摘要
[ Objective] The aim of this study was to provide a basis for study on adiponectin as a candidate gene for fat deposition. [ Method] The promoter sequence of adiponectin was obtained by porcine BAC library screening and primer-walking method. The polymorphisms of adiponectin promoter from 290 pigs, including 5 breeds of Lantang pig, Large spotted pig, Large white pig, Landrace and Duroc, were analyzed with PCR-RFLP. [ Result] At SNP site of adiponectin 5＇-flanking region -1 010 bp （G/A）, GG genotype frequency in Chinese indigenous pigs was significantly higher than that in exotic pigs. At SNP site of adiponectin 5＇-flanking region -394 bp （T/C）, the genotype distribution of Chi- nese indigenous pigs was abundant, while no CC genotype was detected in exotic pigs, and T allele frequency was higher in exotic pigs. [ Conclusion] SNP site mutation of - 1 010 bp （G/A） may lead to changes of the gene transcription level, while SNP site of -394 bp （T/C） properly has no relationship with gene transcription level and fat deposition.
[目的]为脂联素基因作为脂肪沉积候选基因的研究提供依据。[方法]通过猪BAC文库筛选及引物步移法(primer-walking)的测序方法获得脂联素基因的启动子序列,具体步骤:通过已经发表的部分猪脂联素基因cDNA序列设计特异引物用于BAC克隆的筛选。以超级池DNA为模板进行第1次PCR反应,筛出含阳性克隆的超级池;以挑出的阳性超级池的3维池DNA为模板进行第2次PCR反应,根据阳性板池、列池、行池序号得到可能的阳性克隆号;依照阳性克隆号从工作文库中取出相应的BAC克隆,平板划线37℃倒置培养20 h。待菌落长好后,做菌体PCR鉴定,以确认克隆的正确性。将获得的BAC阳性克隆平板划线,37℃摇菌过夜培养,进行BAC的提取、纯化与酶切回收。先以M13引物向BAC两端测序,再以发表的cDNA序列外显子1设计测序引物采用primer-walking的方法,获得连续的DNA序列。采用PCR-RFLP技术对蓝塘猪、大花白猪、大白猪、长白猪和杜洛克猪5个品种共290头猪的脂联素基因启动子区的多态性进行分析。[结果]脂联素基因的5′侧翼区-1 010 bp(G/A)的SNP位点,本地猪种中GG基因型频率明显高于外来猪种;-394 bp(T/C)的SNP位点,本地猪种基因型分布较为丰富,而在外来猪种中却没有检测到CC基因型,且外来猪中T等位基因频率较高。[结论]脂联素基因上游-1 010 bp(G/A)的SNP位点突变可能会引起该基因转录水平的改变,而-394 bp(T/C)的SNP位点与基因转录水平及与脂肪沉积可能无关。
基金
Supported by 948 Project from Ministry of Agriculture(2006-G50)~~
