藏山羊SFRS18基因克隆、表达及其与肌内脂肪含量相关性研究

doi:10.16431/j.cnki.1671-7236.2015.11.029

›› 2015, Vol. 42 ›› Issue (11): 3016-3025.doi: 10.16431/j.cnki.1671-7236.2015.11.029

藏山羊SFRS18基因克隆、表达及其与肌内脂肪含量相关性研究

廖红海^1,2, 林亚秋¹, 李倩^1,2, 邬杨楠¹, 王永^1,2

1. 西南民族大学生命科学与技术学院, 成都 610041;
2. 青藏高原动物遗传资源保护与利用四川省重点实验室, 成都 610041

收稿日期:2015-03-23 出版日期:2015-11-20 发布日期:2015-11-26
通讯作者: 王永 E-mail:wangyong010101@swun.cn
作者简介:廖红海(1988-),男,仡佬族,贵州正安人,硕士生,研究方向:动物遗传育种与繁殖,E-mail:liaohonghaivip@163.com
基金资助:
四川省"十二五"畜禽育种攻关项目(2011NZ0099-36);四川省科技创新产业链示范工程重大项(2014NZ0003);西南民族大学研究生"创新型科研项目"(CX2015SZ069)

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

LIAO Hong-hai^1,2, LIN Ya-qiu¹, LI Qian^1,2, WU Yang-nan¹, WANG Yong^1,2

1. College of Life Science and Technology, Southwest University for Nationalities, Chengdu 610041, China;
2. Key Laboratory of Sichuan Province for Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Exploitation, Chengdu 610041, China

Received:2015-03-23 Online:2015-11-20 Published:2015-11-26

摘要/Abstract

摘要： 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SFRS18 mRNA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1299 bp,开放阅读框(ORF)长为1272 bp,编码423个氨基酸,蛋白分子结构式为C₂₀₀₃H₃₄₂₄N₇₆₀O₆₉₆S₄,分子质量为49.42 ku,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SFRS18 mRNA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。

关键词: 藏山羊; SFRS18基因; 克隆; 肌内脂肪

Abstract: 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 fluorescence quantitative PCR.The results showed that 1299 bp for SFRS18 cDNA,1272 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 was 11.20.SFRS18 protein was unstable and hydrophilic,without signal peptide.There were 103 phosphorylation sites,2 N-glycosylation sites and 39 O-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.

Key words: Tibetan goat; SFRS18 gene; clone; intramuscular fat (IMF)

中图分类号:

廖红海, 林亚秋, 李倩, 邬杨楠, 王永. 藏山羊SFRS18基因克隆、表达及其与肌内脂肪含量相关性研究[J]. , 2015, 42(11): 3016-3025.

LIAO Hong-hai, LIN Ya-qiu, LI Qian, WU Yang-nan, WANG Yong. Cloning,Expression Analysis of SFRS18 and Correlation Analysis between its mRNA Expression and Intramuscular Fat Content in Tibetan Goat[J]. , 2015, 42(11): 3016-3025.

参考文献

[1] Zimowska G,Shi J,Munguba G,et al.Pinin/DRS/memA interacts with SRp75,SRm300 and SRrp130 in corneal epithelial cells[J].Invest Ophthalmol Vis Sci,2003,44:4715-4723.
[2] Graveley B R.Sorting out the complexity of SR protein functions[J].RNA,2000,6(9):1197-1211.
[3] Shen H,Green M R.RS domains contact splicing signalsand promote splicing by a commonmechanism in yeast through humans[J]. Genes & Development,2006,20(13):1755-1765.
[4] Kim J J,Zhao H,Thomsen H,et al.Combined line-cross and half-sib QTL analysis of crosses between outbred lines[J].Genet Res,2005,85(3):235-248.
[5] Wang X,Xue C,Wang X,et al.Differential display of expressed genes reveals a novel function of SFRS18 in regulation of intramuscular fat deposition[J]. Int J Biol Sci,2009,5(1):28-33.
[6] Calkins C,Dutson T,Smith G,et al.Relationship of fiber type composition to marbling and tenderness of bovine muscle[J].Journal of Food Science,1981,46(3):708-710.
[7] Wang Y H,Bower N I,Reverter A,et al.Gene expression patterns during intramuscular fat development in cattle[J].Journal of Animal Science,2009,87(1):119-130.
[8] Dellaire G,Makarov E M,Cowger J J M,et al.Mammalian PRP4 kinase copurifies and interacts with components of both the U5 snRNP and the N-CoR deacetylase complexes[J].Molecular and Cellular Biology,2002,22(14):5141-5156.
[9] 王欢欢.SFRS18对脂肪细胞分化与PPARγ对猪IMF含量影响的研究[D].南京:南京农业大学,2012.
[10] Wang X X,Chen J,Liu H L,et al.The pig pi60 co-activator family:Full length cDNA cloning,expression and effects 208 on intramuscular fat content in longissimus dorsi muscle[J].Domestic Animal Endocrinology,2008,35(2):208-216.
[11] 刘凌云,薛绍白,柳惠图.细胞生物学[M].北京:高等教育出版社,2002.
[12] 邵伟,樊玉杰,徐永镇.SR蛋白家族在RNA剪接中的调控作用[J].生命科学,2010,7(22):711-716.
[13] Ghosh G,Adams J A.Phosphorylation mechanism and structure of serine-arginine protein kinases[J].FEBS Journal,2011,278(4):587-597.
[14] Sanford J R,Ellis J,Caceres J F.Multiple roles of arginine/serine-rich splicing factors in RNA processing[J].Biochem Soc Trans,2005,33:443-456.
[15] 魏琳琳,高晋生,王晋霖,等.绵羊LPIN1基因的克隆和mRNA表达研究[J].畜牧兽医学报,2013,44(9):1371-1379.
[16] Lin S,Fu X D.SR proteins and related factors in alternative splicing[J].Adv Exp Med Biol,2007,623:107-122.
[17] Dellaire G,Makarov E M,Cowger J J,et al.Mammalian PRP4 kinase copurifies and interacts with components of both the U5 snRNP and the N-CoR deacetylase complexes[J].Mol Cell Biol,2002,22:5141-5156.
[18] Salma N,Xiao H,Mueller E,et al.Temporal recruitment of transcription factors and SWI/SNF chromatin-remodeling enzymes during adipogenic induction of the peroxisome proliferator activated receptor g nuclear hormone receptor[J].Mol Cell Biol,2004,24(11):4651-4663.
[19] Norris A W,Chen L,Fisher S J,et al.Muscle-specific PPAR gamma deficient mice develop increased adiposity and insulin resistance but respond to thiazolidinediones[J].J Clin Invest,2003,112(4):608-618.
[20] Kokta T A,Dodson M V,Gertler A,et al.Intercellular signaling between adipose tissue and muscle tissue[J].Domest Anim Endocrinol,2004,27(4):303-331.

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

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