斑马鱼ca15b的克隆及在原始生殖细胞中的特异表达

CLONING, IDENTIFICATION AND EXPRESSION ANALYSIS OF CA15B, A NOVEL GENE SPECIFICALLY EXPRESSED IN PRIMORDIAL GERM CELLS OF ZEBRAFISH

高保真PCR克隆获得了ca15b基因的全长,利用胚胎整体原位杂交等技术研究了ca15b基因在斑马鱼早期发育过程中的动态表达。结果发现,ca15b在斑马鱼早期发育过程中存在显著的原始生殖细胞(Primordial germ cell,PGC)特异表达模式。ca15b是一个母源性表达的基因:在分裂期的胚胎中,其mRNA集中分布于位于分裂沟的生殖质(Germ plasm);从囊胚期开始,可以观察到其在PGC中的特异表达;在原肠胚中,其mRNA在体细胞中急剧降解,仅特异表达于PGC,这一表达特征一直持续到受精后1d的胚胎。将体外合成的包含5′UTR和3′UTR的ca15b全长mRNA注射到斑马鱼胚胎后,仅能增强原肠期之前胚胎中ca15b的整体杂交信号;在原肠胚期之后,注射的mRNA在体细胞中快速降解。这提示在ca15b mRNA上可能存在某种转录后调控其在早期胚胎体细胞中降解而在PGC中稳定存在的机制。

Primordial germ cells （PGCs） are the ancestor cells of adult gametes at embryonic stage. PGCs-specific gene expression plays a fundamental role in germ cell induction, development, migration and differentiation. Understanding the functional role of PGCs-specifically expressed genes could provide us some tools to perform gene and embryo ma-nipulations. Screening a public database of zebrafish （http：//www.zfin.org） we found a PGCs-specific candidate gene, carbonic anhydrase 15b （ca15b）. To confirm this, whole-mountin situ hybridization and other technologies were uti-lized to analyze the expression ofca15bduring zebrafish early development. We observed thatca15b is a maternally expressed gene and it is highly expressed in germ plasm during cleavage stage. The expression ofca15b at mRNA level was specifically detected in the PGCs at blastula stage. At gastrula stage,ca15b is still strictly expressed in PGCs while its expression is undetectable in somatic cells. This specific expression pattern ofca15b persisted until one day post-fertilization. More importantly, the post-transcriptional regulation elements that mediate PGCs-specific expression ofca15b may locate in its coding sequence.