鸡胰岛素降解酶基因环状转录本克隆及其表达规律
收稿日期: 2019-05-30
修回日期: 2019-11-17
网络出版日期: 2019-12-09
基金资助
国家自然科学基金项目编号(31672412);国家自然科学基金项目编号(31972550);广东省自然科学基金项目编号(2017A030307002);农业部鸡遗传育种与繁殖重点实验室开放项目资助编号(201501)
Cloning and expression analysis of chicken circular transcript of insulin degrading enzyme gene
Received date: 2019-05-30
Revised date: 2019-11-17
Online published: 2019-12-09
Supported by
Supported by the National Natural Science Foundation of China Nos(31672412);Supported by the National Natural Science Foundation of China Nos(31972550);the Natural Science Foundation of Guangdong Province No(2017A030307002);the Opening Project of Key Laboratory of Chicken Genetics, Breeding and Reproduction, Ministry of Agriculture of China No(201501)
本课题组前期通过高通量测序发现,鸡胰岛素降解酶(insulin degrading enzyme, IDE)基因可能存在一个环状转录本。为确定IDE基因环状转录本(circIDE)的真实存在,探究其表达规律,本研究以吉林芦花鸡为研究对象,通过PCR扩增和测序验证了circIDE的真实存在,通过RNase R处理和反转录证实了circIDE的环形结构,通过qRT-PCR分析circIDE和IDE mRNA的时空表达规律,并对比分析了circIDE和线性IDE mRNA在正常体型芦花鸡和GHR突变的矮小体型芦花鸡中的表达差异。结果表明：鸡circIDE全长为1332 nt,由IDE基因外显子2~11环化形成。RNase R耐受性分析表明,鸡circIDE具备环形分子的一般特征,不易被RNase R降解。与oligo-d(T)18引物相比,随机引物对circIDE具有较高的反转录效率,进一步说明circIDE是一个不含poly(A)的环状结构分子。组织表达谱结果表明,circIDE在1周龄和12周龄正常体型芦花鸡肝脏和心脏高表达,在胸肌和腿肌中低表达;circIDE在肝脏组织的时序表达谱结果表明,circIDE在鸡6周龄之前为低表达,在8周龄以后表现为高表达;正常与矮小体型芦花鸡品系间circIDE表达量对比分析表明,正常体型芦花鸡circIDE表达水平高于矮小体型芦花鸡,其中在肝脏组织中差异显著(P<0.05)。本研究证实了鸡IDE基因存在一个环状转录本circIDE,并初步揭示了circIDE的表达规律,circIDE在正常体型和矮小体型芦花鸡肝脏组织中表达量存在差异,本研究结果为深入开展鸡circIDE的生物学功能及其在鸡生长发育过程中的作用机制奠定了基础。
冷奇颖, 郑嘉辉, 徐海冬, PatriciaAdu-Asiamah, 张颖, 杜炳旺, 张丽 . 鸡胰岛素降解酶基因环状转录本克隆及其表达规律[J]. 遗传, 2019 , 41(12) : 1129 -1137 . DOI: 10.16288/j.yczz.19-157
Insulin-degrading enzyme (IDE) is a highly conserved metallopeptidase that functions in the catabolism of bioactive peptides. In our previous study, we identified a putative circular transcript in that chicken insulin-degrading enzyme (IDE) gene through analyzing a high throughput sequencing result. Here we set to confirm the circular transcript of IDE (circIDE) and explore its expression regularity in normal barred Plymouth chicken. The circIDE was confirmed by PCR amplification and sequencing. The circular structure of circIDE was determined by RNase R processing and reverse transcription experiments. Then we analyzed the spatiotemporal expression pattern of circIDE and IDE mRNA and compared the differential expression of circIDE and IDE mRNA in the normal barred Plymouth chicken and the dwarf ones. The results showed that the full length of chicken circIDE was 1332 nt, divided form exon 2-11 of the IDE gene. RNase R tolerance analysis showed that chicken circIDE had the general characteristics of circular molecule, and was highly resistant to RNase R. The random primers had higher transcription efficiency than the oligo-d(T)18 primers, confirming that circIDE is a circular structured molecule without poly(A). circIDE was highly expressed in the liver and heart tissues but less in the muscle tissues of leg and breast in normal chickens at the age of 1 and 12 weeks. The expression profile of circIDE in liver tissue showed that circIDE level was lower in1 to 6 weeks and then became higher after 8 weeks of age. The expression of circIDE in liver tissue was significantly higher in normal chicken than that in dwarf barred Plymouth chicken (P<0.05). This study confirmed a circIDE strucutre in chicken IDE gene and uncovered its expression regularity. We demonstrated that the expression level of circIDE in the liver tissue was higher in normal barred Plymouth chicken compared to dwarf species. This study paves the way for further understanding the biological function of chicken circIDE, including its roles in regulating chicken growth and development.
Key words： chicken; insulin degrading enzyme; circRNA; alternative splicing
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