鸡ITM2A基因生物学特性及其在成肌细胞增殖分化中的作用
Biological Characteristics of Chicken(Gallus gallus)ITM2A Gene and Its Role in Myoblast Proliferation and Differentiation
贾其辉 1曹玉珠 1邢雨欣 1马乘霖 1管宏波 1王倩 1康相涛 2田亚东 2李转见 2刘小军 2李红2
作者信息
- 1. 河南农业大学动物科技学院,郑州 450046
- 2. 河南农业大学动物科技学院,郑州 450046;河南省家禽育种国际联合实验室,郑州 450046;河南省鸡种质资源创新与利用重点实验室,郑州 450046
- 折叠
摘要
整合膜蛋白(integral membrane protein 2A,ITM2A)在肌肉发育等多种生物学过程中发挥重要作用.本课题组前期研究显示,ITM2A基因被定位与鸡(Gallus gallus)体重显著相关,但ITM2A在鸡骨骼肌增殖分化过程中的生物学功能并不清楚.本研究运用在线软件对鸡ITM2A蛋白质进行生物信息学分析;选取6只7日龄AA肉鸡(Arbor Acres Plus),采集心、肝、脾、肺、肾和胸肌、腿肌等器官和组织样品用于组织表达谱分析,选取不同发育时期胸肌和腿肌组织用于时序表达分析.利用qPCR技术检测ITM2A的相对表达水平,使用PCR技术扩增ITM2A基因CDS,构建重组质粒过表达载体.分离鸡原代成肌细胞(chicken primary myoblasts,CPMs),检测CPMs诱导分化不同时期ITM2A的相对表达量.在CPMs中过表达ITM2A,检测细胞增殖、细胞分化相关标志基因的表达,评估ITM2A的生物学功能.生物信息学分析结果显示,ITM2A蛋白由251个氨基酸残基组成,主要由α螺旋(38.25％)和β折叠(37.75％)组成;属于不稳定、亲水性、非分泌蛋白,具有1个跨膜结构,主要定位于细胞膜,可能与鳍芽起始因子同源物(fin bud initiation factor homolog,FIBIN)、核糖体蛋白质侧翼柄亚基 P0(ribosomal protein lateral stalk subunit P0,RPLP0)、连环蛋白α样1(catenin alpha like 1,CTNNAL1)等蛋白存在相互作用;与火鸡(Meleagris gallopavo)同源性最高,亲缘关系最近.组织表达分析结果显示,ITM2A在检测组织中广泛表达,在肺脏和脾脏中表达最高,胸肌、腿肌中表达最低.时序表达分析显示,随孵化天数的增加,在胸肌和腿肌中的ITM2A表达逐渐下降,在孵化后1周时达到最低.与对照组相比,过表达组ITM2A表达量极显著高于对照组(P＜0.01),细胞增殖相关标志基因相对表达水平无显著变化,细胞分化相关标志基因肌球蛋白重链(myosin heavy chain,MYHC)、生肌决定因子(myoblast determination,MYOD)和成肌细胞融合因子(myomarker,MYMK)表达量显著上调(P＜0.05).ITM2A基因的表达在CPMs增殖时期呈下降趋势,随着诱导天数的增加ITM2A表达呈上升趋势.本研究表明,ITM2A能够促进鸡成肌细胞的分化,为进一步研究ITM2A影响鸡肌肉发育的分子机制提供了依据.
Abstract
The integral membrane protein 2A(ITM2A)plays an important role in various biological processes such as muscle development.Previous research results showed that the ITM2A gene is significantly related to chicken(Gallus gallus)body weight.However,the biological function of ITM2A in the proliferation and differentiation process of chicken skeletal muscle is not clear.In this study,bioinformatics analysis of chicken ITM2A protein was performed using online software.Six 7-day-old Arbor Acres Plus(AA)broilers were selected,and samples of heart,liver,spleen,lung,kidney,breast muscle,leg muscle were collected for tissue expression profile analysis.Breast muscle and leg muscle tissues at different developmental stages were selected for temporal expression analysis.The relative expression of ITM2A were detected using qPCR,and the ITM2A gene coding sequence was amplified using PCR technology to construct a recombinant plasmid for overexpression.Chicken primary myoblasts(CPMs)were isolated,and the relative expression of ITM2A at different stages of CPM induction differentiation were measured.The biological function of ITM2A was evaluated by assessing the expression of cell proliferation and differentiation marker genes in CPM cells overexpressing ITM2A.The bioinformatics analysis results showed that the ITM2A protein consisted of 251 amino acid residues,predominantly composed of α-helices(38.25％)and β-sheets(37.75％).It was an unstable,hydrophilic,non-secretory protein with a transmembrane structure,mainly located on the cell membrane,and might interact with proteins such as fin bud initiation factor homolog(FIBIN),ribosomal protein lateral stalk subunit P0(RPLP0),and catenin alpha like 1(CTNNAL1).It exhibited the highest homology with turkeys(Meleagris gallopavo)and the closest evolutionary relationship.Tissue expression analysis revealed that ITM2A was widely expressed in the examined tissues,with the highest expression in the lung and spleen and the lowest expression in pectoral and leg muscles.Temporal expression analysis showed that ITM2A expression gradually decreased with increasing days post-hatch,reaching its lowest point at 1 week post-hatch.Compared to the control group,the expression of ITM2A in the experimental group was extremely significantly increased(P＜0.01).There were no significant changes in the relative expression levels of cell proliferation-related marker genes,while the expression levels of cell differentiation-related marker genes,such as myosin heavy chain(MYHC),myoblast determination(MYOD),and myomarker(MYMK),were significantly upregulated(P＜0.05)in the overexpression group.The expression of the ITM2A gene showed a decreasing trend during CPM proliferation and an increasing trend with induction days.This study demonstrated that ITM2A could promote the differentiation of chicken myoblasts,providing fundamental information for further elucidating the molecular mechanisms through which ITM2A influences muscle development in chickens.
关键词
鸡/整合膜蛋白(ITM2A)基因/成肌细胞/表达分析Key words
Chicken/Integral membrane protein 2A(ITM2A)gene/Myoblasts/Expression analysis引用本文复制引用
基金项目
河南省重大科技专项(221100110200)
中原学者首席科学家工作室(30601985)
出版年
2024
国家科技期刊平台
NSTL
万方数据