昆明犬IGF2基因克隆、生物信息学分析及其时空表达研究

doi:10.16431/j.cnki.1671-7236.2022.03.008

摘要/Abstract

摘要： 【目的】克隆昆明犬胰岛素样生长因子2（IGF2）基因并研究其序列特征及时空表达规律，为工作犬体型及生长发育相关研究提供基础资料。【方法】采用PCR法扩增昆明犬IGF2基因CDS区，运用生物信息学分析预测昆明犬IGF2蛋白的结构与功能；利用实时荧光定量PCR技术检测IGF2基因在2.5月龄昆明犬心脏、肝脏、脾脏、肺脏、肾脏及大腿内侧肌肉和不同年龄段肝脏中的表达情况。【结果】昆明犬IGF2基因CDS区全长717 bp，编码238个氨基酸；系统进化树分析显示，昆明犬IGF2基因与赤狐的遗传距离最近，与鸡的遗传距离最远。生物信息学分析表明，IGF2蛋白分子质量为26.46 ku，理论等电点为9.61，无信号肽和跨膜结构，属于亲水性非分泌蛋白；主要分布于细胞核（47.8%）和线粒体（17.4%），存在23个磷酸化位点；该蛋白的二级结构以α-螺旋和无规则卷曲为主，三级结构与二级结构预测结果相符。组织表达谱分析显示，IGF2基因在2.5月龄昆明犬肝脏中相对表达量最高，且极显著高于肾脏、肺脏及肌肉组织（P<0.01）；仔犬期（2.5月龄）肝脏中的IGF2基因的表达量极显著高于幼犬期（6月龄）、青年期（1岁）及成年期（1.7和2.5岁）（P<0.01）。【结论】本研究成功克隆昆明犬IGF2基因，并发现IGF2在多个组织广泛表达，在肝脏中的表达量最高，可为深入探讨昆明犬IGF2基因在生长发育中的调控机理提供参考。

关键词: 胰岛素样生长因子2(IGF2); 昆明犬; 克隆; 序列分析; 组织表达

Abstract: 【Objective】 This study was aimed to clone insulin-like growth factor 2 (IGF2) gene of Kunming dogs and obtain its sequence characteristics and spatio-temporal expression, to provide basic data for the study of body size and growth characteristics of working dogs.【Method】 The IGF2 gene CDS region of Kunming dogs was amplified by PCR technique, the structure and function of IGF2 protein in Kunming dogs were analyzed and predicted by bioinformatics method.The expression levels of IGF2 gene in heart, liver, spleen, lung, kindey and medial thigh muscle and liver at different growth stage in Kunming dogs were detected by Real-time quantitative PCR.【Result】 The CDS region of IGF2 gene in Kunming dogs was 717 bp which encoded 238 amino acids.The genetic tree analysis showed that the genetic distance of IGF2 gene from Kunming was closest to that of Red fox, and the genetic distance was farthest from that of chicken.The theoretical molecular weight and isoelectric point of IGF2 protein was 26.46 ku and 9.61, respectively.It had no signal peptide and transmembrane structure and belonged to hydrophilic non-secretory protein.The results of subcellular localization showed that IGF2 protein was mainly located in the nucleus (47.8%) and mitochondria (17.4%), and had 23 phosphorylation sites.The secondary structure of the protein was mainly composed of alpha helix and random coil.The prediction of tertiary structure was consistent with the results of secondary structure.The mRNA expression level of IGF2 gene in liver of 2.5 months old Kunming dogs was the highest, and was extremely significantly higher than that in kidney, lung and muscle (P<0.01).The expression of IGF2 gene in the liver of puppies (2.5-month-old) was extremely significantly higher than that of puppies (6-month-old), youth (1-year-old) and adults (1.7 and 2.5-year-old) (P<0.01).【Conclusion】 Kunming dog IGF2 gene was successfully cloned, and it was revealed that IGF2 was widely expressed in many tissues, which was highest in liver.The results could provide a basis for further study on the regulation mechanism of IGF2 gene in the growth and development of Kunming dogs.

Key words: insulin-like growth factor 2 (IGF2); Kunming dogs; cloning; sequence analysis; tissue expression

中图分类号:

韦云芳, 李飞翔, 星云, 黄庆国, 李居东, 万九生, 陈方良. 昆明犬IGF2基因克隆、生物信息学分析及其时空表达研究[J]. 中国畜牧兽医, 2022, 49(3): 866-875.

WEI Yunfang, LI Feixiang, Xing Yun, Huang Qingguo, LI Judong, WAN Jiusheng, CHEN Fangliang. Study on Cloning, Bioinformatics Analysis and Spatio-temporal Expression of IGF2 Gene in Kunming Dogs[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(3): 866-875.

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