摘要
[目的]采用RT-PCR法克隆与分析瘦素受体Ob-RbcDNA序列。[方法]从160日龄初情期的苏姜猪的下丘脑中提取组织总RNA,根据GenBank中猪Ob-RbmRNA全序列设计引物,用反转录-聚合酶链式反应(RT-PCR)进行cDNA扩增,获得1条343 bp的片段,将PCR产物克隆于pGEM-Teasy载体后进行测序分析。[结果]用紫外分光光度计检测所提取的RNA质量,OD260/OD280=1.9,证明所提RNA的质量较好,无降解和蛋白质污染;用1%的琼脂糖检测所提RNA,有3条清晰的条带,分别是28S、18S、5S,28S与18S浓度比约为2∶1,说明所提的RNA的完整性较好。所获Ob-RbcDNA序列用Blast程序与在GenBank中发表的猪Ob-RbcDNA序列(AF092422)比较表明,其同源性为100%,说明所扩增的序列是正确的。[结论]该研究为进一步探索Ob-Rb基因组织表达和作用机理奠定了理论基础。
[ Objective ] The study aimed to clone and analyze the Ob-Rb cDNA sequence of leptin receptor by using RT-PCR method. [ Method] The total RNA of porcine was extracted from hypothalamus of 160-day-old Sujiang pig at puberty. A pair of primers was designed according to the Ob-Rb mRNA whole sequence reported in GenBank previously. A cDNA fragment in length 343 bp was synthesized and amplified by RT-PCR. The PCR product was cloned into pGEM-T easy vector and then sequenced. [ Result] When the extracted RNA was detected by ultraviolet spectrophotometer, OD260/OD280 was 1.9, proving that the extracted RNA had good quality without degradation and protein pollution. When the extracted RNA was detected by 1% agarose, it had 3 clear bands, being 28S, 18S and 5S with the eoncn, ratio of 28S and 18S of 2:1, showing that the extracted RNA had good integrity. The comparison on the obtained Ob-Rb cDNA sequence with reported Ob-Rb eDNA sequence of porcine from GenBank(AF092422) by Blast procedure indicated that its homology was up to 100%, demonstrating that this amplified sequence was correct. [ Conclusion] This study laid the theoretical base for further exploring the tissue expression and action mechanism of Ob-Rb gene.
出处
《安徽农业科学》
CAS
北大核心
2009年第35期17404-17405,共2页
Journal of Anhui Agricultural Sciences