野生型斑马鱼胚胎中hoxd3基因mRNA的表达谱  

Expression pattern of hoxd3 gene during early development of wild-type zebrafish embryos

作　　者：舒莉萍[1,2] 何志旭[1,3] 姚冬静[3] 马健娟[3] 李涛[3] 叶芝旭[3] 

机构地区：[1]贵阳医学院组织工程与干细胞实验中心,贵州贵阳550004 [2]贵阳医学院免疫教研室,贵州贵阳550004 [3]贵阳医学院儿科教研室,贵州贵阳550004

出　　处：《浙江大学学报（医学版）》2012年第1期69-74,共6页Journal of Zhejiang University(Medical Sciences)

基　　金：国家自然科学基金面上项目(30960412);贵州省科技基础条件平台项目([2009]4005);贵州省优秀科技教育人才省长专项资金(S2008-4)

摘　　要：目的:为探讨hoxd3基因在斑马鱼发育过程中与血管形成可能的作用机制,用hoxd3基因的反义mRNA探针进行斑马鱼全胚胎原位杂交,观测hoxd3基因在野生型斑马鱼胚胎发育过程中的表达情况。方法:提取斑马鱼胚胎总RNA,经RT-PCR扩增斑马鱼hoxd3基因片段,将得到的hoxd3基因片段和pCS2+质粒经EcoRⅠ、XbaⅠ双酶切后插入pCS2+质粒中,挑选阳性克隆,通过双酶切、菌落PCR以及测序鉴定;将pCS2+-hoxd3重组子经EcoRⅠ酶切线性化,经T3RNA体外转录体系合成地高辛标记的hoxd3基因的反义mRNA探针。用hoxd3基因的反义mRNA探针进行斑马鱼全胚胎原位杂交。结果:构建和鉴定了pCS2+-hoxd3重组质粒,经斑马鱼全胚胎原位杂交检测hoxd3基因的表达,发现在Tuebingen野生型斑马鱼受精后24 h～72 h胚胎的中脑后脑交界处以及后脑中可以观察到hoxd3基因的表达。结论:hoxd3基因在Tuebingen野生型斑马鱼胚胎发育早期的神经系统高表达,未发现在血管生成区域的表达。Objective： To investigate the expression pattern of hoxd3 gene during early embryogenesis and angiogenesis of wild-type zebrafish. Methods： Total RNA was extracted from embryos of zebrafish in different development stages by trizol.The cDNA of hoxd3 gene was amplified by RT-PCR.The RT-PCR product was ligated to pCS2＋ vector by T4 DNA ligatase polymerase and sequenced.T3 RNA polymerase in vitro transcription system was used to obtain the probe of digoxin-labeled anti-sense mRNA of hoxd3 gene.The expression pattern of hoxd3 was detected by whole embryo in situ hybridization（WISH） with anti-sense mRNA probe. Results： pCS2＋-hoxd3 plasmid was successfully constructed,which was used to prepare anti-sense mRNA probe of hoxd3 in vitro.Expression pattern of hoxd3 gene was detected by WISH during zebrafish early embryogenesis and angiogenesis.It was observed that hoxd3 mRNA was expressed at the junction region of midbrain and hindbrain in wild-type zebrafish in embryos at 24～72h postfertilization（hpf）. Conclusion： hoxd3 gene is mainly expressed in nervous system of wide-type zebrafish embryos.

关 键 词：寡核苷酸类 反义/遗传学 原位杂交 基因表达 斑马鱼 胚胎发育 遗传载体 质粒/遗传学 转染 内皮 血管/细胞学 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]