摘要
从大熊猫脑垂体中提取总RNA ,利用RT PCR技术 ,首次扩增出大熊猫垂体泌乳素 (PRL)cDNA序列 (GenBank登录号 :AY16 12 85 )。结果表明 ,大熊猫PRLcDNA的开放阅读框为 6 87bp ,编码含 2 2 9个氨基酸残基的前体蛋白。将克隆的PRLcDNA片段构建到表达质粒pGEX 4T 1中 ,转化大肠杆菌BL2 1,在异丙基硫代半乳糖苷 (IPTG)的诱导下表达PRL蛋白。SDS PAGE电泳结果表明 ,表达的PRL蛋白为不可溶蛋白。蛋白质同源性比较显示 ,大熊猫与猪、猫的同源性大于 95 % ,与人、牛、山羊的在 70 %～ 80 %之间 ,与小鼠、大鼠的分别为 4 5 9%和 5 2 %。大熊猫aa64为丝氨酸 ,是亲水性氨基酸 ;而猫、牛、山羊 (脯氨酸 )和人 (丙氨酸 )aa64是疏水性氨基酸。在二级结构上 ,aa64在第一α螺旋与第二α螺旋之间 ,极性的差别可能导致蛋白空间结构的不同。
The giant panda ( Ailuropoda melanoleuca ) is an endangered species and indigenous to China.It has been proposed that it has a highly specialized reproductive pattern with low fecundity,but little is known (about) its basic reproductive biology at molecular level.In this study,the pituitary prolactin (PRL) cDNA of giant panda was amplified by RT-PCR from pituitary total RNA and then cloned,sequenced and submitted to GenBank (GenBank accession No.AY161285).The sequence analysis revealed that the giant panda prolactin cDNA contains a 687-nucleotide open reading frame encoding the prolactin prohormone of 229 amino acid residues.The signal peptide contains 30 amino acid residues and the mature prolactin is composed of 199 amino acid residues.Then the DNA fragment amplified was subcloned into pGEX-4T-1 procaryotic expression plasmid and protein expression was induced by IPTG in Escherichia coli BL21.SDS-PAGE analysis revealed the PRL protein is infusible.The multiple sequence alignments revealed that the homology of giant panda is 95% to cat and pig,80%～70% to human,cow and goat,52% to rat and 45.9% to mouse at the amino acid level.The 64th amino acid of giant panda prolactin is hydrophilic serine instead of hydrophobic proline of cat,goat,and cow or hydrophobic alanine of human.
基金
国家自然科学基金
成都大熊猫繁育研究基金会资助项目
Oakland -China野生动物保护基金项目资助
