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目的:分段克隆昆明种小鼠磷酸二酯酶(PDE)β亚单位编码基因pde6bCDS序列全长,分析比较昆明种小鼠与C57BL/6J小鼠PDEβ亚单位编码基因pde6b序列的差异。方法:设计覆盖pde6bCDS区的引物序列,通过逆转录-聚合酶链式反应扩增并克隆入载体pMD18-T中,转化大肠杆菌,酶切鉴定后测序。通过生物信息检索、序列拼接并应用生物信息学相关软件进行序列分析。结果:克隆了野生型昆明种小鼠的pde6bCDS全长。昆明种小鼠与C57BL/6J小鼠pde6bCDS区(NM_008806)相比较存在如下不同:昆明种小鼠第706位碱基为A,而数据库中序列NM_008806为G;第1149位碱基前者为T,后者为C。昆明种小鼠与C57BL/6J小鼠pde6b编码的蛋白序列差异并不明显,仅有第236位氨基酸残基为甘氨酸(G)突变成丝氨酸(S),为相对保守性替换关系。结论:克隆了昆明种小鼠pde6bCDS区全长序列;pde6b基因在不同种属小鼠之间编码序列存在差异,表现出多样性特点,但蛋白序列保守性较高。
OBJECTIVE: To clone the pde6bCDS sequence of PDE β subunit of Kunming mice in stages and analyze the differences of pde6b sequence between Kunming mice and PDEβ subunit of C57BL / 6J mice. Methods: Primer sequences covering the pde6bCDS region were designed and amplified by reverse transcription - polymerase chain reaction and cloned into pMD18 - T vector. The recombinant plasmid was transformed into E. coli and identified by restriction enzyme digestion and sequencing. Through bioinformatics search, sequence stitching and application of bioinformatics software for sequence analysis. Results: The full-length pde6bCDS of wild-type Kunming mice was cloned. Kunming mice and C57BL / 6J mice pde6bCDS area (NM_008806) compared with the following differences: Kunming mice at the 706th base A, and the database sequence NM_008806 G; the first 1149 base T, The latter is C. The difference of the protein sequence of pde6b between Kunming mice and C57BL / 6J mice was not obvious. The only amino acid residue at position 236 was the mutation of glycine (G) to serine (S), which was a conservative substitution. CONCLUSION: The full-length sequence of pde6bCDS in Kunming mice was cloned. The coding sequence of pde6b gene in different species of mice was different, showing diversity, but the protein sequence was highly conserved.