草原兔尾鼠卵透明带3(ZP3)cDNA保守序列的克隆与序列分析

Cloning and Sequencing Analysis of Lagurus lagurus Zona Pellucida 3 cDNA Conservative Fragment

根据 Gene Bank数据库中 5种啮齿目动物卵透明带 3 (ZP3 ) c DNA的序列 ,利用 DNAMAN和 NIH NCBI BLAST同源性分析程序设计了特异性引物。从 6周龄的未孕雌性的草原兔尾鼠卵巢中用柱离心法得到总 RNA,利用反转录技术得到 c DNA,通过特异性引物进行 PCR扩增草原兔尾鼠的 ZP3 c DNA保守序列。将扩增产物连接到 p MD1 8- T载体上 ,通过蓝白斑筛选得到阳性克隆 ,酶切鉴定后进行测序。将其与 Gene Bank中啮齿目动物 ZP3 c DNA进行同源性比较 ,获得了草原兔尾鼠 ZP3 c DNA的保守基因序列 ,与啮齿类同类基因比较 ,具有较高的同源性。

The sequences of five Rodent zona pellucida 3 (ZP3) cDNAs were obtained from the Gene Bank. According to the sequences, the special primers were designed by DNAMAN and NIH NCBI BLAST. The ovaries were prepared by dissecting the nonpregnant female Lagurus lagurus distributed in Xinjiang. The total RNA was abstracted from the ovaries and cDNA of Lagurus lagurus ZP3(lZP3) was cloned and ligated into pMD18 T Vector. The correction of the recombinant vector pMD18 lZP3 was tested by blue and white spot screen and restriction enzyme digestion. Finally, the nucleotide sequence of the cloned lZP3 cDNA was defined by PE 377 DNA Sequencer (Using RV M and reverse M13 47 as the primers). The results showed that the conservative sequence of the cloned lZP3 was high similar to five published Rodent ZP3 cDNAs.