藏山羊SFRS18基因克隆、表达及其与肌内脂肪含量相关性研究

Cloning,Expression Analysis of SFRS18 and Correlation Analysis between its mRNA Expression and Intramuscular Fat Content in Tibetan Goat

试验旨在克隆藏山羊SFRS18(splicing factor arginine/serine-rich 18)基因CDS序列,并进行生物信息学分析,同时分析其组织表达特征以及与肌内脂肪含量进行相关性分析,为深入研究该基因在山羊肌内脂肪沉积中的作用积累数据。采用RT-PCR技术获得藏山羊SFRS18基因序列,结合生物信息学分析蛋白的理化性质、结构和不同物种的同源性,实时荧光定量检测SFRS18mRNA表达情况,并将表达量与肌内脂肪含量相关联。结果表明,藏山羊SFRS18基因cDNA序列长为1 299bp,开放阅读框(ORF)长为1 272bp,编码423个氨基酸,蛋白分子结构式为C2003H3424N760O696S4,分子质量为49.42ku,等电点pI=11.20,SFRS18蛋白为不稳定的亲水性蛋白,无信号肽;有103个磷酸化位点,2个N-糖基化位点和39个O-糖基化位点;亚细胞定位于在细胞核(82.6%)、细胞质(8.7%)、细胞骨架(4.3%)和质膜(4.3%),属于非跨膜蛋白;预测二级结构由0.71%α-螺旋和99.29%无规则卷曲组成;藏山羊核苷酸序列和氨基酸序列与牛、绵羊和水牛的相似性最高(99%),系统进化树分析表明藏山羊与牛亲缘关系最近;实时荧光定量PCR结果显示,SFRS18基因在藏山羊的不同组织中都存在表达,其中在脾脏中表达水平最高,在背最长肌中表达水平最低;在藏山羊背最长肌和腿肌中SFRS18mRNA表达与肌内脂肪含量均呈显著正相关(r=0.081,P<0.05;r=0.373,P<0.05)。SFRS18基因可以作为调节山羊脂肪沉积的候选基因。

In the present study,SFRS18 gene CDS region of Tibetan goat was cloned and analyzed by bioinformatics method,further its expression regulation in organs or tissues of goat were investigated and the correlation between SFRS18 mRNA expression and intramuscular fat（IMF）content in muscles was analyzed.Thereby providing data for further investigation on the gene functions in goat IMF deposition.The RT-PCR technique was employed to amplify the SFRS18 gene of Tibetan goat.Physicochemical property,structure and homology of SFRS18 encoded protein were analyzed by bioinformatic methods,and the tissue expression specificity as well as the development stage expression specificity of this gene were analyzed using the Real-time fluores-cence quantitative PCR.The results showed that 1 299 bp for SFRS18 cDNA,1 272 bp for ORF and 423 amino acids for protein encoded.The formula of SFRS18 protein was C2003H3424N760O696S4,and the molecular weight of SFRS18 protein was 49.42 ku,and its theoretical isoelectric point was11.20.SFRS18 protein was unstable and hydrophilic,without signal peptide.There were 103 phosphorylation sites,2N-glycosylation sites and 39O-glycosylation sites within the SFRS18 protein.This protein respectively located in nuclear（82.6%）,cytoplasmic（8.7%）,cytoskeletal（4.3%）and plasma membrane（4.3%）through the sub-cellular level prediction.It was an untransmembrance protein.Moreover,the secondary structure of the SPRS18 protein was composed of 99.29% random coil and 0.71% α-helix,indicating an unconventional protein.The nucleotide sequence and the deduced amino acids sequence of Tibetan goat SFRS18 shared 99% homology with the SFRS18 mRNA of Bos taurus,Ovis aries and Bubalus bubalis.Accordingly,the close genetic relationship between Tibetan goat and Bos taurus was indicated by the phylogenetic tree analysis.The results of Real-time PCR exhibited that the SFRS18 gene was expressed in various tissues at different levels.The mRNA expression level of the SFRS18 gene was the highest in spleen and the lowest in longissimus dorsi.The results of correlation analysis showed that the mRNA expression level of the SFRS18 gene in longissimus doris and in crureus was correlated positively with IMF content（r=0.081,P〈0.05;r= 0.373,P〈0.05）.The SFRS18 gene was involved in the regulation of IMF deposition in goat.