藏黄牛褪黑激素合成酶AA-NAT基因cDNA克隆及序列的比较分析

Molecular cloning and nucleotide sequence analysis of Tibetan cattle melatonin synthetase AA-NAT genes

哺乳动物季节性繁殖主要受褪黑激素调控,而褪黑激素的分泌受其2个合成酶HIOMT和AA-NAT调控.分布于青藏高原的藏黄牛和牦牛是典型的季节性繁殖动物,普通黄牛繁殖则季节性不明显.比较分析它们三者的AA-NAT序列,从而进一步研究它们繁殖的季节性.采用RT-PCR技术,从2个公藏黄牛个体的松果体组织总RNA中分别克隆到AA-NAT基因的表达序列,并对其进行生物信息学分析.结果表明:藏黄牛AA-NAT基因的表达序列全长为624bp,开放阅读框(ORF)为621 bp,编码207个氨基酸,预测其表达蛋白分子量为52.3005kDa,等电点为5.06,不具有信号肽,有两个具有较高可能性的跨膜区,存在4个O连接糖基化位点,而不存在N连接糖基化位点,有3个潜在的Ser和3个Thr磷酸化位点.藏黄牛该基因表达序列核苷酸及编码氨基酸与亲缘关系较远的牦牛一致,而与亲缘关系较近的普通黄牛却有一定差异,这可能与繁殖的季节性有关.

The mammal seasonal breeding is regulated by melatonin, and the secretion of melatonin is decided by its synthetases HIOMT and AA-NAT. Bos Taurus＇ reproductive seasonality is not distinct, whereas Tibetan cattle and yak, distributing generally in Tibet plateau, are typical ones. The AA-NAT sequences among Tibetan cattle, Bos Taurus and yaks are compared and analysed, therefore reproductive seasonality of the aforementioned three are further investigated. Aimed gene is amplified by RT-PCR, using the total RNA isolated from there Tibet cattle＇s pineal bodies, and then is analyzed through sequence comparation and bioinformation. The result shows that the full length of the expression sequence is 624 bp containing an ORF of 621 bp. The predicted protein sequence shows that the protein is composed of 207 amino acids with a pI at 5.06, without signal peptides, with two more possible transmembrane domains, with four O-glycosylation sites, without N-glycosylation sites, with three larvaceous Ser and three Thr phosphorylation sites, and its estimated molecular weight is 52.3005 kDa. The sequence of nucleotide and coding amino acid of Tibet cattle is consistent with yak, while different with Bos Taurus, thereby the cause of aforesaid conclusion may be connected with breeding seasonality.