Cloning and tissue expression analysis of CIDEc gene in Longlin goat

Abstract

[Objective] To investigate the biological characteristics and expression pattern of DFF45-like effector C gene (CIDEc) in Longlin goat, and to provide theoretical basis for revealing the regulation mechanism of CIDEc gene on goat fat.

[Method] Total RNA was extracted from longissimus dorsi muscle, heart, liver, spleen, kidney, abdominal fat and subcutaneous fat of Longlin goat, and abdominal fat and subcutaneous fat of Nubia goat. The sequence of CIDEc gene coding region (CDS) of Longlin goats was amplified by PCR. Online softwares such as ExPASy, TMHMM Server v.2.0, ProtScale, NPS@SOPMA and SSWISS-Model were used for bioinformatics analysis, and real-time fluorescence quantitative PCR was used to detect the expression of CIDEc gene in different tissues and organs of Longlin goat and Nubia goat.

[Result] The CDS sequence of Longlin goat CIDEc gene was 741 bp, encoding 244 amino acid residues. The molecular weight of the encoded protein was 26.09 kD, the instability coefficient was 48.44, the fat index was 100.7, and the theoretical isoelectric point (pI) was 5.28. It was an acidic protein with no transmembrane structure and strong hydrophilicity. The CDS sequence of Longlin goat CIDEc gene had only one mutation compared with the CDS sequence of common goat CIDEc gene(XM_018038444.6) published by NCBI, that was, the mutation at site 560 G was changed to T, which belonged to the synonym mutation. The CDS sequence of CIDEc gene of Longlin goat was the highest in similarity with that of sheep (99.0%), and the lowest in similarity with that of mice (79.6%). The phylogenetic tree constructed based on the similarity of CIDEc gene CDS sequence also showed that Longlin goat was the most closely related to sheep, and closely related to mice. In the secondary structure of Longlin goat CIDEc protein, there were 32.38% of equenxes, 12.70% of quturns, 13.11% of extended chains and 41.80% of irregular coils. The CIDEc gene was expressed in all 7 tissues and organs of Longlin goats, and the CIDEc gene was highly expressed in abdominal fat and subcutaneous fat, which was extremely significantly higher than that in other organs and tissues( P<0.01). The relative expression level of CIDEc gene in adipose tissue (subcutaneous adipose tissue and abdominal adipose tissue) of Nubia goat was significantly higher than that of Longlin goat( P<0.05).

[Conclusion] CIDEc gene is expressed in all organs and tissues of Longlin goat, especially in adipose tissue, and its expression level in adipose tissue (subcutaneous fat and abdominal fat) of Nubian goat is significantly higher than that in Longlin goat. CIDEc protein is an important regulator of fat metabolism and is closely related to lipid storage in animals.

Abstract

摘要：【目的】 明确隆林山羊诱导细胞凋亡DFF45样效应因子c基因(CZDÆc)的生物学特性及其表达规律, 为揭示 CZDÆc基因对山羊脂肪代谢的调控机制提供理论依据。 【方法】提取隆林山羊背最长肌、心脏、肝脏、脾脏、肾脏、腹脂 和皮下脂肪及努比亚山羊腹脂和皮下脂肪的总RNA, PCR扩增隆林山羊C/DÆc基因编码区(CDS)序列, 使用ExPASy、TMHMM Server v.2.0、ProtScale、NPS@SOPMA和SWISS-MODEL等在线软件进行生物信息学分析, 并利用实 时荧光定量PCR检测CfflÆc基因在隆林山羊和努比亚山羊不同组织器官中的表达情况。 【结果】隆林山羊CfflÆc基因 CDS序列全长为741 bp, 共编码244个氨基酸残基, 其编码蛋白分子量26.09 kD, 不稳定系数48.44, 脂肪指数100.7, 理 论等电点(pI)5.28,属于酸性蛋白, 不存在跨膜结构, 亲水性较强。隆林山羊C7DÆc基因CDS序列与NCBI已公布的山 羊C/DÆc基因(XM_018038446.1)CDS序列相对比仅有1处碱基发生突变, 即第560位点G突变为T, 属于同义突变。隆 林山羊与绵羊的CZDÆc基因CDS序列相似性最高(99.0%), 与小鼠的相似性较低(79.6%);基于CZDÆc基因CDS序列 相似性构建的系统发育进化树也显示隆林山羊与绵羊的亲缘关系最近, 与小鼠的亲缘关系较远。在隆林山羊CIDEc 蛋白的二级结构中：a-螺旋占32.38%, P-转角占12.70%, 延伸链占13.11%, 无规则卷曲占41.80%。C/DÆc基因在隆林 山羊7个组织器官中均有表达, 且在腹脂和皮下脂肪中高表达, 极显著高于在其他器官组织的相对表达量( P<0.01); CZDÆc基因在努比亚山羊脂肪组织(皮下脂肪和腹脂)中的相对表达量显著高于在隆林山羊脂肪组织中的相对表达 量( P<0.05)。 【结论】C/DÆc基因在隆林山羊各器官组织中均有表达, 以在脂肪组织中的表达水平较高, 且其在努比亚 山羊脂肪组织(皮下脂肪和腹脂)中的表达水平显著高于在隆林山羊中的表达水平。可见, CIDEc蛋白是脂肪代谢的 重要调节剂, 与动物体内脂质存储密切相关。