猪MORC2基因克隆、生物信息学分析及组织表达定位

【目的】克隆猪的Microrchidia家族CW锌指蛋白2（Microrchidia family CW-type zinc finger 2，MORC2）基因，利用生物信息学手段分析其序列特征，并检测MORC2基因在猪不同组织中的表达情况和卵巢中的定位。【方法】以猪卵巢cDNA为模板扩增和克隆MORC2基因完整CDS区，并进行相似性比对及系统发育树构建；利用生物信息学软件对猪MORC2蛋白序列进行预测；使用实时荧光定量PCR检测MORC2基因在猪不同组织中的表达情况；利用免疫组化方法检测猪MORC2蛋白在猪卵巢中的定位情况。【结果】猪MORC2基因CDS区序列全长3 102 bp，编码1 033个氨基酸。猪MORC2蛋白氨基酸序列与人、黑猩猩、恒河猴、小鼠、牛、绵羊、鸡和斑马鱼的相似性分别为94.8%、94.6%、94.9%、91.9%、93.8%、93.9%、80.8%和64.3%。系统进化树表明，猪与灵长类亲缘关系最近，与反刍动物和啮齿类次之，与斑马鱼（鱼类）亲缘关系最远。猪MORC2蛋白分子质量为117.44 ku，理论等电点为8.16，半衰期为30 h，属于不稳定蛋白。MORC2蛋白的平均疏水性为―0.736，为亲水性蛋白，不含跨膜结构和信号肽。猪MORC2蛋白有174个磷酸化位点、81个糖基化位点；亚细胞定位属于核蛋白，细胞质次之，线粒体中有少量表达；含有经典的MORC蛋白家族结构：GHKL-ATPase、zf-CW和CC结构域。组织表达谱结果显示，MORC2基因在猪各组织中广泛表达，其中在肝脏中表达量最多，显著高于其他组织（P＜0.05），在心脏、肺脏和肌肉中表达量较少，显著低于其他组织（P＜0.05）。免疫组化结果显示，MORC2蛋白在健康猪卵泡颗粒细胞和膜细胞中均有表达且表达量较高，在闭锁的卵泡颗粒细胞和膜细胞中表达量较少。【结论】试验成功获得猪MORC2基因完整CDS区序列，该基因在猪各组织中广泛表达，MORC2蛋白主要在健康猪的卵泡颗粒细胞和膜细胞中表达。研究结果为进一步研究MORC2蛋白调控猪卵巢发育和卵泡闭锁的分子机制提供理论依据。

Cloning,Bioinformatics Analysis and Tissue Expression Location of Porcine MORC2 Gene

【Objective】 The aim of this study was to clone porcine Microrchidia family CW-type zinc finger 2 (MORC2) gene,analyze the sequence characteristics using bioinformatics,and detect the expression of MORC2 gene in different tissues and the localization of MORC2 gene in ovary of pigs.【Method】 The complete CDS region of MORC2 gene was amplified and cloned with porcine ovary cDNA as template,and the similarity comparison and phylogenetic tree construction were carried out.The sequence of porcine MORC2 protein were predicted by bioinformatics software.The expression of MORC2 gene in different tissues of pigs were detected by Real-time quantitative PCR.The location of MORC2 protein in porcine ovary was detected by immunohistochemical method.【Result】 The length of MORC2 gene CDS was 3 102 bp,encoding 1 033 amino acids.The amino acid sequence of porcine MORC2 protein had 94.8%,94.6%,94.9%,91.9%,93.8%,93.9%,80.8% and 64.3% similarity with Human sapiens, Pan troglodytes, Macaca mulatta,Mus musculus,Bos taurus,Ovis aries,Gallus gallus and Danio rerio,respectively.Phylogenetic trees revealed that pig was most closely related to primates,ruminants and rodents were next,and the farthest related was Danio rerio.The molecular weight of porcine MORC2 protein was 117.44 ku,the isoelectric point was 8.16,the half-life was 30 h,and there were no transmembrane structure and signal peptides,it was an unstable hydrophilic protein.There were 174 phosphorylation modification sites and 81 glycosylation modification sites of MORC2 protein,it was mainly located in cell nuclear,less in cytoplasm and mitochondria.MORC2 protein contained the classical MORC protein family structure (GHKL-ATPase,zf-CW and CC domain).Tissue expression profile results showed that MORC2 gene was widely expressed in 11 tissues of pigs,the expression was the highest in liver,which was significant higher than that in other tissues (P＜0.05),and the expression were the lowest in heart,lung and muscle,which was significant lower than that in other tissues (P＜0.05).Immunohistochemical results showed that MORC2 protein was expressed in both granulosa cells and membrane cells of healthy pigs,and the expression was higher,but less in atretic granulosa cells and membrane cells.【Conclusion】 The CDS of porcine MORC2 gene was obtained,it was widely expressed in various tissues.MORC2 protein was expressed in both granulosa cells and membrane cells of healthy pigs.The results provided a theoretical basis for further research on the molecular mechanism of MORC2 protein regulating porcine ovarian development and follicular atresia.