B6-Co小鼠Ankrd55和Ddx4突变候选基因克隆及测序  

Mutation Screening of Two Candidate Genes in B6-Co Mouse Affected with Cornea Opacity： Ankrd55 and Ddx4

作　　者：李瑶[1] 吴刘成[1] 卢泽艳[1] 郑良凤[1] 葛良玉[1] 邵义祥[1,2] 

机构地区：[1]南通大学实验动物中心,南通226001 [2]南通大学比较医学研究所,南通226001

出　　处：《实验动物与比较医学》2012年第5期371-375,共5页Laboratory Animal and Comparative Medicine

基　　金：国家自然科学基金项目（30671081）;江苏省自然科学基金项目（BK2010279）;南通大学自然科学基金项目（112052）

摘　　要：目的对B6-Co小鼠Ankrd55和Ddx4基因进行克隆测序分析，寻找是否存在突变位点。方法以小鼠Ankrd55和Ddx4基因的mRNA序列设计引物，以mRNA为模板，采用RT-PCR和PCR技术分段进行目的基因扩增，将目的片段连接在T载体上，转化至感受态细胞，筛选阳性克隆，提取质粒DNA分子，电泳检测，EcoRE酶切释放目的片段，测序、分析、比对。结果Ddx4基因位于小鼠13号染色体第113412349的碱基由c转换成A，导致编码氨基酸的密码子改变，产物脯氨酸（P）变成谷氨酰胺（Q）。结论Ddx4基因发生单碱基突变，表明该突变可能与B6－co小鼠的EOB表型相关，但是有待于进一步研究。Objective To find mutational site of Ankrd55 and Ddx4 gene, clone and sequence these gene in B6-Co mutant strain mouse with cornea opacity phenotype. Methods The primers were designed according to mRNA. The target gene was amplified by PCR and RT-PCR in which the PCR templates were mRNA, and then recombinant vectors were transformed to competent cells after DNA fragments were connected to the T vector. The positive clones were selected and plasmid DNA extrac tion was detected by electrophoresis. The fragments by EcoRI digestion for identification of positive clones and at the end sequencing. Results The C base in sequence of Ddx4 chromosome 13 113 412 349 was replaced by A by sequence alignment with genome data base, with coding protein Pro replace- ment by Gin. Conclusion There is a single base mutation in Ddx4 gene, and the mutation may relate to the EOB phenotype of B6-Co mice. And it needs to be studied further.

关 键 词：B6-Co小鼠 角膜混浊 PCR体外扩增:基因克隆 

分 类 号：Q78[生物学—分子生物学] R772.2[医药卫生—眼科]