| 摘要: |
| 【目的】克隆青海湖裸鲤慢收缩骨骼肌型肌钙蛋白I基因(TNNI1)和快收缩骨骼肌型肌钙蛋白I基因(TNNI2)，分析其在不同组织及盐碱胁迫环境下皮肤和腹侧肌肉组织中的表达水平，为揭示青海湖裸鲤生长缓慢及恢复青海湖裸鲤鱼类资源提供基础数据。【方法】以青藏高原特有鱼类青海湖裸鲤为研究材料，利用RACE技术克隆TNNI1和TNNI2基因全长cDNA序列，进行氨基酸序列同源性比对；利用实时荧光定量PCR法检测2个基因在青海湖裸鲤鳃、眼、脑、脾脏、肝脏、肠、肾、心、皮肤、腹侧肌肉组织的表达情况，以及盐碱胁迫下腹侧肌肉和皮肤组织中的表达规律。【结果】青海湖裸鲤TNNI1 cDNA序列全长为1 211 bp，其中开放阅读框540 bp，共编码179个氨基酸；TNNI2 cDNA序列全长为737 bp，其中开放阅读框531 bp，共编码176个氨基酸。序列同源性分析发现，青海湖裸鲤TNNI1氨基酸序列与安水金线鲃的同源性高达92.13%，TNNI2氨基酸序列与鲤鱼的同源性最高为92.61%。系统发育树结果显示，从TNNI1氨基酸序列来看，青海湖裸鲤与金线鲃属鱼类、鲫鱼、鲤鱼的亲缘关系较近；从TNNI2氨基酸序列来看，青海湖裸鲤与鲤鱼的亲缘关系最近，其次是鲫鱼和金线鲃属鱼类。TNNI1和TNNI2基因在青海湖裸鲤不同组织中均有表达，且在腹侧肌肉中相对表达量最高。盐碱胁迫条件下，TNNI1和TNNI2基因在肌肉组织中的相对表达量均显著下调；在皮肤组织中TNNI1的相对表达量极显著下调（P＜0.01），而TNNI2极显著上调（P＜0.01）。【结论】成功克隆了青海湖裸鲤TNNI1、TNNI2基因全长；TNNI1、TNNI2基因在腹侧肌肉组织中高表达，表明其与肌肉组织发育密切相关；盐碱胁迫条件下TNNI1、TNNI2基因在腹侧肌肉组织中的表达均显著下调，提示2个基因表达量降低可能是青海湖裸鲤生长缓慢的重要因素。 |
| 关键词: 青海湖裸鲤 肌钙蛋白 TNNI1基因 TNNI2基因 盐碱胁迫 |
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| 基金项目:国家自然科学基金项目“青海湖裸鲤鳞片缺失发育的分子机理”（31660745）；国家自然科学基金项目“青海湖裸鲤耐盐碱关键蛋白的鉴定及其调控机理的研究”（31960741） |
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| Cloning of TNNI1 and TNNI2 genes of Gymnocypris przewalskii and expression analysis in response to salinity-alkali stress |
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XU Baoke,A Linlin,ZHANG Haichen,et al
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| Abstract: |
| 【Objective】The slow skeletal muscle type troponin I gene (TNNI1) and the rapid skeletal muscle type troponin I gene (TNNI2) of Gymnocypris przewalskii were cloned and their expression levels in different tissues and in skin and muscle under saline stress were analyzed to provide data for revealing slow growth and recovering resources of G.przewalskii.【Method】The full-length cDNA sequences of TNNI1 and TNNI2 of the endemic fish G.przewalskii in the Qinghai-Tibet Plateau were cloned by RACE technology,and then the amino acid sequence homology comparison was performed.Real-time PCR technology was used to detect the expressions of two genes in gills,eyes,brain,spleen,liver,intestine,kidney,heart,skin and muscle as well as in skin and muscle under saline-alkali stress.【Result】The cDNA sequence of TNNI1 in G.przewalskii was 1 211 bp and the open reading frame was 540 bp,encoding 179 amino acids.The cDNA sequence of TNNI2 of G.przewalskii was 737 bp and the open reading frame was 531 bp,encoding 176 amino acids.Sequence homology analysis found that the homology of TNNI1 between G.przewalskii and Sinocyclocheilus anshuiensis was 92.13%,and the homology of TNNI2 between G.przewalskii and Cyprinus carpio was 92.61%.The phylogenies tree showed that G.przewalskii was closely related to Sinocyclocheilus,C.carpio and Cyprinus auratus according to TNNI1 and it was closely related to C.carpio,C.auratus and Sinocyclocheilus in order according to TNNI2.TNNI1 and TNNI2 differentiated during the evolution of vertebrates.Both genes were expressed in different tissues with the highest levels in muscle.Under saline-alkali stress,both genes in muscle were significantly downregulated,TNNI1 was significantly downregulated in skin (P<0.01),and TNNI2 was significantly upregulated in skin (P<0.01).【Conclusion】This study successfully cloned full length genes of TNNI1 and TNNI2 of G.przewalskii.TNNI1 and TNNI2 genes were highly expressed in muscle,indicating that they were closely related to muscle development.Under saline-alkali stress,TNNI1 and TNNI2 genes were significantly downregulated in muscle,suggesting that expression decrease may be an important reason for its slow growth. |
| Key words: Gymnocypris przewalskii troponin TNNI1 gene TNNI2 gene saline stress |
