Martins Rute S T, Deloffre Laurence A M, Mylonas Constantinos C, Power Deborah M, Canário Adelino V M
Centro de Ciências do Mar, CIMAR-Laboratório Associado, University of Algarve, Faro, Portugal.
Reprod Biol Endocrinol. 2007 May 30;5:19. doi: 10.1186/1477-7827-5-19.
DAX1 (NR0B1), a member of the nuclear receptors super family, has been shown to be involved in the genetic sex determination and in gonadal differentiation in several vertebrate species. In the aquaculture fish European sea bass, Dicentrarchus labrax, and in the generality of fish species, the mechanisms of sex determination and differentiation have not been elucidated. The present study aimed at characterizing the European DAX1 gene and its developmental expression at the mRNA level.
A full length European sea bass DAX1 cDNA (sbDAX1) was isolated by screening a testis cDNA library. The structure of the DAX1 gene was determined by PCR and Southern blot. Multisequence alignments and phylogenetic analysis were used to compare the translated sbDAX1 product to that of other vertebrates. sbDAX1 expression was analysed by Northern blot and relative RT-PCR in adult tissues. Developmental expression of mRNA levels was analysed in groups of larvae grown either at 15 degrees C or 20 degrees C (masculinising temperature) during the first 60 days, or two groups of fish selected for fast (mostly females) and slow growth.
The sbDAX1 is expressed as a single transcript in testis and ovary encoding a predicted protein of 301 amino acids. A polyglutamine stretch of variable length in different DAX1 proteins is present in the DNA binding domain. The sbDAX1 gene is composed of two exons, separated by a single 283 bp intron with conserved splice sites in same region of the ligand binding domain as other DAX1 genes. sbDAX1 mRNA is not restricted to the brain-pituitary-gonadal axis and is also detected in the gut, heart, gills, muscle and kidney. sbDAX1 mRNA was detected as early as 4 days post hatching (dph) and expression was not affected by incubation temperature. Throughout gonadal sex differentiation (60-300 dph) no dimorphic pattern of expression was observed.
The sbDAX1 gene and putative protein coding region is highly conserved and has a wide pattern of tissue expression. Although gene expression data suggests sbDAX1 to be important for the development and differentiation of the gonads, it is apparently not sex specific.
DAX1（NR0B1）是核受体超家族的成员，已被证明参与多种脊椎动物物种的遗传性别决定和性腺分化。在水产养殖鱼类欧洲海鲈（Dicentrarchus labrax）以及大多数鱼类物种中，性别决定和分化的机制尚未阐明。本研究旨在在mRNA水平上对欧洲海鲈DAX1基因及其发育表达进行表征。
通过筛选睾丸cDNA文库分离出全长欧洲海鲈DAX1 cDNA（sbDAX1）。通过PCR和Southern印迹确定DAX1基因的结构。使用多序列比对和系统发育分析将翻译后的sbDAX1产物与其他脊椎动物的产物进行比较。通过Northern印迹和相对RT-PCR分析成年组织中的sbDAX1表达。在孵化后前60天在15摄氏度或20摄氏度（雄性化温度）下生长的幼虫组中，或在选择用于快速（大多为雌性）和缓慢生长的两组鱼中分析mRNA水平的发育表达。
sbDAX1在睾丸和卵巢中作为单一转录本表达，编码一个预测的301个氨基酸的蛋白质。不同DAX1蛋白中存在可变长度的聚谷氨酰胺延伸，位于DNA结合域。sbDAX1基因由两个外显子组成，由一个283 bp的单一内含子隔开，在配体结合域的相同区域具有保守的剪接位点，与其他DAX1基因相同。sbDAX1 mRNA不限于脑-垂体-性腺轴，在肠道、心脏、鳃、肌肉和肾脏中也可检测到。sbDAX1 mRNA最早在孵化后4天（dph）被检测到，其表达不受孵化温度的影响。在整个性腺性别分化过程（60 - 300 dph）中未观察到二态性表达模式。
sbDAX1基因和推定的蛋白质编码区域高度保守，具有广泛的组织表达模式。尽管基因表达数据表明sbDAX1对性腺的发育和分化很重要，但它显然不是性别特异性的。
