GALNTL5基因在不同月龄湖羊附睾中的表达模式及其miRNA的预测

Expression pattern of GALNTL5 and its target miRNA screening in epididymis at different ages of Hu sheep

[目的]本文旨在探究不同发育阶段湖羊附睾(头、体、尾)中GALNTL5(polypeptide N-acetylgalactosaminyltransferase-like protein 5)的表达规律及其microRNA(miRNA)预测。[方法]选取3、9、24月龄(M)雄性湖羊各5只,屠宰后采集附睾(头、体、尾)组织。采用RT-qPCR和Western blot技术检测不同发育阶段湖羊附睾中GALNTL5 mRNA和蛋白表达水平;用免疫组化技术对其进行定位分析;用RNAhybrid和miRanda 2种软件分别预测GALNTL5对应的miRNA并取交集,同时检测候选miRNA在附睾尾中的表达规律。[结果]GALNTL5 mRNA和蛋白在附睾体、尾中表现为性成熟后(9 M和24 M)显著高于性成熟前(3 M)(P<0.05);GALNTL5 mRNA在附睾头中表现为24 M显著高于3 M和9 M(P<0.05),但3 M与9 M之间差异不显著(P>0.05)。除了GALNTL5蛋白在9 M与24 M附睾尾中差异显著外,其余均差异不显著(P>0.05);GALNTL5蛋白在附睾头、体中的表达规律一致;GALNTL5定位于附睾主细胞、基细胞及精子中。预测获得11个候选miRNA,按评分高低进行排序,选取miR-487a-5p、miR-485-5p、miR-329b-5p和miR-27a这4个miRNA进行验证,发现miR-487a-5p表达水平表现为性成熟后(9 M和24 M)显著高于性成熟前(3 M)(P<0.05),而miR-485-5p和miR-27a呈现相反趋势。[结论]GALNTL5在精子成熟过程中扮演着重要的角色;miR-485-5p和miR-27a可能与GALNTL5存在靶向关系。

[Objectives]The aim of this study was to investigate the expression pattern of polypeptide N-acetylgalactosaminyltransferase-like protein 5(GALNTL 5)in the epididymis caput,corpus and cauda of Hu sheep at different developmental stages and screen its candidate target miRNA.[Methods]Five Hu sheep at 3,9,24 month(M)were randomly selected,respectively and the caput,corpus and cauda of the epididymis were collected.The GALNTL 5 mRNA and protein expression pattern in the caput,corpus and cauda of Hu sheep at three developmental stages were examined by RT-qPCR and Western blot.The location of GALNTL5 was detected by immunohistochemical.The target miRNA were predicted by RNAhybrid and miRanda software.Meanwhile,the expression levels of candidate miRNA of GALNTL 5 were also detected.[Results]The mRNA and protein expression of GALNTL 5 in the corpus and cauda were significantly higher in the sexually mature(9 M and 24 M)groups than the pre-pubertal(3 M)group(P<0.05).The mRNA expression of GALNTL 5 in the caput was significantly higher in 24 M than 3 M and 9 M(P<0.05),but there was no significant difference between 3 M and 9 M(P>0.05).However,there was no significant difference of GALNTL5 protein between 9 M and 24 M except that in cauda(P>0.05).The expression patterns of GALNTL5 protein in the caput and corpus were similar.GALNTL5 was expressed in the ram epididymis,and was mainly localized in principal cells,basal cells and sperm.Eleven candidate miRNA were obtained from the intersection of the results by the two software.The scores were ranked from high to low and the first four miRNA(miR-487a-5p,miR-485-5p,miR-329b-5p and miR-27a)were selected for candidate miRNA.The expression level of miR-487a-5p in the cauda was significantly higher in the sexually mature(9 M and 24 M)groups than the pre-pubertal(3 M)group(P<0.05),while miR-485-5p and miR-27a showed an opposite tendency with miR-487a-5p(P<0.05).[Conclusions]GALNTL 5 plays an important role in the process of sperm maturation.Meanwhile,miR-485-5p and miR-27a may have a targeted regulatory relationship with GALNTL 5.