广西巴马小型猪GHR基因克隆及部分序列的PCR-SSCP分析

Cloning and PCR-SSCP analysis of growth hormone receptor (GHR) gene of Bama minipig in Guangxi

为了探讨广西巴马小型猪矮小的分子机理,以广西巴马小型猪肝脏RNA为模板,扩增巴马小型猪生长激素受体(GHR)基因的全序列,将该扩增片段连接到pMD18-T载体后经过转化测序。采用PCR-SSCP方法,对4个品种120头猪的第10外显子的部分序列进行多态性分析。结果表明,克隆的广西巴马小型猪GHR基因全长为1917bp(GenBank登录号为EF041823),与GenBank(X54429)的序列同源性为99%,且第10外显子部分序列SSCP检测没有多态性。经序列比对后,发现广西巴马小型猪生长激素受体胞内域编码区发生7处突变,导致相应氨基酸发生置换,这有可能影响生长激素的胞内运输和生长激素受体的下游信号转导,但经SSCP检测发生突变的位点并没有引起单核苷酸构象的改变。该结论为进一步研究广西巴马小型猪的矮小机理与GHR基因的关系奠定了基础。

To discuss the molecular mechanism of dwarfism for Bama minipig in Guangxi, the whole sequence of growth hormone receptor （GHR） gene of Bama minipig were amplified and cloned to pMD18-T vector through RT-PCR with total RNA of liver. The polymorphisms of partial sequence of 10^th exon for 120 minipigs of four varieties were analyzed by PCR-SSCP method. The results showed that 1917 bp of whole sequence for GHR gene of Bama minipig from Guangxi was successfully cloned（GenBank ID： EF041823）, and the sequence homology to X54429 in GenBank was 99%. The SSCP analysis indicated that there was no polymorphism in partial sequence of 10^th exon. By sequence alignment, seven substitutions of amino acids were found in the coding region of cytoplasmic domain of GHR, which possibly influenced the intracellular translocation and downstream signal transduction of GHR, while the mutation sites did not lead to the conformation changes of single nucleotide. The results laid a foundation on further research of relationship between dwarfism mechanism and GHR gene of Bama minipig from Guangxi.