近交培育五指山小型猪GHR基因的克隆及其真核表达载体的构建

Cloning and Eukaryotic Expression Vector Construction of Growth Hormone Receptor Gene from Chinese Inbreeding Wuzhishan Pig(WZSP)

目的探讨近交培育五指山猪矮小的分子机理,为五指山猪实验动物化奠定基础。方法近交系五指山猪肝脏组织提取总RNA,然后RT-PCR成功扩增出GHR基因,克隆入pGEM-Teasy载体进行测序,并与正常猪GHR基因序列进行比对;然后经SalⅠ和XbaⅠ双酶切回收后与同样经过双酶切回收的pEGFP-C1真核表达载体连接,构建五指山猪GHR基因真核表达载体。结果测序结果表明:五指山猪GHR基因编码区包括639个氨基酸;经过酶切和测序验证五指山猪GHR基因真核表达载体构建成功。结论经序列比对后发现GHR信号肽编码区发生两处氨基酸替换,可能会影响到生长激素的胞内运输;生长激素受体胞内域编码区发生多处突变,并且导致相应氨基酸发生替换,可能会影响生长激素受体的下游信号转导。五指山猪GHR基因真核表达载体的成功构建为进一步研究GHR的功能和下游信号转导奠定了基础。

Objective To disclose the molecular mechanism of dwarfism for Chinese inbreeding Wuzhishan pig （WZSP）. Methods Growth hormone receptor gene form WZSP were cloned to pGEM-T vector through RT-PCR, which based on the total RNA obtained from WZSP＇s liver tissues. The segment containing WZSP GHR gene was linked with the segment of pEGFP-C1 after digestion by Sal I and Xba I enzyme and purification. Results The results of sequencing showed that the coding sequence of GHR gene for WZSP contained 639 amino acids including the signal peptide of 18 amino acids. The eukaryotic expression vector named WZSPGHR-pEGFP-C1 was constructed successfully after enzyme digestion and sequencing. Conclusion Five substitutions of amino acids were found in the cytoplasmic domain of GHR, which possibly influenced GHR signal transduction. The study laid a foundation for the research of function and signal transduction of GHR.