NOBOX基因的cDNA克隆及其特性分析

Cloning and characterization of porcine NOBOX gene

摘要:

NOBOX(新生儿卵巢同源基因)是一个卵母细胞特异性表达的同源基因, 在早期滤泡发生中起重要的作用。本研究结合电子克隆的方法, 从猪卵母细胞中成功地克隆了NOBOX基因的全长cDNA序列(GenBank Accession No. FJ587509)。猪NOBOX基因的cDNA全长为1768 bp, 包含1419 bp的开放阅读框。生物信息学分析表明NOBOX基因编码了472个氨基酸, 分子量为51.08 kD, 等电点为5.73。该蛋白定位于细胞核中, 含有一个保守的结构域——cd00086。借助Clustalw 软件, 采用N-J算法构建了NOBOX蛋白的系统进化树, 分析了不同物种间的进化关系。应用实时荧光定量PCR技术分析该基因在母猪不同组织、细胞及4种孤雌激活胚胎的表达模式, 结果表明该基因在母猪各组织中均有不同程度的表达, 其中在心脏、肾脏和卵母细胞中表达水平较高, 推测其可能在心脏、肾脏和卵母细胞中发挥着重要的作用; NOBOX基因在胚胎发育阶段的表达水平高于G-V期的卵母细胞, 表明在胚胎发育阶段pNOBOX的表达增强。

Abstract:

Newborn ovary homeobox gene (NOBOX) is an oocyte-specific homeobox gene that plays a critical role in early folliculogenesis and represents a candidate gene for nonsyndromic ovarian failure. We used in silico approach in combination with rapid amplification of cDNA ends (RACE) to clone the full-length cDNA of NOBOX (GenBank Accession No. FJ587509) from porcine oocytes. It contains 1768 bp nucleotides, with an open reading frame (ORF) of 1419 bp. The putative porcine NOBOX gene encodes 472 amino acids with the molecular weight of 51.08 kD and pI of 5.73. Bioinformatics prediction indicates that this protein contains a cd00086 homeodomain. Real-time PCR analysis showed that the NOBOX gene is expressed in various tissues, oocytes and embryos cells (4-cell, 8-cell, morula and blastocyst) at different expression levels. The expression levels of this gene in heart, kidney and oocytes are higher than that in other tissues, which suggested that the NOBOX protein might play an important role in those tissues. The expression of NOBOX in developmental stages is higher than that in GV-stage oocytes, which suggested that the expression of pNOBOX was enhanced in developmental stages.