斑马鱼NUP98基因的克隆及其时空表达图谱

The Cloning of NUP98 Gene and its Expression Pattern during Developmental Stages in Zebrafish

目的:斑马鱼NUP98基因的克隆及其在个体早期发育过程中的表达情况研究。方法:提取斑马鱼胚胎的总RNA,制备地高辛标记的NUP98 RNA反义探针,WISH(整体胚胎原位杂交)研究NUP98在斑马鱼早期发育过程中的表达;提取斑马鱼胚胎各时相和成鱼各组织的RNA,实时定量PCR检测斑马鱼胚胎各时相和成鱼各组织中的表达。结果:成功克隆斑马鱼NUP98基因,通过实时定量RT-PCR和原位杂交,获得NUP98基因在斑马鱼早期发育过程中的表达情况:NUP98在2-cell、32-cell、oblong、shield期、12 h前普遍性表达(0.75 h、1.7 h、3.7 h、6 h、12 h);24 h以后在眼部、头部表达较多,特别是在脊索表达较高;斑马鱼NUP98在0、0.5 h、6 h、12 h、24 h、48 h表达逐渐降低,到72 h和96 h表达有所增加,但是仍低于24 h其表达水平;NUP98在成鱼眼、脑、鳔、肾、肝、睾丸、胆囊、卵巢、鳍、心、肠、肌肉、腮、皮肤的表达中,眼的表达最高,明显高于其他组织,腮、卵巢、肠的表达次之,肌肉、鳔、胆囊、睾丸、皮肤、脑的表达紧随其后,鳍、肝、心、肾的表达最低。结论:NUP98基因可能在个体脑部、脊索及眼部的早期发育过程中起到了重要作用;NUP98基因可能具有抑制肿瘤发生的作用,该基因的调节异常对白血病的发生发展可能有重要影响。这些研究结果为进一步研究NUP98基因在造血系统中的作用,评估其是否适合作为血液系统恶性肿瘤的新的治疗靶点等奠定了理论基础。

Objective： To clone ofNUP98 gene, and to investigate its expression pattern during the early developmental stages in zebrafish. Methods： The total RNA of different stages of zebrafish embryos was extracted; digoxigenin-labeled antisense RNA probe of NUP98 was prepared; WISH （whole embryo mount in situ hybridization） methods was used to detect the expression patterns of NUP98 in early developmental stages in zebrafish, the total RNA of different stages of zebrafish embryos and adult tissues were extracted; real-time PCR was used to detect the expression patterns of NUP98 in early developmental stages and adult tissues in zebrafish. Results The NUP98 probe was synthesized successfully, and the pictures and data of early expression of NUP98 gene by WISH and Real-Time PCR in zebrafish were obtained： NUP98 transcript was distributed ubiquitously before the first 12 hours （during 2-cell stage to 12 hours）; after a 24h post fertilization （hpf）, there was obvious expression in the eyes and brain, especially in the notochord; the NUP98 expression of zebrafish embryo was gradually reduced during 0 to 48 h, which had a little increase during the 72 to 96 h, but less than the first 24 hour; in the adult tissues, the expression of eye was the highest, much higher than the tissues; the expression of gillo, vary, gut was the second higher level; and the expression of muscle, swimbladder, gallbladder, testis, skin, brain was followed by upside; the expression of fm, liver, heart and kidney was the lowest. Conclusion： NUP98 gene probably plays an important role in the early development of the brain, notochord and eyes of zebrafish, and the gene may play a role as a tumor suppressor, which dysregulation reacts, on leukemia development and progression. All these research results establish theoretical foundation for the further functional study ofNUP98 gene in the hematopoietic system and the evaluation for whether it is appropriate for being a new therapeutic target of hematologic system malignant tumor.