文昌鱼Pax1/9基因上游调控区的克隆及分析

Cloning and Analysis of the AmphiPax1/9 Upstream Regulatory Region

脊椎动物Pax1/9是一重要的发育调控基因亚家族,文昌鱼基因组中仅有单一的该亚家族直系同源基因Amphi-Pax1/9,为检验此基因上游调控元件的功能在脊椎动物体内是否具有通用性,将文昌鱼Pax1/9基因上游约4.6 kb的侧翼序列与绿色荧光蛋白(GFP)报告基因连接,构建重组质粒表达载体(pAmphiPax1/9-AcGFP),显微注射斑马鱼胚胎,并以斑马鱼Pax1和Pax9的上游同源序列为阳性对照.结果表明,阳性对照斑马鱼胚胎中GFP能够表达,但注射pAm-phiPax1/9-AcGFP质粒的斑马鱼胚胎中无明显的GFP表达,这一现象可能是由于Pax1/9上游调控序列在二物种间存在较大的进化差异,启动元件具有物种特异性.

Pax1/9 is a crucial developmental regulatory gene subfamily in vertebrates. Amphioxus possesses a single orthologous gene ArnphiPax1/9 of the subfamily. In order to investigate if the ArnphiPax1/9 upstream sequence contains regulatory elements and promotes gene expression in zebrafish, a recombinant expression vectors（pAmphiPax1/9-AcGFP） inserted with a 4. 6 kb DNA segment of ArnphiPax1/9 upstream region was constructed, and then,microinjected the vectors into zebrafish embryos at one-cell stage using homologous sequences from zebrafish Pax1 and Pax9 as positive controls. The results showed that no GFP expression is observable in the embryos injected with pAmphiPax1/9-AcGFP,whereas the expression appears clearly in the positive controls. The observations suggest that the AmphiPax1/9 upstream region was unable to promote gene expression in zebrafish, which might be owing to the deep divergence between the two species.