改良红鲫Dmc1基因编码阅读框的克隆及表达

Molecular Cloning and Expression of the Open Reading Frame of Dmc1 Gene for Improved Red Crucian Carp

Dmc1基因是一个在减数分裂前期Ⅰ表达的基因,其表达产物为减数分裂时同源染色体配对所必需。本实验根据普通红鲫Dmc1基因编码阅读框(ORF)序列设计引物,克隆了改良红鲫Dmc1基因(命名为IRCC-Dmc1)的ORF序列并将之插入到cDNA5-FRT/TO载体中,构建了改良红鲫Dmc1基因表达载体FRT/TO-IRCC-Dmc1-Myc。以FRT/TO-IRCC-Dmc1-Myc质粒转染HEK293T细胞,蛋白印迹杂交实验检测到改良红鲫Dmc1基因在HEK293T细胞中有着正确的表达。本文为将来进一步研究改良红鲫Dmc1基因的功能打下了基础。

Dmcl （disrupted meiotic cDNA）gene is specifically expressed in the premeiotic prophase I during miosis. In this study, we obtained the open reading frame （ORF） of Dmcl gene for IRCC （named IRCC-Dmel ） through PCR ampli- fication, using primers based on the ORF of norulal Red Crucian Carp. The IRCC-Dmcl was inserted into cDNAS- FRT/TO and the recombinant plasmid FRT/TO-IRCC-Dmcl-Myc was constructed. HEK293T cells were transfected with FRT/TO-IRCC-Dmcl -Myc through calcium phosphate method. The data of western blot demonstrated that IRCC-Dmel has strong expression in HEK293T cells. Our study has established a solid foundation for the functional study of IRCC-Dmcl gene