中文摘要：

目的采用吗啡啉修饰反义寡核苷酸显微注射方法下调斑马鱼Tbx1基因表达,研究斑马鱼Tbx1基因功能下调对其他两个T盒基因Tbx20和Tbx2表达的影响。方法采用吗啡啉修饰的反义寡核苷酸显微注射方法抑制斑马鱼Tbx1基因表达,分别将2.5、5、8、10 ng吗啡啉反义寡核苷酸在斑马鱼0-4细胞期注入胚胎,并构建Tbx20,骨形成蛋白2b（Bmp2b）和Tbx2反义RNA探针,进行整体原位杂交,观察Tbx1基因下调对Tbx20、Bmp2b及Tbx2表达的影响。结果Tbx1吗啡啉寡核苷酸显微注射组胚胎表现出鳃弓、耳囊、心血管系统和胸腺的发育异常。Tbx1基因下调导致Tbx20的表达出现改变,Tbx20在心脏的表达与对照组相比明显下调,神经元的表达范围明显缩小;Tbx1基因功能下调会导致Bmp2b在心脏和咽囊的表达减低,Bmp2b在后部咽囊的表达较前部咽囊减低得更为明显;Tbx1基因功能下调胚胎,Tbx2在鳃弓的表达模式发生改变,48 hpf,Tbx2在鳃弓的表达出现从后向前逐渐减低,鳃弓的表达范围较对照组明显缩小。结论Tbx1在发育过程中,会对其他T盒基因,如Tbx20和Tbx2具有激活或抑制的调控作用。Tbx1对Tbx20的作用可能是通过影响Bmp2b的途径,继发地影响Tbx20的表达。Tbx1基因功能下调,会改变Tbx2在鳃弓的表达模式。

英文摘要：

Objective Tbx1,one of the genes mapped within the del22q11 locus in human,is important for aortic arch formation and also contributes to the development of the outflow tract.Tbx1 currently represents the most promising candidate gene for DiGeorge syndrome.The hierarchical interaction between different T-box genes was revealed to play a central role of early heart patterning and morphogenesis.The aim of this study was to explore the effect of Tbx1 knock-down on the expressions of two other T-box genes,Tbx2 and Tbx20 in zebrafish.Methods Wild-type（AB＊ strain） zebrafish stocks were obtained from the International Zebrafish Research Center（IZRC）.The following morpholino oligos（Gene Tools LLC,Corvallis,OR,USA） were designed against the translational start codon,and the sequence was 5′CAGGTCGCGTTTAACATTTAGGTTA 3′.The positive control and standard control morpholino were designed for control microinjections.The morpholino oligos at the indicated concentrations were microinjected into single-to-four-cell stage zebrafish embryos.The RNA antisense probes of Tbx20,Bmp2b and Tbx2 used for in situ hybridization were prepared.Whole-mount in situ hybridization was used to monitor the expressions of Tbx20,Bmp2b and Tbx2.Stained embryos were examined under Olympus BX61 and SZX12 microscopes,and photographed with a DP70 digital camera.The experiments involving control and experimental embryos were conducted in parallel.Results Tbx1 morpholino was injected into fertilized eggs in various doses to determine an optimal dose.Embryos presented a consistent phenotype with doses between 5-10 ng MO/embryo（57%,n=100）.While injection of the same amount of control-MO could not produce the specific effect,demonstrating that these results were not due to an injection artifact.Tbx1 morphant embryos were characterized by defects in the pharyngeal arches,otic vesicle,aortic arches and thymus.During zebrafish embryogenesis,the expression of Tbx20 was found in the anterior lateral plate mesoderm,heart field,branchiomotor neuro