遗传 ›› 2019, Vol. 41 ›› Issue (12): 1129-1137.doi: 10.16288/j.yczz.19-157
冷奇颖, 郑嘉辉, 徐海冬, PatriciaAdu-Asiamah, 张颖, 杜炳旺, 张丽()
收稿日期:
2019-05-30
修回日期:
2019-11-17
出版日期:
2019-12-20
发布日期:
2019-12-09
作者简介:
冷奇颖,硕士研究生,专业方向:动物遗传育种与繁殖。E-mail: lengqiying1995@163.com
基金资助:
Qiying Leng, Jiahui Zheng, Haidong Xu, Patricia Adu-Asiamah, Ying Zhang, Bingwang Du, Li Zhang()
Received:
2019-05-30
Revised:
2019-11-17
Online:
2019-12-20
Published:
2019-12-09
Supported by:
摘要:
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.
Qiying Leng, Jiahui Zheng, Haidong Xu, Patricia Adu-Asiamah, Ying Zhang, Bingwang Du, Li Zhang. Cloning and expression analysis of chicken circular transcript of insulin degrading enzyme gene[J]. Hereditas(Beijing), 2019, 41(12): 1129-1137.
表2
引物序列信息"
引物名称 | 引物序列(5°→3°) | 复性温度(℃) | 扩增产物长度(bp) | 位置 | 用途 |
---|---|---|---|---|---|
circIDE-D | F: AGATAAAGAACGACCACG | 50 | 265 | Exon10 | 环状验证 |
R: CAATTCCAGTCCACGATA | Exon2 | ||||
circIDE-full | F: TCTTCAGAAGGAGACAA | 46 | 1326 | Exon2 | 全长克隆 |
R: CGAATGTTTTCTGGTC | Exon11 | ||||
circIDE-DL | F: GAAGAAGTATTGGCTGCTG | 54 | 198 | Exon11 | circIDE定量 |
R: TTCCAGTCCACGATATTCTC | Exon2 | ||||
IDE-DL | F: TGGCGTATCTCAAGACCCTG | 57 | 175 | Exon22 | IDE mRNA定量 |
R: GCTGGTGCCAAGTTCACATC | Exon24 | ||||
GAPDH | F: AGGACCAGGTTGTCTCCTGT | 57 | 153 | Exon7, 8 | 内参基因定量 |
R: CCATCAAGTCCACAACACGG | Exon9 |
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