猪sall4b基因的克隆及表达分析

Cloning and gene expression of sall4b gene in pig

摘要:

sall4基因是sall基因家族的一个成员，在胚胎发育、器官形成和干细胞多能性的维持以及重建中都起到重要作用，有sall4a和sall4b两种剪切突变体类型。目前猪的sall4基因序列尚未获得。鉴于其在多能性细胞调控中的作用，对猪的sall4基因进行了克隆测序，并对其在各组织及胚胎中的表达进行了初步研究。通过5￠和3￠RACE克隆得到猪sall4基因cDNA全长序列 (2 372 bp)，序列分析证明此基因编码的蛋白结构更接近于小鼠和人Sall4B亚型，同源性可达70％~80％，而与其他物种的Sall4A相比则缺少一段含锌指结构域的片段，同源性降至30％~55％。Real-time PCR证明猪sall4b基因广泛表达于猪的各种器官，其中除卵巢组织呈高量表达之外，脾、肺、心和睾丸表达量也相对较高；在早期胚胎发育过程中除4-细胞阶段相对表达量较低，其他阶段呈高量表达。免疫荧光跟踪Sall4在猪早期胚胎中的表达情况发现Sall4在着床前胚胎中全程表达并定位于细胞核中，在囊胚阶段基因表达趋向于定位在内细胞团中。表达分析证明sall4b基因与多能性紧密相关，预示着猪sall4b基因将可能作为新的重编程因子用于诱导猪多能干细胞的体系中。

Abstract:

sall4, a member of sall4 gene family, plays important roles in embryo development; organogenesis as well as pluripotency maintenance and re-establishment. There are two isoforms of Sall4, Sall4A and Sall4B. The sequence of porcine sall4 gene is still not reported. Because of its distinct role in maintaining the pluripotent state of stem cells, we cloned and sequenced porcine sall4 gene and assessed its expression in pig tissues and embryos. One 2 372 bp nucleotide sequence representing the full-length cDNA of pig sall4 was obtained by 5￠and 3￠RACE. Analyses of putative protein sequence showed a 70% to 80% identity with isoform Sall4B of human and mouse. Comparing with Sall4A, the identity reduced to 30% to 55% because of the loss of a zinc-finger domain-rich fragment. Assessment of sall4b expression in porcine tissues by Real-time PCR showed that it expressed most strongly in ovary and stronger in spleen, lung, heart and testis. For preimplantation embryos, the expression level was lower in 4-cell embryos compared with other stages. Immuno-fluorescence analysis of Sall4 on porcine preimplantation embryos indicated that it expressed in all the preimplantation embryos and located in nucleus, in blastocyst it preferentially limited in ICM cells. Expression pattern in early embryos suggest that pig sall4b is associated with pluripotency and might be a new and useful reprogramming factor for establishing pig induced pluripotent stem cell lines.