斑马鱼fem-1c cDNA克隆与表达分析

为进一步探究鱼类性别决定的相关机理, 增加对鱼类性控基因表达和功能的认识, 克隆斑马鱼fem-1c 基因并对其进行表达分析。研究采用RACE-PCR方法从斑马鱼卵巢组织cDNA中克隆了fem-1c的cDNA全长序列, 其大小为2701 bp, 编码618个氨基酸。生物信息学分析显示, 斑马鱼FEM-1C蛋白包含9个ANK结构域、2个TPR结构域和2个低复杂性区域, 与其他脊椎动物的FEM-1C蛋白序列保守性较高。脊椎动物的fem-1c与tmed7、trim36等邻近的45个基因具有保守的同线性关系。半定量RT-PCR实验结果显示斑马鱼fem-1c在受精后17d开始表达, 并特异地表达于成体卵巢组织中。RNA原位杂交结果显示, fem-1c基因mRNA定位于卵巢组织的Ⅰ期和Ⅱ期卵母细胞胞质中。fem-1c的时空表达特征暗示其在斑马鱼卵巢分化中具有重要作用。  

THE CLONING AND EXPRESSION ANALYSIS OF ZEBRAFISHFEM-1C,A MEMBER OFFEM-1 FAMILY

Feminization-1 (fem-1) gene was first discovered in Caenorhabditis elegans, and it plays an important role in nematode sex determination. The mutation in fem-1c could feminize the nematode sex differentiation. There are three conservative members of fem-1 family in vertebrate, namely fem-1a, fem-1b and fem-1c. We cloned the entire cDNA sequence of fem-1c from zebrafish ovary cDNA library using RACE-PCR. The full length of zebrafish fem-1c cDNA was 2701 bp, encoding a 618-amino acid protein. We used online bioinformatic software and predicted that the FEM-1C protein contained 9 ANK domains, 2 TPR domains and 2 low complexity regions. Zebrafish FEM-1C shared a highly conservative protein sequence with its homologues in other vertebrates. The sequence analysis also revealed that in vertebrates there was a conservative synteny between fem-1c and its neighborhood genes, especially the nearest 45 genes such as tmed7 and trim36. We conducted the expression analysis in different tissues of adult zebrafish using RT-PCR and found that fem-1c was exclusively expressed in the ovary. The expression analysis in the gonad of larvae at different development stages showed that the expression of fem-1c began on the 17th day post-fertilization. The in situ hybridization results showed that fem-1c was expressed in cytoplasm at stage I and Ⅱ of oocyte. This specific expression pattern suggested that fem-1c might play a role in zebrafish ovary development.