牙鲆amhr2基因的分子特征及组织表达

Molecular Characteristics and Expression Analysis of amhr2 Gene in Bastard Halibut Paralichthys olivaceus

为探讨牙鲆AMH受体AMHR2的分子特征及其在性腺发育中的作用,采用分子克隆和生物信息学方法克隆鉴定了牙鲆amhr2基因,通过半定量PCR和实时荧光定量PCR以及化学原位杂交技术测定了amhr2基因在牙鲆不同组织中的表达谱。试验结果显示:通过克隆验证拼接得到的牙鲆amhr2 cDNA序列其开放阅读框长为1536 bp,编码511个氨基酸;牙鲆AMHR2蛋白的近N-端含有长度为19个氨基酸(1~19位氨基酸)的信号肽结构,氨基酸序列对比发现其与狭鳞庸鲽的相似度最高,为89.96%;进化树分析得出,AMHR2牙鲆在进化上与狭鳞庸鲽、大菱鲆的亲缘关系最近,进化地位相一致,说明牙鲆amhr2基因与其他物种具有一定的保守性;定量结果显示,amhr2基因主要在牙鲆卵巢与精巢中表达,且在卵巢中的表达水平显著高于精巢;原位杂交结果表明,amhr2基因主要在牙鲆支持细胞、精巢初级精母细胞以及次级精母细胞中表达,并在卵巢间质细胞、Ⅱ~Ⅴ期卵母细胞以及卵泡膜中表达。牙鲆AMHR2氨基酸序列与狭鳞庸鲽亲缘关系最近,amhr2基因主要在性腺组织中表达,且在牙鲆精巢和卵巢的生殖细胞和体细胞中均有表达,可能在牙鲆的性腺发育中有重要的调控作用。

In order to investigate the molecular characteristics of anti-Müllerian hormone(AMH)and its typeⅡreceptor AMHR2 and its role in gonadal development,the amhr2 gene was cloned in bastard halibut Paralichthys olivaceus with gonads from stageⅢto stageⅣand body weight of(720±50)g and identified by molecular cloning and bioinformatics methods.The expression profile of amhr2 gene was determined in gonad,brain,stomach,muscle,liver,kidney and heart of bastard halibut by semi-quantitative PCR and real-time fluorescence quantitative PCR and chemical in situ hybridization(ISH).The results showed that the amhr2 cDNA sequence obtained by cloning and splicing was 1536 bp in length and encoded 511 amino acids.The amino acid sequence analysis showed that the near N-terminus of the Amhr2 protein contained a signal peptide structure of 19 amino acids(1st—19th amino acids)and the amino acid sequence comparison showed that it had the maximal similarity with Hippoglossus stenolepis(89.96%).The evolutionary tree analysis revealed that the amhr2 gene of bastard halibut was the most closely related to H.stenolepis and turbot Scophthalmus maximus,and its evolutionary status was consistent,indicating that the amhr2 gene of bastard halibut is conserved with other species.The quantitative results showed that the amhr2 gene was mainly expressed in the ovaries and testes,with significantly higher expression level in the ovaries than that in the testes.The in situ hybridization results showed that amhr2 gene was mainly expressed in the sertoli cells,primary spermatocytes and secondary spermatocytes of the testis,and expressed in the interstitial cells,Ⅱ—Ⅴoocytes and follicular membrane in the ovary.The amhr2 gene is most closely related to the H.stenolepis and expressed primarily in gonad and in both germ cells and somatic cells of the testis and ovary,which may be important for the regulation of gonadal development in the bastard halibut.