体节特异性表达基因terra的调控与功能研究

Zebrafish terra gene encodes a member of DM transcription factor family. Unlike other members of this family that are expressed in the gonads, terra is specifically expressed in the forming somites. This study cloned terra genomic gene of 3154 bp, which consists of 4 exons and 3 introns. Transfection of human cells with terra-GFP expressing constructs identified 74KGFKR78 of Terra as a nuclear localization signal, K77 and R78 of which are key residues for the signal. A 7.8 kb promoter sequence of terra is fused to GFP and injection of the resulted construct leaded to GFP expression only in a few somatic cells of the zebrafish embryos. This suggests that this sequence is insufficient for controlling correct spatiotemporal expression of terra, and that other important regulatory elements may exist in introns or regions outside the gene. In attempt to study function of terra using double-stranded RNA interfering approach, we unexpectedly found that double-stranded RNA can activate defense system in embryonic cells of zebrafish, which causes nonspecific degradation of endogenous mRNAs and eventually leads to abnormal development, even death of the embryos. A transgene construct consisting of a-actin promoter and full-length coding sequence of terra was injected into zebrafish embryos to generate transgenic fish. We identified 5 transgenic germlines out of 140 founder fish. Although the transgenic embryos expressed terra at a high level in the somites, the embryos showed no defects in somites or altered expression pattern of somite-specific markers such as myoD and snail. When the binding domain of Terra (BDT) was fused to GFP and the fusion gene (BDT-GFP) was placed under the control of a-actin promoter, the injected embryos showed GFP expression in the nuclei of somatic cells. However, we were unable to identify any transgenic fish after screen of more than 250 F0 fish. We speculate that there exist other members of DM transcription factor family in the genome of zebrafish that are involved in reproductivity, and overexpression of the BDT may competitively inhibit function of such members and thus result in failure of differentiation of BDT-expressing primordial germ cells. In order to study the potential function of Terra in somitogenesis, we have replaced a-actin promoter with a heat-inducible hsp70 promoter to control the expression of the BDT-GFP. The identification of the transgenic fish is underway.

通过突变和转基因技术，鉴定负责斑马鱼terra基因的体节特异性表达的调控元件；通过活逡煳槐泶锓ǎ治雠咛シ⒂讨衪erra基因和其它与体节形成和分化有关的基因之间相互作用关系；用显性干扰等位基因法，分析terra活性受抑制后胚胎的表型变化。这些研究对诓鱰erra在体节发生中的作用以及体节形成和分化的分子机制有重要的意义。.....................................