牦牛IGFBP4和IGFBP5基因的克隆及其在各组织和不同生长阶段肝中的差异表达分析

doi:10.11843/j.issn.0366-6964.2021.07.009

摘要/Abstract

摘要： 旨在克隆牦牛胰岛素样生长因子结合蛋白4和5（insulin-like growth factor binding protein 4 and 5，IGFBP4 and IGFBP5）基因，并检测其在牦牛不同组织及不同生长阶段肝中的表达水平。本研究选取1日龄、15月龄和5岁龄健康雌性麦洼牦牛共9头（每组各3头），体重分别约为（12.35±1.85）、（98.88±2.50）和（268.55±27.82） kg，采集5岁龄牦牛心、肝、脾、肺、肾、小肠组织及3个不同生长阶段牦牛肝组织。本试验克隆IGFBP4和IGFBP5基因，并对其序列进行生物信息学分析，采用qRT-PCR方法、Western blot技术与免疫组化技术检测其在5岁龄牦牛6种组织及1日龄、15月龄和5岁龄3个不同生长阶段肝中的表达差异。结果表明，获得牦牛IGFBP4和IGFBP5基因的CDS序列分别为777和816 bp，分别编码258和271个氨基酸，GenBank序列号分别为MT012934和MT003005。在同源性比较中，牦牛IGFBP4和IGFBP5序列均与黄牛的同源性最高，分别为99.6%、99.5%。组织表达谱分析显示，IGFBP4和IGFBP5基因均在5岁龄牦牛肝中表达量最高，与肾、心、小肠、脾、肺相比差异极显著（P<0.01），且6种组织中IGFBP4基因的表达量均极显著高于IGFBP5基因（P<0.01）。不同生长阶段肝中差异表达结果显示，随着年龄的增长，IGFBP4和IGFBP5的mRNA与蛋白水平在牦牛肝脏组织中的表达量均呈现上升趋势，即5岁龄>15月龄>1日龄，在5岁龄时表达量最高，与1日龄和15月龄均存在极显著差异（P<0.01），且IGFBP4的表达量极显著高于IGFBP5（P<0.01）。结果提示，IGFBP4和IGFBP5可能协同调控牦牛肝的生长发育，这为深入研究IGFBP4和IGFBP5在牦牛肝生长发育过程中的作用以及表达调控提供了一定的基础数据。

关键词: IGFBP4, IGFBP5, 牦牛, 克隆与表达, 肝

Abstract: This study aimed to clone IGFBP4 and IGFBP5 genes of yak, and detect their expression level in different tissues and liver at different growth stages of yak. In this study, 9 healthy female Maiwa yaks with triplicates were selected from 1 day old, 15 months old and 5 years old with body weight of (12.35±1.85), (98.88±2.50) and (268.55±27.82) kg, respectively. The heart, liver, spleen, lung, kidney and intestine of 5 years old yak and the liver of yak at 3 growth ages were collected. The IGFBP4 and IGFBP5 genes were cloned and their sequences were analyzed by bioinformatics method. In addition, the expressions of IGFBP4 and IGFBP5 were detected by qRT-PCR, Western blot and immunohistochemistry in 6 tissues and liver at 3 growth stages of yak. The results indicated that the CDS sequence of IGFBP4 and IGFBP5 genes were 777 and 816 bp (GenBank accession number:MT012934 and MT003005), encoding a total of 258 and 271 amino acids, respectively. The homology of IGFBP4 and IGFBP5 genes of yak compared with the cattle reached 99.6% and 99.5%, respectively. At the same time, compared with the kidney, heart, intestine, spleen and lung, the expression of IGFBP4 and IGFBP5 genes were the highest in the liver of 5 years old yak (P<0.01). The expression of IGFBP4 gene were significantly higher than that of IGFBP5 gene in these tissues (P<0.01). With the increase of age, the mRNA and protein expression of IGFBP4 and IGFBP5 in yak liver were showed an upward trend in the liver of yak, which was the expression of 5 years old >15 months old >1 day old. Compared with 1 day old and 15 months old, the expression level of IGFBP4 and IGFBP5 in yak liver were the highest in 5 years old (P<0.01). Moreover, the expression of IGFBP4 was extremely significant higher than that of IGFBP5(P<0.01). The results suggest that IGFBP4 and IGFBP5 may co-regulate liver growth and development of yak. It will benefit further study of the role and expression regulation of IGFBP4 and IGFBP5 in the growth and development of yak liver.

Key words: IGFBP4, IGFBP5, yak, cloning and expression, liver

中图分类号:

S823.85

傅芳, 王利, 罗晓林, 官久强. 牦牛IGFBP4和IGFBP5基因的克隆及其在各组织和不同生长阶段肝中的差异表达分析[J]. 畜牧兽医学报, 2021, 52(7): 1858-1868.

FU Fang, WANG Li, LUO Xiaolin, GUAN Jiuqiang. Cloning of Yak IGFBP4 and IGFBP5 and Their Expression Patterns in Different Tissues and Liver at Different Growth Stages[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(7): 1858-1868.

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