摘要
为了解斑马鱼胚胎发育过程中FGF3基因的时空性表达情况,并探讨其对胚胎发育的调控作用,该研究分别提取2,4,8,12,24,36,48,72 hpf斑马鱼胚胎的总RNA,经逆转录成cDNA,实时荧光定量PCR检测FGF3基因mRNA表达量;扩增FGF3基因特异片段,构建pGEM-T/FGF3基因片段重组质粒,经克隆及测序验证后,合成地高辛标记的反义RNA探针,以整体原位杂交法检测斑马鱼胚胎FGF3基因的空间性表达。结果显示:FGF3基因在2 hpf胚胎就有表达,并持续至胚胎孵化,12 hpf胚胎FGF3表达量达到高峰(P<0.01);胚胎发育过程中FGF3表达部位以头、尾、咽弓为主。由此得出结论 ,FGF3主要在胚胎发育早期表达,其表达可能与胚胎脑、眼、耳、咽弓及尾部器官的发育调控有关。
The current study aimed to study the temporal and spatial expression ofFGF3 gene during zebrafish em- bryonic development. Total RNA was extracted from zebrafish embryos at 2, 4, 8, 12, 24, 36, 48, 72 hours post fertiliza- tion （hpf）, respectively, and was reversely transcripted to cDNA. FGF3 mRNA levels were then determined by Real-time quantitative PCR. The results showed that FGF3 gene expression started at as early as 2 hpf, and was present throughout the whole embryonic development. The expression level of FGF3 reached the highest （P〈0.01） at 12 hpf. Whole- mount in situ hybridization was then used to determine the spatial expression ofFGF3 gene at the time points of 8, 12, 24, 36, 48, 72 hpf. Digoxigenin labeled antisense FGF3 RNA probe was synthesized by T7 RNA polymerase from pGEM-T/FGF3 gene fragment recombinant plasmid. Our results showed that FGF3 was mainly expressed in the head, tail and pharyngeal arches of zebrafish embryos during embryonic development. In conclusion, FGF3 gene was expressed in a temporal dynamic pattern during zebrafish embryonic development and reached the highest at about 12 hpf. The spatial expression was mainly in the head, tail and pharyngeal arches, indicating an association of FGF3 with the regulation of embryonic brain, eyes, ears, pharyngeal arches and tail development.
出处
《中国细胞生物学学报》
CAS
CSCD
北大核心
2012年第4期355-360,共6页
Chinese Journal of Cell Biology
基金
国家自然科学基金(No.30972509)资助项目~~
关键词
斑马鱼
胚胎发育
成纤维细胞生长因子3
实时荧光定量PCR
整体原位杂交
zebrafish
embryonic development
fibroblast growth factor 3
Real-time quantitative PCR
whole-mount in situ hybridization
