陆地棉脂肪酶基因克隆及氨基酸序列分析

根据陆地棉(Gossypium hirsutum L.)EST序列设计一对引物,采用RT-PCR方法从萌发的棉籽中获得了脂肪酶(triacylglycerol acylhydrolases,EC 3.1.1.3)基因,该基因编码483个氨基酸;比对结果显示,棉籽脂肪酶与拟南芥、水稻、蓖麻等脂肪酶相似性较低,具有由Ser-Asp-His组成的三联体催化活性中心,在亲核Ser残基周围有GXSXG保守序列.软件预测显示该脂肪酶为可溶性蛋白,分子量约为55.4 kD,等电点为9.07,不含N端信号肽,亚细胞定位可能是过氧化物酶体或胞质溶胶.

Cloning and Amino Acid Sequence Analysis of the Lipase Gene from Gossypium hirsutum

A lipase gene was cloned from cotton seedlings after germination 24 hours.The cDNA amplified by RT-PCR is 1 466 bp and contains a 1 449 bp putative open reading frame.Similarity search revealed that the deduced amino acid sequence shares a low level of identity(47%~38%) with the lipases from Arabidopsis thaliana,Oryza sativa and Ricinus communis.The deduced lipase protein contains 483 amino acids and possesses a putative catalytic triad(SDH) and a conserved pentapeptide(GXSXG) surrounding the nucleophilic serine residue.The calculated molecular mass is 55.4 kD and the predicted pI is 9.07.According to the prediction by software,the protein is soluble,has no signal peptide at N terminus,and probably targets cytosol or peroxisome.