc-11bhsd2 Pcc-cyp11a1 Pcc- cyp17a1 Pcc-star) F 1 ( 18 ), 3~5 (Cyprinus carpio var. singuonensis), (1600±175) g, 2, (450±15) g (n=300), 3, HCG [400 IU/kg( )] LRH-A [6 µg/kg( )] [1 mg/kg( ), 0.7%NaCl ], 6~8 h, 20, 3~4 d, F 1 (120 m 40 m 80 cm ) (30 m 12 m 1.5 m) ( 600 ) 1 1~2 0.1% (m/m) (Artemia naupli), : 10~15, ph 7.5~8.5, 28,, 2, : (25±1), 14 h 10 h (.. ) 1.2 PccF 1 F , (, ), PccF 1, % 1.3 PccF 1 RNA F 1 (n=11) (n=9), F 1 (n=10) (n=10),, GSI (gonadosomatic index, %)= / 100, HSI(hepatosomatic index, %)= / 100, [6] RNA 1.4 PccF 1 [6], Pcc-tubulin, 3, 1 (Pcc-dmrt1a Pcc-dmrt1b Pcc-dmrt1c Pcc-foxl2 Pcc-nr5a1b Pcc-sox9a Pcc-vasa); 2 (Pcc-esr1 Pcc-er alpha2 Pcc-esr2a Pcc-esr2b Pcc-ar); 3 (Pcc-3bhsd Pcc- 11bhsd2 Pcc-cyp11a1 Pcc-cyp17a1 Pcc-star) [6] PCR, 1

3 Tab. 1 表 1 本研究中用到的性腺分化相关基因的定量引物 qrt-pcr primers of sex-differentiation related genes in the present study qrt primer (5 3 ) sequence (5 3 ) /bp product length Pcc-tubulin-F Pcc-tubulin-R Pcc-dmrt1a-F Pcc-dmrt1a-R Pcc-dmrt1b-F Pcc-dmrt1b-R Pcc-dmrt1c-F Pcc-dmrt1c-R Pcc-amh-F Pcc-amh-R Pcc-dax1-F Pcc-dax1-R Pcc-foxl2-F Pcc-foxl2-R Pcc-nr5a1b-F Pcc-nr5a1b-R Pcc-sox9a-F Pcc-sox9a-R Pcc-vasa-F Pcc-vasa-R Pcc-esr1-F Pcc-esr1-R Pcc-er alpha2-f Pcc-er alpha2-r Pcc-esr2a-F Pcc-esr2a-R Pcc-esr2b-F Pcc-esr2b-R Pcc-ar-F Pcc-ar-R Pcc-3bhsd -F Pcc-3bhsd -R Pcc-11bhsd2-F Pcc-11bhsd2-R Pcc-cyp11a1-F Pcc-cyp11a1-R Pcc-cyp17a1-F Pcc-cyp17a1-R Pcc-cyp19a1a-F Pcc-cyp19a1a-R Pcc-star-F Pcc-star-R CCAGGGCTGTGTTTGTAGACC CAATAGTGTAGTGTCCACGGGC GTCGCCTGTCCAGCCATAA CGATGGAGTTGATGGAGAAGGT GGTCGCCATAACGTGTCC GATGGAGTCGACAGAGAAGGTT TATCACCTCAGAACCACCGTC TCGGAGAAAACCTGGTATTGC CTCACAGAGTGGAGGTGACA AACAGCAAAACGGATCGGTA ACTTCGGAGACACAAGAAACCA TTCAGGTGTGACAGTGTACCAAGT TTTATTTCAGTCAAAAGTCTCGTC CAAAGCCATTGCGTCATC GATAAAACCCAGAGGAAACGCT GCTGGAAGGATGCGAGGA CTGAGGACGGCAGTGAGCA GCTCTTGCTCTCCAGGGCT TACAGGACCCAAGGTTGTCTATG TGTTTCTGGACAGGAGTAGGCT TACGGCATCAGTAAATCGGGT GTGCTCCATTCCTTTGTTGCTC TAGGCAGCCCTCGCATCTT TGCCTGCTGAGAAGATACCACA TATCATTATGGTGTGTGGTCGTG ATCCTGCCAGAGAATCGTGTC CACACTCTACGCCGTCTCTGC CCCTAACAGCTTTGCATGAGTAA ATGGCAGGTTTGATGGAGGG TCCACAAGTGAGGGCTCCATA GAGGAATGGAATCCGCAATG CAGAAGTGAGAAGGGCAGAACG GTTTGGCATCATACGGGGC TGGGGTTGAGGAGAGAGGAGT AGGAGCCCCGAAGGAAAC ACGACCCATAGCGTACAGACC GGCTGCAGATATTCCCAAATAAAG TCAGAAGTGCGTCCAAGAGGT AGACTGGCCTGGTGGTTGC AGTGCTCAGCTCCCCGTG CCACATCCGAAGAAGAAGC CTGGTTACTGAGGATGCTGAT

4 5 : SPSS(18.0 ), = 2 Ct [7], ± ( x ±SD) t, P<0.05, P< PccF 1 PccF 1 (P<0.01, 2), GSI (P<0.01), HSI (P<0.05) PccF 1 (P<0.01, 2), GSI (P<0.01), HSI (P>0.05) (n=202), 3 (n=67, 67, 68) , 114 ; 3 (n=100 / ) , 286 ; PccF 1 (43.6±3.0)%, PccF 1 (4.7±1.2)%,, V ;, I~II Tab. 2 表 2 实验室和池塘养殖彭泽鲫 F 1 生物学指标 The bioindicator for F 1 offspring of Carassius auratus var. pengze cultured in laboratory and pond conditions culture environment sex /g body weight /cm total length /% GSI /% HSI laboratory pond male 7.755± ± ± ±1.068 female 7.978±1.124 ** 7.392±1.406 ** 2.167±1.195 ** 4.565±2.549 * male ± ± ± ±0.684 female ± ** 38.45±4.05 ** 2.087±0.591 ** 6.556± : ** (P<0.01), * (P<0.05). Note: ** stands for highly significant difference between the males and the females under identical culture environment (P<0.01), while * stands for significant difference (P<0.05). 2.2 PccF 1 PccF 1 1, Pccdax1 Pcc-dmrt1a Pcc-nr5a1b Pcc-sox9a (P<0.01), Pcc-dmrt1b Pcc-dmrt1c (P<0.05), Pcc-vasa (P<0.01) Pcc-er alpha2 Pcc-esr2a (P<0.01), Pcc-ar (P<0.05), Pcc-esr1 Pcc-esr2b (P<0.01) (P<0.05) Pcc-3bhsd Pcc-11bhsd2 Pcc-cyp11a1 Pcccyp17a1 Pcc-star (P<0.01), Pcc-cyp19a1a (P>0.05) 2.3 PccF 1 PccF 1 2,, Pcc-dax1 Pcc-nr5a1b Pcc-sox9a (P<0.05), Pcc-amh (P<0.05), Pcc-dmrt1b Pcc-dmrt1c Pcc-vasa (P<0.05), Pcc-dmrt1a (P>0.05) Pcc-esr1 Pcc-er alpha2 Pcc-ar (P<0.01), Pcc-esr2b (P<0.05), Pcc-esr2a (P>0.05) Pcc-cyp11a1 (P<0.01), Pcc-11bhsd2 Pcc-cyp17a1 Pcc-cyp19a1a Pcc-star (P<0.01), Pcc-3bhsd (P<0.05)

5 PccF 1 * (P<0.05), ** (P<0.01). Fig.1 Relative gonadal differentiation related gene expressions to tubulin between male and female PccF 1 offspring under laboratory culture condition * stands for significant difference between the males and females (P<0.05), while ** stands for highly significant difference (P<0.01). 2 PccF 1 * (P<0.05), ** (P<0.01). Fig.2 Relative gonadal differentiation related gene expressions to tubulin between male and female PccF 1 offspring under pond culture condition * stands for significant difference (P<0.05) between the males and females, while ** stands for highly significant difference (P<0.01).

6 5 : [8], [9] [8], (Perca flavescens),, [10], GSI, [11] vtg zp2, [12],, HSI ( ), [1], amh dax1 dmrt1 [13] foxl2 NR5A1b [14] sox9a [13] sox9a NR5A1b amh [14], (sox9a, dmrt1, amh) (dax1) NR5A1/foxl2- cyp19a1a [1], foxl2 NR5A1 Pcc-cyp19a1a E2 [15], [16], nr5a [17] dax1 [18] [3, 15] amh dmrt1 foxl2 [1], amh, amh [19], [1], 20 d,, 7 g,, I~II, V,, F 1 Pcc-dax1, Pcc-dmrt1a, Pccnr5a1b Pcc-sox9a ( 1), HPG (E2/T) [20], E2 [14 15] Pcc-foxl2 Pcc-cyp19a1a F 1, [20] PccF 1 ( 2), Pcc-ers1 Pcc-amh Pcc-dmrt1s Pcc-foxl2 Pcc-zp2 (Pcc-3bhsd Pcc- 11bhsd2 Pcc-cyp17a1 Pcc-cyp19a1a Pcc-star, 2),, Pcc-cyp19a1a (Pcc-amh Pcc- dmrt1s Pcc-foxl2),,,, [21], E2 [3, 15] 参考文献 : [1] Devlin R H, Nagahama Y. Sex determination and sex differentiation in fish: An overview of genetic, physiological, and environmental influences[j]. Aquaculture, 2002, 208(3 4): [2] Li M H, Yang H H, Li M R, et al. Antagonistic roles of Dmrt1 and Foxl2 in sex differentiation via estrogen production in tilapia as demonstrated by TALENs[J]. Endocrinology, 2013, 154(12): [3] Segner H, Casanova-Nakayama A, Kase R, et al. Impact of

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8 5 : 993 Analysis of different gene expression profiles of sex differentiationrelated genes between male and female pengze crucian carp (Carassius auratus var. pengze) under different culture modes ZHENG Yao 1, CHEN Jiazhang 1, BING Xuwen 1, WANG Zaizhao 2 1. Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences; Scientific Observing and Experimental Station of Fishery Resources and Environment in the Lower Reaches of the Changjiang River, Ministry of Agriculture; Key Open Laboratory of Ecological Environment and Resources of Inland Fisheries, Chinese Academy of Fishery Sciences, Wuxi , China; 2. College of Animal Science and Technology, Northwest A&F University, Shaanxi Key Laboratory of Molecular Biology for Agriculture, Yangling , China Abstract: Pengze crucian carp (Carassius auratus var. pengze, Pcc) is naturally gynogenic, and gynogenic Pcc produced by artificial breeding can theoretically produce solely female offspring. In laboratory culture, a higher proportion of male fish occurred in F 1 progenies compared with pond culture conditions. A higher proportion of males was found in the F 1 progeny under laboratory culture (43.6%) compared with pond (4.7%) conditions. To determine the cause of this variant sex ratio, ovarian gene expression profiles were detected and compared between the male and female F 1 progenies for different culture conditions. Results showed that expressions of most testicular sex differentiation-related, steroid receptor, and steroidogenic genes in PccF 1 offspring were significantly higher than those in ovaries under laboratory culture conditions, except for Pcc-vasa, Pcc-esr1, and Pcc-esr2b. With respect to pond culture conditions, expressions of most testicular sex differentiation-related and steroid receptor genes in PccF 1 offspring were significantly higher than those in ovaries, except for Pcc-amh, Pcc-dmrt1b, Pcc-dmrt1c, Pcc-foxl2, Pcc-vasa, and Pcc-esr2b. Amounts of the steroidogenic genes had reversed expression patterns (male<female). The gene expression profiles for steroidogenesis and transcriptional regulation of aromatase were different in the gonads of F 1 progenies between laboratory and pond culture conditions. The variant sex ratio may be attributed to the differential gene expression of both steroidogenic enzymes and transcription factors of aromatase under different culture densities, which may be associated with the regulation and/or synthesis of endogenous steroid hormone levels and related to different sex ratios. Key words: gynogenesis; Carassius auratus var. pengze; sex differentiation; steroid receptor; steroidogenic enzyme; gene expression Corresponding author: WANG Zaizhao. zzwang@nwsuaf.edu.cn