小鼠生长激素受体基因环状转录本的克隆及其表达规律

doi:10.16288/j.yczz.21-156

遗传 ›› 2021, Vol. 43 ›› Issue (9): 890-900.doi: 10.16288/j.yczz.21-156

小鼠生长激素受体基因环状转录本的克隆及其表达规律

张为露(), 冷奇颖, 郑嘉辉, AliHassanNawaz, 焦振海, 王府建, 张丽()

广东海洋大学滨海农业学院,湛江 524088

收稿日期:2021-04-27 修回日期:2021-07-21 出版日期:2021-09-20 发布日期:2021-08-19
通讯作者: 张丽 E-mail:178052526@qq.com;zhangli761101@163.com
作者简介:张为露,在读硕士研究生,专业方向：动物遗传育种与繁殖。E-mail: 178052526@qq.com
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(2020A1515011576);粤桂联合基金资助编号(2020B1515420008)

Cloning and expression of circular transcript of mouse growth hormone receptor gene

Weilu Zhang(), Qiying Leng, Jiahui Zheng, Ali Hassan Nawaz, Zhenhai Jiao, Fujian Wang, Li Zhang()

College of Coastal Agricultural Sciences, Guangdong Ocean University, Zhanjiang 524088, China

Received:2021-04-27 Revised:2021-07-21 Online:2021-09-20 Published:2021-08-19
Contact: Zhang Li E-mail:178052526@qq.com;zhangli761101@163.com
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(2020A1515011576);Guangdong-Guangxi Joint Fund No(2020B1515420008)

摘要/Abstract

摘要：

根据文献报道并通过circBase数据库查找,发现小鼠生长激素受体基因(growth hormone receptor, GHR)存在8个环状转录本。为确定GHR基因环状转录本(circGHR)的真实存在,探究其表达规律,本研究以昆明小鼠(Mus musculus)为研究对象,通过PCR扩增和测序检测circGHR的真实存在,并筛选出一个circGHR作为目标分子,通过RNase R处理和反转录证实了circGHR的环形结构,利用qRT-PCR分析circGHR和GHR mRNA的时空表达规律。结果表明：小鼠circGHR全长为820 nt,由GHR基因外显子2~8环化形成。RNase R耐受性分析表明,小鼠circGHR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circGHR具有较高的反转录效率,进一步说明circGHR是一个不含poly(A)的环状结构分子。组织表达谱结果表明,circGHR在1周龄和7周龄小鼠肝脏和肾脏高表达,在胸肌和腿肌中低表达;circGHR在肝脏和胸肌组织的时序表达谱结果表明circGHR表达无显著差异;circGHR在腿肌组织的时序表达谱结果表明,circGHR在小鼠5周龄之前为低表达,在7周龄以后表现为高表达。本研究结果证实了小鼠GHR基因存在一个环状转录本circGHR,并初步揭示了circGHR的表达规律,为后期深入开展小鼠circGHR的生物学功能及其在小鼠生长发育过程中的作用机制奠定基础。

关键词: 小鼠, 生长激素受体, 环状RNA

Abstract:

Based on reports in the literature and search results on the circBase database, 8 circular transcripts of the mouse growth hormone receptor (GHR) gene were identified. In order to confirm the existence of the circular transcripts of the GHR gene (circGHRs) and to explore their expression patterns, the Kunming mouse (Mus musculus) was used as a research animal. This study detected the existence of circGHRs by RT-PCR amplification and sequencing, one of which was selected as circGHR for detailed analysis. The circular structure of circGHR was confirmed by RNase R treatment and reverse transcription. The spatiotemporal expression of circGHR and GHR mRNA was analyzed by qRT-PCR. The results showed that the full length of mouse circGHR was 820 nt, which was formed by circularization of exons 2-8 of the transcript of the GHR gene. RNase R tolerance analysis shows that mouse circGHR has the general characteristics of circular molecules and is not easily degraded by RNase R. Compared with oligo-d(T)18 primers, random primers have higher reverse transcription efficiency for circGHR, which further shows that circGHR is a poly(A)-free cyclic structure molecule. Tissue expression profile results show that circGHR is highly expressed in the liver and kidney of 1 week-old and 7-week old Kunming mice, but is low in pectoral muscles and leg muscles. The time-series expression profile of circGHR does not show any significant difference between the liver and pectoral muscle tissue. The circGHR expression in the leg muscle was low before 5 weeks of age but increased after 7 weeks of age. This study confirmed the existence of a circular transcript circGHR of the mouse GHR gene, and initially revealed the expression pattern of circGHR. The results of the study laid a foundation for in-depth developmental studies on the biological functions of the mouse circGHR and its mechanism of action regarding the growth and development of mice.

Key words: mouse, growth hormone receptor, circular RNA

张为露, 冷奇颖, 郑嘉辉, AliHassanNawaz, 焦振海, 王府建, 张丽. 小鼠生长激素受体基因环状转录本的克隆及其表达规律[J]. 遗传, 2021, 43(9): 890-900.

Weilu Zhang, Qiying Leng, Jiahui Zheng, Ali Hassan Nawaz, Zhenhai Jiao, Fujian Wang, Li Zhang. Cloning and expression of circular transcript of mouse growth hormone receptor gene[J]. Hereditas(Beijing), 2021, 43(9): 890-900.

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引物名称	引物序列(5ʹ→3ʹ)	复性温度(℃)	扩增产物长度(bp)	位置	用途
circGHR1	F：TAGTTTGACCGGGATTCGTGG	59	171	Exon7	circGHR1 环状验证
circGHR1	R: GGAACGACACTTGGTGAATCG	59	171	Exon4	circGHR1 环状验证
circGHR2	F：GGTGAGATCCAGACAACGGA	59	134	Exon8	circGHR2 环状验证
circGHR2	R: GACACTTGGTGAATCGAGGC	59	134	Exon4	circGHR2 环状验证
circGHR3	F：GGGATTCGTGGAGACATCCAA	59	343	Exon7	circGHR3 环状验证
circGHR3	R: GACTGCCAGTGCCAAGGTTA	59	343	Exon2	circGHR3 环状验证
circGHR4	F：GACCGGGATTCGTGGAGACATC	61	422	Exon7	circGHR4 环状验证
circGHR4	R: ACGACACTTGGTGAATCGAGG	61	422	Exon4	circGHR4 环状验证
circGHR5	F：CCATCCCATATGGTGGATCTGT	59	142	Intron1	circGHR5 环状验证
circGHR5	R: ATGGGAAAGGAGGTGATGGC	59	142	Intron1	circGHR5 环状验证
circGHR6	F：GCTGGACCAAAAATGTTTCACTGTT	60	176	Exon6	circGHR6 环状验证
circGHR6	R: CGTTGGCTTTCCCTTTTAGCA	60	176	Exon4,5	circGHR6 环状验证
circGHR7	F：CAGCGAAGTCCTCCGTGTAATA	59	100	Exon8	circGHR7 环状验证
circGHR7	R: CAGGGCATTCTTTCCATTCCTG	59	100	Exon6	circGHR7 环状验证
circGHR8	F: CAGTCACCAGCAGCACATTTT	58	70	Exon2	circGHR8 环状验证
circGHR8	R: AGGTTAAGAAGACCTGACAAAGAT	58	70	Exon2	circGHR8 环状验证

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
circGHR-D	F：GGGATTCGTGGAGACATCCAA	59	343	Exon7	环状验证
circGHR-D	R: GACTGCCAGTGCCAAGGTTA	59	343	Exon2	环状验证
circGHR-full	F：GTCTCAGGTATGGATCTTTGTCAGG	57	820	Exon2	全长克隆
circGHR-full	R: CTTCTTCACATGCTTCCAATATGTTC	57	820	Exon8	全长克隆
circGHR-DL	F：GGGATTCGTGGAGACATCCAA	59	343	Exon7	circGHR定量
circGHR-DL	R: GACTGCCAGTGCCAAGGTTA	59	343	Exon2	circGHR定量
GHR-DL	F：GGGTGAGATCCAGACAACGG	57	285	Exon8	GHR mRNA定量
GHR-DL	R: TCACCTCCTCCAACTTCCCT	57	285	Exon11	GHR mRNA定量
GAPDH	F：AGGTTGTCTCCTGCGACTTCA	57	184	Exon6	内参基因定量
GAPDH	R: TGGTCCAGGGTTTCTTACTCC	57	184	Exon7	内参基因定量

小鼠生长激素受体基因环状转录本的克隆及其表达规律

Cloning and expression of circular transcript of mouse growth hormone receptor gene

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