五指山小型猪生长激素及其受体基因的克隆与序列分析

Cloning and sequence analysis of growth hormone and receptor gene from Chinese Wuzhishan miniature pig

动物生长发育调控是一个高度复杂而精细的生理过程，受多种因素如神经、体液、遗传、营养及环境等的影响。其中，动物神经内分泌生长轴各因子（激素、受体、结合蛋白等）及其基因对动物的生长发育起着关键的作用。正常情况下，下丘脑接受体内外的信息，生长激素释放激素（growth hormone releasing hormone，GHRH）和生长抑素（somatostatin，SS），调节垂体生长激素（growth hormone，GH）的分泌，GH通过与生长激素结合蛋白（growth hormone binding protein，GHBP）结合而运输，与靶器官上的生长激素受体（growth hormone receptor，GHR）结合，促使类胰岛素生长因子（insulin—like growth factors，IGFs）的产生并进入血液循环，IGFs再通过其结合蛋白（insulin—likeg rowth factor binding protein，1GFBP）转运到全身组织细胞，促使组织细胞的生长与分化。五指山猪（Wuzhishan Pig，WZSP）是一种原产于中国海南省山区的小型猪。五指山猪成年体重仅为35kg，是正常猪体重的15％～30％，是一种频临灭绝的珍稀物种。五指山猪具有以下特征：体型小，性成熟早，肉质好，耐近交和遗传稳定。为了开发和应用这一珍稀猪种的遗传资源，中国农业科学院北京畜牧兽医研究所从1989年就已经开始对五指山猪进行近交系培育，现在已经获得五指山猪近交系18代（近交系数为0．974），但五指山猪的矮小机理至今不明。为了探讨五指山猪矮小的分子机理，对五指山猪生长激素及其受体基因进行了克隆和序列分析。从纯种近交16代五指山猪的垂体和肝组织提取总RNA，根据GenBank中猪生长激素及其受体基因的序列（登录号分别为X53325，NM214254）设计特异性引物，对五指山猪生长激素及其受体基因进行扩增，然后对PCR产物进行回收、克隆测序。测序结果表明，五指山猪生长激素基因开放阅读框长651bp，编码216个氨基酸，其中包含26个氨基酸的信号肽；生长激素受体基因编码区编码639个氨基酸，其中包含18个氨基酸的信号肽。运用生物学软件DNAssist2．2将五指山猪和普通猪的生长激素基因编码区进行比对发现，五指山猪生长激素基因的编码区有4个突变：26号位置发生C→T突变，该突变导致缬氨酸代替丙氨酸；65号位置发生G→A突变，该突变导致谷氨酰胺替换精氨酸；114号位置发生T→C突变，252号位置发生A→G突变，但是这两个突变都未引起所编码氨基酸的变化。五指山猪和普通猪生长激素受体基因比对发现，1143号位置的G→T突变引起天冬氨酸替换谷氨酸；1225号位置的G→T突变导致丝氨酸替换丙氨酸；1666号位置C→G突变导致缬氨酸替换亮氨酸；1739号位置C→G突变导致丙氨酸被甘氨酸所代替；1801号位置G→A突变导致缬氨酸被异亮氨酸代替；1248号位置G→A突变，1287号位置G→A突变和1827号位置G→C突变均为同义突变，未引起相应编码氨基酸的变化。五指山猪生长激素基因有两处氨基酸替换，这两处替换发生在信号肽区，对成熟蛋白的结构和功能没有影响，但是有可能会影响蛋白的运输。五指山猪生长激素受体基因突变都发生在生长激素受体的胞内结构域，这些突变可能改变生长激素受体的空间构像，从而对生长激素受体的胞内信号转导产生一定的影响，影响生长激素一生长激素受体一胰岛素样生长因子轴的信号级联，削弱生长激素的促生长作用。

Growth hormone （GH） is a single-strand polypeptide hormone secrcted by acidophil in anterior pituitary. The growth promoting effects of GH are achieved through GH＇s diverse and pleiotropic effects on cellular metabolism and differentiation, and are mediated chiefly via the activation of growth hormone receptor （GHR） on cell surface. A single GH molecule contains two GHR-binding sites and binds two GHR molecules, sequentially induce functional GHR dimerization and hence activate downstream signal transduction. Wuzhishan pig （WZSP） was a kind of miniature pig and mainly distributed in the mountain area of Hainan province in China. The mature body-weight is only 35 kg, which is 15～30 percent of normal weight of other pigs and is one of the rare and endangered breeds. WZSP F18 has been obtained by inbreeding method presently （Inbreeding coefficient, 0. 974）. But the reason of WZSP＇s dwarfism is unknown. Cloning and sequence analysis of WZSP＇s GH and GHR cDNA can provide the basis for understanding the molecular mechanism of WZSP＇s dwarfism. In this study, a novel cDNA encoding the WZSP GH was isolated from pituitary tissue using polymerase chain reaction with special primers. The nucleotide sequence predicted a mature protein of 217 amino acids and a 26 amino acid signal peptide. Although two mutations at position 26 and 65 caused the substitution of amino acid, they lay in the sequence encoding signal peptide that could be cut off during the mature process, which not affect the structure of mature protein. The mutations in signal peptide may be influential to the membrane transport of WZSP＇s GH. cDNA encoding the WZSP GHR was isolated from liver tissue using RT-PCR. The nucleotide sequence predicted a mature protein of 639 amino acids and a 18 amino acid signal peptide. Five amino acid substitutions of WZSP GHR gene were located in cytoplasmic domain, which was possible to influence GHR downstream signal transduction and blocked GH effect.