牦牛MPC1和MPC2基因克隆及其组织表达特征分析

Cloning and tissue expression characteristics of MPC1 and MPC2 genes in Bos grunniens

【目的】明确牦牛线粒体丙酮酸载体编码基因(MPC1和MPC2)的分子生物学特性及其组织表达特征,为揭示MPC基因在牦牛适应高原环境和能量代谢中的作用机制提供理论依据。【方法】选取麦洼牦牛为研究对象,PCR扩增牦牛MPC1和MPC2基因编码区(CDS)序列,利用在线生物信息学分析软件预测其蛋白结构及理化性质,并以实时荧光定量PCR检测MPC1和MPC2基因在不同年龄(0.5、1.5和2.5岁)牦牛各组织中的表达情况。【结果】牦牛MPC1基因CDS序列长330 bp,共编码109个氨基酸残基;MPC2基因CDS序列长384 bp,共编码127个氨基酸残基。基于MPC1和MPC2基因CDS序列相似性构建的系统发育进化树显示,牦牛分别与黄牛和水牛的亲缘关系最近,与绵羊的亲缘关系最远。牦牛MPC1和MPC2蛋白均为稳定的亲水性蛋白,丙氨酸和亮氨酸含量较高,有跨膜结构但无信号肽,均存在1个MPC结构域;其二级结构以α-螺旋为主(分别占57.80%和55.12%),三级结构模型为5xpd.1.A。MPC1和MPC2基因在2.5、1.5和0.5岁牦牛心脏中的相对表达量均最高,其次是肾脏和肝脏,而在脾脏中的相对表达量最低;MPC1和MPC2基因在不同年龄段牦牛各组织中的表达趋势不一致,可能与基因表达的组织特异性及二者在牦牛不同生长发育阶段发挥的作用不同有关。【结论】MPC1和MPC2基因在反刍动物间具有高度的保守性,其表达存在组织特异性和时序性,尤其在心脏中高表达,且编码蛋白中富含丙氨酸和亮氨酸,说明MPC1和MPC2基因在牦牛改善肉品质及适应高原环境的过程中扮演着重要角色,但二者在牦牛不同生长发育阶段发挥的作用有所差异。

【Objective】To clarify the molecular biological and tissue expression characteristics of mitochondrial pyruvate carrier encoding genes(MPC1 and MPC2)in yak(Bos grunniens),and provide basic data for further research on the mechanism of MPC genes in the plateau adaptation and energy metabolism of yak.【Method】Maiwa yak was selected as the research object,the coding region(CDS)sequence of MPC1 and MPC2 genes were cloned by PCR.The protein struc‐ture and physicochemical properties were predicted by online bioinformatics analysis software,and their expression levels in different tissues of different ages yak(0.5-year-old,1.5-year-old and 2.5-year-old)were detected by real-time fluores‐cence quantitative PCR.【Result】The length of the CDS sequence obtained by cloning the yak MPC1 gene was 330 bp,and the number of encoded amino acids residues was 109,the length of CDS region of MPC2 gene was 384 bp,and the number of encoded amino acids was 127.Phylogenetic tree based on CDS sequence similarity of MPC1 and MPC2 genes showed that yak was most closely related to Bos taurus and Bubalus bubalis respectively,and most far from Oris aries.MPC1 and MPC2 proteins were stable hydrophilic proteins,which dominated by alanine and leucine,which had trans‐membrane structure and no signal peptide,had one MPC domain.The secondary structure was dominated byα-helix(57.80%and 55.12%),and the model of the tertiary structure was 5xpd.1.A.Among the 0.5-year-old,1.5-year-old and 2.5-year-old the expression levels of MPC1 and MPC2 genes were the highest in the heare of yak,followed by spleen and liver,and the lowest in the spleen.However,the expression patterns of MPC1 and MPC2 genes in different tissues of yak at three ages were inconsistent that may be related to the tissue specificity of gene expression and their different roles in different growth and development stages of yak.【Conclusion】The MPC1 and MPC2 genes exhibit high conservation across ruminants,with their expression being both tissue-specific and sequential,particularly in the heart.The encoded protein is notably abundant in alanine and leucine,suggesting a crucial role of the MPC1 and MPC2 genes in meat quality and plateau adaptation,but their functions vary during different growth stages of yak.