牙鲆(Paralichthys olivaceus)抗缪勒氏管激素Ⅱ型受体基因(amhr2)表达特征分析及功能初探

doi:10.11978/2023040

Abstract

Abstract:

Anti-Mullerian hormone receptor Ⅱ (Amhr2) is the specific receptor of Anti-Mullerian hormone (Amh) in the TGF-β pathway. It is critical for gonadal differentiation and development in fish and determination of the sex in some fish species. However, the expression and function of amhr2 in fish has been poorly studied. To clarify the expression characteristics and function of amhr2, a 1536 bp CDS encoding 511 amino acids, was obtained from Paralichthys olivaceus, an important commercially cultured marine fish in China. The flounder Amhr2 protein was clustered with those from other teleosts, with the highly conservative motifs occurring in the C-terminal region. Gene collinearity analysis revealed that the amhr2 collaborates with specificity protein 1 (sp1) upstream, and proline-rich protein 13 (prr13) and poly (rC) binding protein 2 (PCBP2) downstream of the teleost genome. The predicted flounder Amhr2 protein was composed of a signal peptide, two transmembrane domains, and a conservative tyrosine protein kinase domain. Real-time quantitative PCR (qPCR) results showed that amhr2 was mainly expressed in the adult gonads, with significantly higher expression in the testes than in the ovaries (P<0.01). At stages I-V of the testis, the expression of amhr2 remained at a high level, whereas the expression level in the ovaries was significantly higher at stage I than at stages Ⅱ-V (P< 0.05). During the flounder gonadal differentiation period, gynogenesis (control, 20 ± 0.5℃, 100% female) and gynogenesis at high temperature (HT, 28 ± 0.5℃, 100% male) were used to detect the expression of amhr2. The results showed that the expression of amhr2 in the control group was highest at 2 cm total length (TL) and then decreased continuously, while the expression in the HT group first increased and then decreased. The inflection point of amhr2 expression in the HT group was at 6 cm TL, with the highest level. To investigate the effect of Amh on amhr2, we examined the co-localization of Amhr2 and Amh with Hela cells. Examination of subcellular localization showed that both Amhr2 and Amh were mainly located in the cytoplasm. Subsequently, the recombinant protein Amh of the flounder was obtained by prokaryotic expression. After in vitro incubation with the recombinant Amh, the expression of amhr2 in the testes showed no significant difference, whereas the transcription level in the ovaries increased significantly at 12 and 48 h (P<0.05). In addition, a dual luciferase reporter assay in HEK293T cells showed that co-transfection of Amh and Amhr2 could inhibit the promoter activity of the cytochrome P450 aromatase gene (cyp19a) (P<0.01), the key gene for estrogen synthesis. These findings suggest that amhr2 is mainly expressed in the testis, but its expression varies at different stages of the gonadal development. And amhr2 may affect the transcription of cyp19a together with Amh, which plays a role in the onset of the male phenotype formation and gonadal development in the flounder.

Key words: Paralichthys olivaceus, amhr2, gonadal differentiation and development, gene expression, function

LI Ze, WANG Lijuan, ZOU Congcong, SHU Chang, WU Zhihao, ZOU Yuxia, YOU Feng. Expression characteristics and function of Anti-Müllerian hormone receptor Ⅱ gene (amhr2) in Paralichthys olivaceus[J].Journal of Tropical Oceanography, 2024, 43(1): 94-106.

Figures/Tables 12

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引物	序列	NCBI序列号及参考文献
amhr2-CDS-F	ATTCCCAGGACCCTCCCTGTT	XM_020096590.1
amhr2-CDS-R	TCTGCAAAGAGAACAGTGCAAC	XM_020096590.1
amh-CDS-F	ATGCCGGTGGTGAACGTCTT	XM_020080898.1
amh-CDS-R	GCGGCATCCACACTCCTTTG	XM_020080898.1
amhr2-qPCR-F	CGTCGTGTTCCTGCCAAAC	XM_020096590.1
amhr2-qPCR-R	GGCAGCTCATACACCTCCTT	XM_020096590.1
actin- qPCR-F	GGAATCCACGAGACCACCTACA	EU090804 Hu et al, 2014
actin- qPCR-R	CTGCTTGCTGATCCACATCTGC	EU090804 Hu et al, 2014
ef- qPCR-F	AGCCAGAAGCGTTTTGAGGAG	XM_020104638.1 Zhong et al, 2008
ef- qPCR-R	AGATGGGGACGAAAGCAACAC	XM_020104638.1 Zhong et al, 2008
amh-pc3.1-EcoRV-F	CACACTGGACTAGTGGATCCGCCACCATGCCGGTGGTGAACGTC	XM_020080898.1
amh-pc3.1-EcoRV-R	AGCTTGGTACCGAGCTCGGATCCGCGGCATCCACACTCCT	XM_020080898.1
amhr2-pc3.1-BamHI-F	CACACTGGACTAGTGGATCCGCCACCATGATGCTGCAGTGGTGGC	XM_020096590.1
amhr2-pc3.1-BamHI-R	AGCTTGGTACCGAGCTCGGATCCTGAGCTGCTTTCAGAGACATAAGACTG	XM_020096590.1
amhr2-pCS2-BamHI-F	ACGACGACGATAAGGGATCCGCCACCATGATGCTGCAGTGGTGGC	XM_020096590.1
amhr2-pCS2-BamHI-R	AATTCGAATCGATGGGATCCTGAGCTGCTTTCAGAGACATAAGACTG	XM_020096590.1
amh-pET-EcoRV-F	AAGGCCATGGCTGATATGCCGGTGGTGAACGTCTT	XM_020080898.1
amh-pET-EcoRV-R	GAATTCGGATCCGATGCGGCATCCACACTCCTTTG	XM_020080898.1

转染组分(ng)\组别	cyp19a转染对照组	cyp19a+amh共转染组	cyp19a+amhr2共转染组	cyp19a+amh+amhr2共转染组
cyp19a-pGL3	400	400	400	400
amh-pc3.1	0	400	0	200
amhr2-pc3.1	0	0	400	200
TK-pRL	40	40	40	40
pcDNA 3.1	560	160	160	160

Expression characteristics and function of Anti-Müllerian hormone receptor Ⅱ gene (amhr2) in Paralichthys olivaceus

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