鸡胰岛素降解酶基因环状转录本克隆及其表达规律

doi:10.16288/j.yczz.19-157

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Hereditas(Beijing) ›› 2019, Vol. 41 ›› Issue (12): 1129-1137.doi: 10.16288/j.yczz.19-157

• Research Article • Previous Articles Next Articles

Cloning and expression analysis of chicken circular transcript of insulin degrading enzyme gene

Qiying Leng, Jiahui Zheng, Haidong Xu, Patricia Adu-Asiamah, Ying Zhang, Bingwang Du, Li Zhang()

College of Agriculture, Guangdong Ocean University, Zhanjiang 524088, China

Received:2019-05-30 Revised:2019-11-17 Online:2019-12-20 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)

Abstract

Abstract:

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

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.

Figures/Tables 7

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References 29

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年龄(周)	能量(MJ/kg)	粗蛋白(%)	钙(%)	有效磷(%)	赖氨酸(%)	蛋氨酸(%)
0~6	11.66	19.90	1.00	0.37	0.90	0.44
7~12	11.98	18.00	0.90	0.33	0.90	0.34
12~	12.11	16.98	0.86	0.33	0.90	0.34

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
circIDE-D	R: CAATTCCAGTCCACGATA	50	265	Exon2	环状验证
circIDE-full	F: TCTTCAGAAGGAGACAA	46	1326	Exon2	全长克隆
circIDE-full	R: CGAATGTTTTCTGGTC	46	1326	Exon11	全长克隆
circIDE-DL	F: GAAGAAGTATTGGCTGCTG	54	198	Exon11	circIDE定量
circIDE-DL	R: TTCCAGTCCACGATATTCTC	54	198	Exon2	circIDE定量
IDE-DL	F: TGGCGTATCTCAAGACCCTG	57	175	Exon22	IDE mRNA定量
IDE-DL	R: GCTGGTGCCAAGTTCACATC	57	175	Exon24	IDE mRNA定量
GAPDH	F: AGGACCAGGTTGTCTCCTGT	57	153	Exon7, 8	内参基因定量
GAPDH	R: CCATCAAGTCCACAACACGG	57	153	Exon9	内参基因定量

Cloning and expression analysis of chicken circular transcript of insulin degrading enzyme gene

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