斑马鱼腺苷甲硫氨酸脱羧酶基因短片段缺失突变体的构建与鉴定

Constructionand and Identification of Zebrafish Adenosylmethionine Decarboxylase 1 Short Segment Deletion Mutant

目的构建斑马鱼腺苷甲硫氨酸脱羧酶(amd 1)突变体,并运用聚合酶链式反应(PCR)技术对突变的碱基位点设计特异性引物,以建立快速鉴定短片段缺失的方法。方法利用CRISPR/Cas9系统构建斑马鱼amd1突变体;运用整胚原位杂交技术分析AMD1在野生型(WT)和amd1纯合子斑马鱼胚胎中的表达;根据突变体的突变位点设计鉴定引物,用PCR和琼脂糖凝胶电泳检测,并使用二代测序技术验证。结果斑马鱼amd1突变体构建成功;受精后24 h,在WT斑马鱼胚胎中AMD1主要在头部、眼睛、体节及内胚层区域表达,而在amd1纯合子斑马鱼胚胎中AMD1的表达明显降低。运用amd 1-screen-(3)-5′引物进行PCR时,在WT斑马鱼胚胎中可获正常条带;而在amd 1纯合子斑马鱼胚胎中未能获得相应条带。结论斑马鱼amd1突变体被成功构建;amd 1-screen-(3)-5′被确定为最佳鉴定引物。

Objective To construct zebrafish adenosylmethionine decarboxylase 1(amd1)mutant,and design the specific primers for the mutation points by polymerase chain reaction(PCR),so as to establish a rapid identification method for homozygote mutants.Methods Zebrafish amd1 mutant was constructed by CRISPR/Cas9 system.Whole embryo in situ hybridization was used to analyze the expression of AMD1 in wild type(WT)and amd1 homozygotes of zebrafish embryos.The identification primer was designed according to the mutant site,which was detected by PCR and agarose gel electrophoresis,and verified by the second generation sequencing technology.Results Zebrafish amd1 mutants with stable inheritance were screened out.At 24hpf(hours post fertilization),AMD1 was mainly expressed in the head,eyes,somites and endoderm regions in the WT embryos,while the expression of AMD1 in amd1 homozygotes zebrafish embryos was significantly reduced.PCR performed with amd1-screen-(3)-5′primers showed normal bands in WT embryos while no corresponding band was obtained in amd1 homozygous zebrafish embryos.Conclusion The zebrafish amd1 mutant was successfully constructed.The best identification primer was identified as amd1-screen-(3)-5′.