【摘要】 为了解NR5a2在中华鳖(Pelodiscus sinensis)性腺分化过程中的作用,通过RACE技术克隆获得中华鳖NR5a2 cDNA。结果显示:该基因序列全长为2 674 bp,开放阅读框为1 608 bp,编码535个氨基酸,5′非编码区长度为40 bp,3′非编码区长度为1 026 bp。生物信息学分析表明该基因编码的蛋白质分子质量为60.5 ku,等电点为8.57,具有保守的ZnF-C4结构域和HOLI结构域。氨基酸多重序列比对分析表明中华鳖NR5a2基因与西部锦龟(Chrysemys picta bellii)的相似性最高,其次是原鸡(Gallus gallus)。系统进化分析表明中华鳖NR5a2与西部锦龟的亲缘关系最近。实时荧光定量PCR结果表明NR5a2在中华鳖不同组织中均有表达,且卵巢组织中的表达量显著高于精巢组织。31.5℃孵化条件下,不同发育时期的中华鳖胚胎组织中发现NR5a2基因在17期时表达量达到最高,随后显著下降。结果表明NR5a2可能参与中华鳖的性别分化。更多还原

【Abstract】 The complete nucleotide sequence of NR5a2 cDNA in Pelodiscus sinensis was obtained by 3′RACE and 5′RACE in present study to understand the effect of specific gene on gonadal differentiation.The full-length cDNA of NR5a2 contained 2 674 bp.The open reading frame(ORF)was 1 608 bp,encoding a putative protein of 535 amino acids.The length of 5′-UTR and 3′-URT was 40 bp and 1 026 bp,respectively.Bioinformatics analysis showed that the molecular weight of the protein encoded by the NR5a2 was 60.5 ku,and the isoelectric point was 8.57.The ORF contained one conservative ZnF-C4 domain and one conservative HOLI domain.Multiple sequence alignment analysis revealed that the NR5a2 coding region showed mostly similarity in amino acids sequence with Chrysemys picta bellii,followed by Gallus gallus.Phylogenetic tree analysis showed that P.sinensis was more affinity with C.picta bellii.The qRT-PCR analysis showed that NR5a2 was existed in all the examined tissues,and the expression level of ovarian tissue was significantly higher than that of testis.The expression of NR5a2 in the 17 th stage of embryo development was the highest and then significantly decreased.Therefore,the NR5a2 gene might be involved in gender differentiation of P.sinensis.更多还原