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2025年4月6日 星期日
  2018, Vol. 26 Issue (6): 970-977    
  研究论文与报告 本期目录 | 过刊浏览 | 高级检索 |
高邮鸭TNNI1基因克隆及组织表达分析
姬改革1,束婧婷2,单艳菊1,章明3,屠云洁3,巨晓军1,刘一帆1
1. 江苏省家禽科学研究所
2. 中国农业科学院家禽研究所
3. 江苏省家禽科学研究所 家禽遗传育种重点实验室
Molecular Cloning and Tissue Expression Analysis of TNNI1 Gene in Gaoyou Duck (Anas platyrhynchos)
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摘要 摘 要 慢速骨骼肌型肌钙蛋白(slow skeletal muscle troponin I 1, ssTNI, TNNI1)基因主要定位于慢收缩骨骼肌肌纤维,在肌肉发育中具有重要的作用。本研究旨在克隆高邮鸭(Anas platyrhynchos)TNNI1基因并分析其在不同组织中的表达规律。以高邮鸭为实验材料,利用5′和3′ cDNA末端快速扩增(rapid-amplification of cDNA ends, RACE)技术,克隆TNNI1基因全长cDNA序列并进行同源性分析;利用荧光定量PCR技术,检测TNNI1基因在70日龄高邮鸭不同组织中的表达差异和不同发育阶段肌肉组织中的表达规律。结果表明,TNNI1基因全长cDNA序列为965 bp,包括56 bp的5′UTR,345 bp的3′UTR和564 bp的ORF,编码187个氨基酸(GenBank登录号: KY926794)。序列同源性分析发现,与北京鸭、鹌鹑(Coturnix japonica)、鸡(Gallus gallus)和哺乳动物TNNI1基因的同源性分别为97.9%、97.9%、97.3%和87.7%。系统进化树分析表明,高邮鸭与鹌鹑和鸡的亲缘关系最近,与哺乳动物亲缘关系较远。荧光定量PCR结果表明,TNNI1基因在本实验各个组织中均有表达,其中腿肌表达量最高,极显著高于其他组织(P<0.01);其次是心脏、肺、胸肌、腺胃、肾脏和十二指肠组织;而大脑、肝脏、脾脏和下丘脑中表达量最低。21胚龄胸肌TNNI1基因表达量极显著高于腿肌(P<0.01),其他发育阶段均是腿肌表达量显著高于胸肌(P<0.05)。高邮鸭TNNI1基因结构与组织表达特征的研究结果,为进一步研究TNNI1基因在鸭肌肉发育中的作用机制提供了基础资料。
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姬改革
束婧婷
单艳菊
章明
屠云洁
巨晓军
刘一帆
关键词 高邮鸭慢速骨骼肌型肌钙蛋白基因(TNNI1)基因克隆基因表达肌肉组织    
Abstract:Abstract Slow skeletal muscle troponin I 1 (TNNI1) located in slow-twitch myofibers that provides a calcium-sensitive switch for the contraction of striated muscle and plays an important role in muscle growth. The aim of this study was to clone the cDNA full length of TNNI1 gene of Gaoyou duck (Anas platyrhynchos domestica), analyze its sequence characteristics, mRNA expression profile in different tissues/organs, which might provide basis to further study the function of TNNI1 gene in muscle development in duck. Gaoyou duck was selected as materials, the full length of TNNI1 cDNA was cloned from breast muscle by 5' and 3' rapid-amplification of cDNA ends (RACE) and its homologies with TNNI1 gene of other animals was analyzed. The TNNI1 mRNA levels in different tissues/organs at sexual age (70 d), as well as in the breast and leg muscles during the developmental stages were measured using quantitative real-time PCR (qRT-PCR). The results showed that the full length of TNNI1 cDNA sequence was 965 bp, containing a 56 bp 5′UTR, a 345 bp 3′UTR and a 564 bp open reading frame (ORF), encoding a protein of 187 amino acids (GenBank accession number: KY926794). Sequence analysis revealed that the TNNI1 amino acid sequence of the Gaoyou duck is similar with those of Beijing duck (Anas platyrhynchos, 97.9%), quail (Coturnix japonica, 97.9%), chicken (Gallus gallus, 97.3%) and mammals (87.7%). Phylogenetic tree analysis showed that Gaoyou duck has the closest phylogenetic relationship to the Peking duck, quail and chicken, but were far away with mammals TNNI1 in amino acid sequences. The qRT-PCR results showed that TNNI1 were expressed in different organizations. And the mRNA level of TNNI1 in leg muscle were significantly higher than that of other tissues (P<0.01), and the heart were the second. There was no significant difference between the mRNA level of breast muscle and glandular stomach (P>0.05), while the lowest level was in brain, liver, spleen, and hypothalamus. During embryogenesis and post-hatching development, the expression pattern of TNNI1 in breast muscle and leg muscle showed an extreme significant difference as time went on. The mRNA level peak of TNNI1 in breast muscle was at embryonic age of 21 days, and in leg muscle was at embryonic age of 27 days. The mRNA level of TNNI1 gene in breast muscle was significantly lower than that of leg muscle at the embryonic days 27 and the neonatal age of 5 days, but was significantly higher than that of leg muscle at the embryonic age of 21 days. The results of these experiments showed that the amino acid sequence of TNNI1 belonged to the same branch as that of poultry, and was far away from mammals. qRT-PCR results showed that TNNI1 could be expressed in different tissue of Gaoyou duck, and existed some difference. The present study also found that there were significant differences in mRNA level of TNNI1 gene between the breast muscle and the leg muscle at different developmental stages. Therefore, structural characterization and tissue expression of TNNI1 gene in Gaoyou duck lay the foundation for further research on the function of TNNI1 gene in duck muscle development.
Key wordsGaoyou duck    Slow skeletal muscle isoform of troponin I gene (TNNI1)    Gene cloning    Gene expression    Muscle tissue
收稿日期: 2017-11-02      出版日期: 2018-05-21
基金资助:江苏省自然科学基金;江苏省六大人才高峰项目;扬州市现代农业项目;扬州市科技公共服务平台建设项目
通讯作者: 束婧婷     E-mail: shujingting@163.com
引用本文:   
姬改革 束婧婷 单艳菊 章明 屠云洁 巨晓军 刘一帆. 高邮鸭TNNI1基因克隆及组织表达分析[J]. , 2018, 26(6): 970-977.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2018/V26/I6/970
 
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