广灵驴PPARγ基因克隆与组织表达分析

Cloning and tissue expression analysis of PPARγ gene of Guangling donkey

以广灵驴作为试验材料,使用RT-PCR方法对广灵驴PPARγ基因CDS编码区进行克隆然后对序列进行分析,并利用实时荧光定量PCR技术检测PPARγ基因在广灵驴的心、肝、脾、肺、肾、背最长肌和皮下脂肪7种组织中的表达特征.结果表明,广灵驴PPARγ基因含有1个1425 bp的开放阅读框,可编码474个氨基酸残基,其核苷酸序列与马的同源性最高;PPARγ蛋白是一种酸性不稳定的亲水性蛋白,二级结构由α-螺旋、延伸链、β-转角和无规则卷曲4种形式组成,蛋白序列中具有3个结构域和48个磷酸化位点,无信号肽剪切位点以及跨膜区域,主要定位在细胞质和细胞核中;PPARγ基因在广灵驴的7种不同组织中均有表达但存在一定差异,在皮下脂肪中的表达水平最为丰富.本试验成功克隆了广灵驴PPARγ基因,并发现其在皮下脂肪中有较高的表达水平,说明PPARγ基因可能与广灵驴脂肪沉积有关.

In order to clarify the role of peroxisome proliferators-activated receptors(PPARs)in regulating intramuscular fat for improved donkey meat,the coding sequence of PPARγgene was cloned from Guangling donkey by RT-PCR,then the bioinformatics analysis was conducted.The expression characteristics of the PPARγgene in heart,liver,spleen,lung,kidney,longissimus dorsi muscle and subcutaneous fat of Guangling donkey were detected by real-time fluorescence quantitative PCR.The results showed that Guangling donkey PPARγgene contains an open reading frame of 1425 bp,which encodes 474 amino acid residues.The nucleotide sequence has the highest homology with horse.PPARγprotein is a kind of acid-labile hydrophilic protein.Its secondary structure consists of 4 forms:α-helix,extension strand,β-turn and random coil.The protein sequence has 3 domains and 48 phosphorylation sites,without signal peptide cleavage site and transmembrane region.It is mainly located in the cytoplasm and nucleus.The PPARγgene was expressed in 7 different tissues of Guangling donkey,and most abundantly expressed in subcutaneous fat.It can be concluded that the PPARγgene is related to the fat deposition of Guangling donkey.