东北虎促卵泡激素和促黄体激素基因的克隆及其序列分析

CLONING AND ANALYSIS OF THE HOMOLOGY OF THE FOLLICLE-STIMULATING HORMONE AND LUTEINIZING HORMONE GENES IN SIBERICAN TIGER (PANTHERA TIGRIS ALTAICA)

本研究首次从东北虎 (Pantheratigrisaltaica)脑垂体提取总RNA ,利用RT PCR技术扩增出东北虎垂体促性腺激素α亚基、促卵泡激素 (FSH) β亚基和促黄体激素 (LH) β亚基的编码区序列 ,并进行克隆、测序和比较分析。结果表明 ,其α亚基、FSHβ亚基、LHβ亚基基因的开放阅读框分别为 36 3bp、 390bp和 4 2 9bp ,分别编码 12 0、 12 9和 14 2氨基酸的前体蛋白。与已发表的人、牛、绵羊、猪、大鼠、小鼠等物种相应序列比较 ,无论在核苷酸水平 ,还是在氨基酸水平都显示出较高的同源性 (6 4 7%～ 96 6 %) ,其中与猪的同源性最高(86 %～ 96 6 %)。东北虎的基因序列还具有其明显的特异性 ,首次发现LHβ亚基cDNA编码的前体蛋白在信号肽部分比其它物种相应序列多一个亮氨酸残基 (Leu)。

The pituitary gonadotropin follicle stimulating hormone (FSH) and luteinizing hormone (LH) play important roles in regulating gonad function and are essential for normal reproductive function in mammals. In this paper, by comparing the FSH/LH common α, FSH β and LH β cDNA sequences reported from humans, cattle, sheep, pigs, rats and mice, three pairs of primers were designed and synthesized according to the conserved region. Using these primers, the genes encoding α, FSH β and LH β in Panthera tigris altaica were amplified by reverse transcript polymerase chain reaction (RT PCR) from pituitary total RNA and were cloned, sequenced and submitted to GenBank, with accessions Nos. AF408393, AF540937 and AF540935 respectively. It was revealed that the open reading region (ORF) of subunit α from Panthera tigris altaica was 369 bp encoding a precursor containing a 24 amino acid signal peptide and a 96 amino acid mature peptide. Similarly, the ORF of FSH β was 390 bp encoding a 19 amino acid signal peptide and 110 amino acid mature protein. The ORF of the LH β subunit was 429 bp encoding a 141 amino acid protein which had an additional Leu residue in the 21 amino acid signal peptide sequence compared to other species. The mature protein of the LH β subunit contained 121 amino acids just like those of other species. The predicted primary structure of the three subunits were similar to those of other mammals with conserved cysteines (10 for the α subunit and 12 for the β subunit) and putative N glycosylation sites (2 for the α and FSH β subunits, 1 for the LH β subunit). When nucleotide sequences and deduced amino acids were compared with homologous sequences from available mammals including humans, cattle, sheep, pigs, rats and mice, they displayed a fairly high (64 7%～96 6%) degree of conservation. Among all species compared, pigs exhibited the highest degree of amino acid similarity of 96 6%, 91 5% and 86 6% with Panthera tigris altaica . Sequence alignment indicative of some distinct amino acid diversity, which could be related to the species specific characteristics of gonadotropins, was also found in the Panthera tigris altaica sequence, such as position 39 of the α subunit, 20, 24, 68 and 96 of the FSH β subunit and 27 and 119 of the LH β subunit. Our results not only provide an insight into the characteristics of FSH and LH in Panthera tigris altaica that can make a practical contribution to its conservation, but also lay the foundation for further research towards producing recombinant FSH and LH which could increase the efficiency of artificial breeding technology.