真鲷(Pagrus major)vasa基因5′端启动子克隆及表达载体构建

MOLECULAR CLONING AND CONSTRUCTION OF THE 5'FLANKING REGION EXPRESSION VECTOR OF THE vasa GENE OF RED SEABREAM(PAGRUS MAJOR)

采用染色体步移(Genome walking)的方法克隆了真鲷vasa基因5′侧翼序列,采用生物信息学方法分析潜在的顺式作用元件,并与斑马鱼核心启动子进行比对,在此基础上构建了绿色荧光蛋白表达载体。序列分析结果显示:克隆得到的真鲷vasa基因5′侧翼序列序列长度为2762bp,其中包括TATA-box、CAAT-box、SP-1、GAGA-1、OCT-1、v-Myb、Sox-5、SRY、HNF等可能对vasa基因转录调控起重要作用的顺式作用元件。潜在的核心启动子区与斑马鱼核心启动子具有同源性(61.1%),推测可能对转录调控具有重要的作用。采用PCR方法扩增起始密码5′侧翼全长片段,连接入去除CMV启动子的绿色色荧光蛋白载体,最终成功构建真鲷vasa基因5′侧翼表达载体,为进一步研究分析启动子效率和绿色荧光蛋白在PGCs中的特异表达提供了基础。

Red seabream （Pagrus major） is a commercially important marine species in China. With the rapid development of marine aquaculture industry, resource conservation and identification of red seabream is increasingly important. Although sperm cryopreservation of the red seabream is relatively successful, little progress has made on embryo cryopreservation. Primordial germ cell （PGC） is the progenitor of the germ cell lineage, giving rise to either egg or sperm, with the potential to create complete individual organisms after fertilization. Cryopreservation of PGCs provides a new way for resource conservation. Vasa, a member of DEAD-box gene family, is strictly expressed in germ cell lineage. It is widely used as a molecular marker to study origin, migration and differentiation of PGCs. The objective of this study is to inves- tigate potential cis-acting elements that involve in the transcription regulation of vasa gene in red seabream and set up a basis for further research on promoter efficiency and isolation of PGCs with GFP special expression. As the vasa 5＇UTR reported （GenBank： AB378581） is not long enough to design three nested primers, we first amplified the first intron to get enough information. Next, 5＇ flanking region of red seabream vasa was amplified from genomic DNA by Genome Walking. Potential transcript factor binding sites were analyzed by bioinformatics method, the potential core promoter was blasted with the core promoter of zebrafish and finally pvasa-EGFP expression vector was constructed. Sequence analysis revealed that the 5＇ flanking region amplified in this study was 2762bp, and the region contained several potential transcription factor binding sites that may have important function on the gene transcription, such as TATA-box, CAAT-box, SP-1, GAGA-1, Oct-l, v-Myb, Sox-5, SRY and HNF. The potential core promoter of red seabream was similar to that of zebrafish, sug- gesting that it may play important role in the transcription regulation. Based on the above information, full length of the vasa 5＇ flanking region above the start codon was amplified and linked to EGFP report gene for the further research.