为分析猪CRTC2基因分子生物学特征及其功能,本研究通过生物信息学方法对猪CRTC2(CREB regulated transcription coactivator 2)基因进行功能预测,并对CRTC2基因的非同义单核苷酸多态性(Non-synonymous single nucleotide polymorphisms,nsSNPs)位点进行分析,同时采用 qRT-PCR 方法检测其在猪不同组织中的表达情况.结果表明:1)猪CRTC2基因转录本CRTC2-202(ENSSSCT00000007195)对应的基因座上存在190个SNPs,在编码区(Coding sequence,CDS)存在10个错义突变(nsSNPs),其中rs709245010位点导致第148位的脯氨酸(P)被亮氨酸(L)替代(P148L),该突变位点会降低蛋白质的稳定性,从而影响其结构和功能.2)CRTC2的CDS区的m6A修饰位点预测结果显示,其不存在m6A修饰.3)CRTC2基因的序列保守性和蛋白质二级结构稳定性预测结果显示,P148L突变对CRTC2编码蛋白质的α螺旋、β转角等二级结构产生一定影响,分析其可能是影响猪脂肪沉积和代谢的重要潜在功能性位点.4)对与CRTC2互作的基因和蛋白质分别进行KEGG(Kyoto encyclopedia of genes and genomes)富集分析,结果显示富集到胰高血糖素信号通路和Wnt信号通路等多条与脂肪沉积和代谢相关的通路.5)qRT-PCR结果表明,CRTC2在长白猪的肝脏、腹脂和背脂中表达量显著高于其他组织(P＜0.05).综上,本研究表明猪CRTC2基因上的rs709245010错义突变(P148L)可能会影响蛋白质结构和功能,且该基因可能在猪脂肪沉积和代谢过程中发挥重要作用.本研究为探究猪CRTC2基因功能提供参考依据,为进一步解析脂肪沉积和脂质代谢机制提供研究思路.
Molecular biological characteristics and function prediction analysis of CRTC2 gene in pig
In order to explore the molecular biological characteristics and functions of porcine CRTC2 gene,this paper predicted the functions of porcine CRTC2(CREB regulated transcription coactivator 2)by bioinformatics methods.The non-synonymous single nucleotide polymorphisms(nsSNPs)of CRTC2 gene were analyzed,and its expression in different pig tissues was detected by qRT-PCR.The results showed as follows:1)There were 190 SNPs in the locus corresponding to pig CRTC2 transcript CRTC2-202(ENSSSCT00000007195),and 10 missense mutations(nsSNPs)in Coding sequence(CDS).rs709245010 causes proline(P)at position 148 to be replaced by leucine(L)(P148L),and this mutation site reduces the stability of the protein,thereby affecting its structure and function.2)The prediction results of m6A modification site in CDS region of CRTC2 showed that there was no m6A modification.3)The prediction results of sequence conservation and protein secondary structure stability of CRTC2 gene showed that P148L mutation had certain effects on the α helix,β corner and other secondary structures of CRTC2-encoded proteins,and it may be an important potential functional site affecting pig fat deposition and metabolism.4)KEGG(Kyoto encyclopedia of genes and genomes)enrichment analysis was conducted for the genes and proteins interacting with CRTC2 respectively.The results showed that several pathways related to fat deposition and metabolism,such as glucagon signaling pathway and Wnt signaling pathway,were enriched respectively.5)qRT-PCR results showed that the expression level of CRTC2 in liver,abdominal fat and back fat of Landrace pigs was significantly higher than that in other tissues(P＜0.05).In conclusion,this study found that rs709245010 missense mutation(P148L)on pig CRTC2 gene may affect protein structure and function,and this gene may play an important role in pig fat deposition and metabolism.This study provides reference for exploring the function of pig CRTC2 gene,and provides research ideas for further analyzing the mechanism of fat deposition and lipid metabolism.
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