鸡HMIT基因的克隆与表达分析

旨在克隆鸡H+/肌醇转运蛋白（H+/myoinositol transporter,HMIT）基因的转录序列,并检测HMIT基因在鸡不同组织中的表达情况,以及外源胰岛素处理对HMIT基因相对表达量的影响。本研究以AA肉鸡为试验动物,采用RT-PCR技术克隆鸡HMIT基因全编码区序列,采用生物信息学技术分析其编码蛋白的生物学特性,利用荧光定量PCR检测HMIT基因在大脑、肝、腹脂、腿肌、心、肾、空肠和回肠等组织中的表达情况,并检测其在肾和大脑中进行胰岛素处理120和240 min后的表达情况。结果表明,以NCBI预测的鸡HMIT（XM_001232939.5）序列为模板设计引物,成功克隆出鸡HMIT基因（1 694 bp,GenBank登录号：MN708211）。克隆的鸡HMIT编码区全长1 380 bp,相比XM_001232939.5预测的编码序列少561 bp,预测编码含有459个氨基酸组成的蛋白。核苷酸和氨基酸序列比对发现,鸡HMIT在物种间高度同源,共线性分析显示,HMIT所在的1号染色体区域在物种间也是高度保守的。HMIT编码的蛋白质是一种不稳定的亲水性蛋白质,其二级结构和三级结构以无规则卷曲和α-螺旋为主。跨膜结构分析显示,鸡HMIT存在8个明显的跨膜结构域。亚细胞定位结果表明,鸡HMIT蛋白分布于质膜（52.3%）、内质网（21.7%）、液泡（17.4%）、线粒体（4.3%）和高尔基体（4.3%）。实时荧光定量PCR检测结果显示,HMIT基因在鸡大脑和肾表达量最高,且显著高于其他组织（P<0.05）。胰岛素处理240 min后,HMIT在肾和大脑中的表达量都显著低于PBS对照组（P<0.05）。本研究克隆获得了鸡HMIT的全编码区,生物信息学分析及组织表达特性研究为深入探索鸡HMIT基因的生理功能和调控机制奠定基础。

Cloning and Expression Analysis of Chicken HMIT Gene

The aim of this study was to clone the transcriptional sequence of chicken H+/myoinositol transporter (HMIT) gene, and to detect its expression in different tissues of chickens, and to explore the effect of exogenous insulin treatment on the relative expression of HMIT gene. In this study, AA broilers were used as experimental animals. The full-length coding region of chicken HMIT gene was cloned by RT-PCR technique, and the biological characteristics of its encoded protein were analyzed by bioinformatics technology. The expressions of HMIT gene in brain, liver, abdominal fat, leg muscle, heart, kidney, jejunum and ileum were detected by fluorescence quantitative PCR. The expressions of HMIT gene in kidney and brain were studied after insulin treatment for 120 and 240 min. The results showed that the chicken HMIT gene (1 694 bp, GenBank accession number: MN708211) was successfully cloned by using the chicken HMIT sequence(XM_001232939.5) predicted by NCBI as a template. The cloned full-length coding region of chicken HMIT was 1 380 bp, which was less than the predicted coding sequence(XM_001232939.5) of 561 bp, which contained a protein of 459 amino acids. Nucleotide and amino acid sequence alignment showed that chicken HMIT was highly homologous among species. The collinearity analysis showed that the region of chromosome 1 where HMIT was located was also highly conserved among species. The protein encoded by HMIT was an unstable hydrophilic protein, its secondary and tertiary structure were mainly composed of random coil and α-helix. Transmembrane structure analysis showed that there were 8 distinct transmembrane domains in chicken HMIT. The results of subcellular localization showed that chicken HMIT protein was distributed in plasma membrane (52.3%), endoplasmic reticulum (21.7%), vacuole (17.4%), mitochondria (4.3%) and Golgi apparatus (4.3%). The results of real-time fluorescence quantitative PCR showed that the expressions of HMIT gene were the highest in chicken brain and kidney, which was significantly higher than those in other tissues(P<0.05). At 240 min after insulin treatment, the expressions of HMIT in kidney and brain were significantly lower than that in PBS control group (P<0.05). In this study, the full-length coding region of chicken HMIT was cloned, the results of bioinformatics and tissue expression characteristics analysis laid a foundation for further exploration of the physiological function and regulatory mechanism of chicken HMIT gene.