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,EooR(E)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-C0 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 extraction 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 replacement by Gin. Conclusion There is a single base mutation in Ddx4 gene, and the mutation may relate to the EOB phenotype of B6-C0 mice. And it needs to be studied further.
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