猪NR1H3基因可变剪接体的克隆及表达特性研究

doi:10.11843/j.issn.0366-6964.2020.01.006

摘要/Abstract

摘要： 旨在克隆猪NR1H3基因的可变剪接体，并预测编码蛋白的结构与功能，研究其转录本的表达特性。本试验以马身猪为研究对象，采用RT-PCR及克隆测序技术对猪NR1H3基因CDS区进行扩增和克隆，采用生物信息学方法分析NR1H3蛋白的生物学特性，采用qRT-PCR技术检测NR1H3基因在心、肝、脾、肺、肾、胃、小肠、股二头肌、腰大肌和皮下脂肪组织中的表达谱及在脂肪组织中的发育性表达规律。结果表明，本研究成功克隆出猪NR1H3基因的两个转录本NR1H3-1和NR1H3-2，其中NR1H3-2为新发现的转录本（MN082630），其CDS区全长1 203 bp，编码的蛋白质含400个氨基酸，属于中性不稳定蛋白；与NR1H3-1相比，NR1H3-2缺失了长度为95 bp的第9外显子，编码的蛋白质NR1H3-2比NR1H3-1少了一个肝X受体的配体结合结构域；氨基酸的相似性分析发现，猪NR1H3蛋白氨基酸序列与北大西洋小须鲸、山羊、绵羊、抹香鲸、长江江豚等物种的相似性较高。NR1H3-1和NR1H3-2在所检测组织中均有表达，且在不同组织间的表达量具有显著差异（P<0.05）；NR1H3-1转录本在小肠中表达量最高，其次是脾、肝、股二头肌等，在心脏中表达量最低；NR1H3-2则在肝中表达量最高，其次是脾、小肠、胃和股二头肌等，在心脏中表达量最低。在各组织中（除小肠、心、肺），NR1H3-2的表达量均高于NR1H3-1，其中在肝和皮下脂肪组织中，NR1H3-2的表达量极显著高于NR1H3-1（P<0.01），在胃中差异达显著水平（P<0.05）。随着日龄的增加，皮下脂肪组织中NR1H3-1和NR1H3-2的表达量整体呈上升趋势，推测该基因对猪的脂肪沉积有调控作用。本试验成功克隆了猪NR1H3基因的两个可变剪接体，并推测其对猪脂质代谢及脂肪生成具有重要的生理作用。

关键词: 猪, NR1H3基因, 可变剪接, 生物信息学, 表达特征

Abstract: The aim of this study was to clone the alternative splicing isoforms of pig NR1H3 gene, to predict the structures and functions of their coding proteins, and to investigate the expression patterns of each transcript. The Mashen pig was used in this study. The full-length coding sequence (CDS) of NR1H3 gene in Mashen pig was cloned and sequenced, the biological characteristics of NR1H3 protein was analyzed by bioinformatics methods, and the expression patterns of NR1H3 gene in heart, liver, spleen, lung, kidney, stomach, small intestine, biceps femoris, psoas magnus and subcutaneous fat and the chronological expression trends in adipose tissue in Mashen pig were detected by quantitative real-time PCR (qRT-PCR). The results showed that two of the transcripts of NR1H3 gene, NR1H3-1 and NR1H3-2, were successfully obtained. NR1H3-2 was a new transcript (MN082630), and the length of CDS was 1 203 bp, which encoded 400 amino acids. The encoded protein was neutral unstable protein. Compared with the NR1H3-1 transcript, NR1H3-2 had a deletion of the 9th exon with a length of 95 bp. Coding protein NR1H3-2 had a deletion of ligand binding domain of liver X receptors compared with NR1H3-1. The similarity of NR1H3 amino acid sequence of pig to those of Balaenoptera acutorostrata scammoi, Capra hircus, Ovis aries, Physeter catodon, Neophocaena asiaeorientalis asiaeorientalis was higher. NR1H3-1 and NR1H3-2 were expressed in all detected tissues in this study, and the expression differences among different tissues were significant (P<0.05). The highest abundance of NR1H3-1 was in small intestine, followed by spleen, liver and biceps femoris, and the lowest expression level was in heart. The highest abundance of NR1H3-2 was in liver, followed by spleen, small intestine, stomach and biceps femoris, and the lowest expression level was in heart. The abundance of NR1H3-2 in all tissues except for small intestine, heart and lung were higher than that of NR1H3-1, and the expression difference between NR1H3-1 and NR1H3-2 was extremely significant in liver and subcutaneous adipose tissue (P<0.01), and the difference was significant in stomach (P<0.05). With the increase of age, the expressions of NR1H3-1 and NR1H3-2 in adipose tissue in Mashen pig showed an overall upward trend, which could be deduced that NR1H3 gene might play an important role in the regulation of fat deposition in pig. In this study, two transcripts of pig NR1H3 gene were successfully cloned. It was speculated that NR1H3 might play important physiological roles in lipid metabolism and fat deposition in pig.

Key words: pig, NR1H3 gene, alternative splicing, bioinformatics, expression patterns

中图分类号:

S828.2

张雪莲, 晋大鹏, 吴怡琦, 李文霞, 秦本源, 蔡春波, 高鹏飞, 郭晓红, 李步高, 曹果清. 猪NR1H3基因可变剪接体的克隆及表达特性研究[J]. 畜牧兽医学报, 2020, 51(1): 43-54.

ZHANG Xuelian, JIN Dapeng, WU Yiqi, LI Wenxia, QIN Benyuan, CAI Chunbo, GAO Pengfei, GUO Xiaohong, LI Bugao, CAO Guoqing. Molecular Cloning of Alternative Splice Variants of NR1H3 Gene and Their Expression Patterns in Pig[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(1): 43-54.

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