角膜混浊小鼠突变候选基因Map3k1克隆与序列分析

Cloning and Sequence Analysis of Mutant Candidate Gene of Map3k1 in Cornea Opacity Mouse

对具有角膜混浊表型的B6-Co突变系小鼠突变候选基因Map3k1进行克隆及测序分析,寻找该突变系小鼠Map3k1基因的突变位点。以小鼠Map3k1基因的mRNA、全长及上游5 kb序列设计引物,分别以基因组DNA和mRNA为模板,采用PCR和RT-PCR技术分段扩增目的基因,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,质粒DNA分子提取,电泳检测,EcoRⅠ酶切释放目的片段,送上海生工生物工程技术服务有限公司测序。成功扩增出Map3k1基因的20个外显子和上游调控序列,B6-Co小鼠测序结果与基因组数据库序列比对,该基因位于13号染色体第112 559 574的碱基由T突变为A,编码的蛋白质第314氨基酸由亮氨酸变为谷氨酸,但是该基因的表达调控及蛋白质剪切机制仍有待深入研究。

To find mutational site of Map3k1 gene,clone and sequene the Map3k1 gene in B6-Co mutant strain mouse with cornea opacity phenotype,The primers were designed according to mRNA,full length sequence,and upstream about 5 kb sequence of mouse Map3k1 gene.The target gene was amplified by PCR and RT-PCR in which the PCR templates were genomic DNA and mRNA,and then recombinant vectors were transformed to comptent cells after DNA fragments were connected to the T vector.The positive clones were selected and plasmid DNA extraction was detected by electophoresis.The fragments by EcoRⅠdigestion for identification of positive clones and at the end sequencing in Sangon Biotech（Shanghai） Co.,Ltd.20 exons of Map3k1 and its upstream regulatory sequence were amplified.Sequencing results indicated the T base in sequence of Map3k1 chromosome 13 112 559 574 was replaced by A in B6-Co mouse by sequece alignment with genome data base,with coding protein 314 Leu replacement by Glu.It was expected to be investigated ON the mechanism of the gene expression regulation and the protein clipping.