摘要
目的:克隆乌金猪脂肪和肥胖相关基因(FTO)编码区序列(CDS),分析其序列组成特征及在乌金猪不同组织中的表达情况。方法:采用反转录PCR方法从乌金猪脂肪组织中克隆FTO基因CDS,利用生物信息学方法分析其序列组成特征;采用实时PCR方法分析乌金猪FTO基因mRNA在不同组织中的表达情况。结果:克隆的FTO基因全长1518 bp(已提交至GenBank数据库,登录号为JQ031263),编码由505个氨基酸残基构成的蛋白,推测的相对分子质量为58.16×103,等电点为5.18;乌金猪与牛、羊、人和大鼠的FTO蛋白的氨基酸序列同源性分别为91%、90%、89%和83%;进化树分析显示,推测的乌金猪FTO蛋白的氨基酸组成与牛、羊的亲缘性较近,其次是人、大鼠;推测的氨基酸组成中无跨膜区,无信号肽,为亲水蛋白;分析发现该蛋白有22个磷酸化修饰位点,包括10个丝氨酸蛋白激酶磷酸化位点、7个苏氨酸蛋白激酶磷酸化位点、5个酪氨酸蛋白激酶磷酸化位点,200、246、302位氨基酸残基有糖基化位点;二级结构预测发现该蛋白共有201个螺旋、33个伸展链和271个卷曲结构;实时PCR检测的组织表达谱表明,FTO基因mRNA在乌金猪肝脏组织中的表达量最高,在脂肪、肾、脾中也有大量表达,在心脏、肌肉中的表达量最少。结论:为深入探讨乌金猪FTO基因的生物学功能奠定了重要基础。
Objective： To clone the coding domain sequenee（CDS） of fat mass and obesity associated （FTO） gene, and analyze the sequence characteristics as well as expression profile in different tissues of Wujin pig. Methods： The CDS of FTO gene was cloned from Wujin pig adipose tissue using reverse transcript PCR method, its sequence characteristics were analyzed using bioinformatics methods, and tissue expression profile was detected using real-time PCR method. Results： The CDS of cloned FTO gene was 1518 bp（GenBank accession number： JQ031263）, which could encode 505 amino acids. The putative molecular weight was 58.16 kD and isoelectrie point was 5.18. Amino acids homology which was encoded by CDS sequence of FTO gene between Wujin pig and cattle, sheep, human or rat exhibited 91%, 90%, 89% and 83% respectively. The phylogenetic tree showed that the similarity of the deduced amino acid composition of FTO gene for Wujin pigs were cattle and sheep, and next were human and rat. The deduced amino acid composition of FTO protein was no trans-membrane region, no signal peptide and a hydrophilic protein, total 22 phosphorylated modification sites including 10 serine protein kinase phosphorylation sites, 7 threonine protein kinase phosphorylation sites and 5 tyrosine protein kinase phosphory-lation sites, and glyeosylation sites were found in 200, 246, 302 sites. Secondary structure prediction showed there were a total of 201 helixes, 33 extended strands and 271 coils. Tissue expression profile which was detected using real-time PCR showed that mRNA expression of FTO gene in liver tissues of Wujin pig was the highest, next was in fat, kidney, spleen tissue, and the expression in muscle and heart was the least amount. Conclusion： The study had laid an important foundation for further study of biological function of FTO gene in Wujin pig.
出处
《生物技术通讯》
CAS
2014年第3期316-322,共7页
Letters in Biotechnology
基金
国家自然科学基金(30660132
31060331
31260592)
国家转基因重大专项(2009ZX08009-140B)
关键词
乌金猪
FTO基因
生物信息学
组织表达谱
Wujin pig
fat mass and obesity associated gene
bioinformatics
tissue expression profile
