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2025年4月6日 星期日
  2017, Vol. 25 Issue (6): 861-873    
  研究论文与报告 本期目录 | 过刊浏览 | 高级检索 |
尼罗罗非鱼TRIM16和TRIM25基因的克隆及表达分析
郑建美,高风英,卢迈新,刘志刚,曹建萌,可小丽,王淼
中国水产科学研究院珠江水产研究所
Cloning and Expression Analysis of TRIM16 and TRIM25 Genes from Nile Tilapia (Oreochromis niloticus)
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摘要 摘 要 泛素连接酶E3家族在脊椎动物的先天免疫中发挥重要作用,由于RING finger结构域的存在,三重基序(tripartite motif, TRIM)蛋白家族的很多成员被报道具有泛素连接酶活性。为了研究TRIM16和TRIM25基因在尼罗罗非鱼(Oreochromis niloticus)免疫过程中的作用,本研究通过逆转录PCR和RACE获得了尼罗罗非鱼TRIM16和TRIM25基因的cDNA及基因组序列,并进行了基因结构和蛋白二级结构分析,qRT-PCR检测基因的组织分布、在胚胎发育过程中表达变化及其对无乳链球菌(Streptococcus agalactiae)感染的响应。结果表明,TRIM16基因的cDNA(GenBank登录号: KY746714)全长2 314 bp, ORF 1 677 bp,编码558个氨基酸;TRIM25基因的cDNA(GenBank登录号: KY968697)全长2 748 bp,ORF 1 677 bp,编码558个氨基酸,二者基因组序列均无内含子。氨基酸序列分析显示,TRIM16和TRIM25均具有TRIM家族的RING finger结构域、B-box结构域等保守结构。组织分析表明,在所检测的11个组织和器官中,TRIM16和TRIM25基因均有表达,血液中表达最高,肝脏中表达最低,血液中的表达量分别为肝脏(对照组)的60.46和274.07倍。这两个基因在尼罗罗非鱼胚胎发育过程中均有表达。人工感染无乳链球菌后,TRIM16和TRIM25基因在所检测的5个组织和器官中的表达量均存在升高的阶段,TRIM16基因在肠、脾、鳃、肾脏和血液中的最高表达量分别为0 h (对照组)的1.30、2.09、1.61、7.81和6.05倍,TRIM25基因在肠、脾、鳃、肾脏和血液中的最高表达量分别为对照组的11.13、1.22、1.26、61.41和77.80倍。上述结果表明,TRIM16和TRIM25基因在尼罗罗非鱼抗无乳链球菌感染的过程中发挥了重要作用。研究结果为进一步了解罗非鱼的抗感染免疫机制、探索病害防治新途径提供了理论基础。
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郑建美
高风英
卢迈新
刘志刚
曹建萌
可小丽
王淼
关键词 尼罗罗非鱼三重基序(TRIM)蛋白家族基因克隆无乳链球菌    
Abstract:Abstract The ubiquitin ligase E3 family plays an important role in the innate immunity of vertebrate, and many members of tripartite motif (TRIM) family have function as an E3 ligase in the ubiquitin modification based on the existence of the conserved RING finger domain. In order to evaluate the immune function of TRIM16 and TRIM25 genes from Nile tilapia (Oreochromis niloticus), the full-length cDNA sequences of TRIM16 and TRIM25 genes from were obtained by reverse transcription PCR and RACE. In addition, the gene structure and protein secondary structure were also analyzed. qRT-PCR was used for analyzing the expression of TRIM16 and TRIM25 genes in tissues and the process of embryonic development, and the response to Streptococcus agalactiae infection. Results showed that the TRIM16 gene (GenBank accession No. KY746714) was 2 314 bp in total length which contained an ORF of 1 677 bp encoding 558 amino acid residues. The 5'-UTR and 3'-UTR were 13 and 624 bp, respectively. TRIM25 gene (GenBank accession: No. KY968697) was 2 748 bp in total length which contained an ORF of 1 677 bp encoding 558 amino acid residues. The 5'-UTR and 3'-UTR were 21 and 1 050 bp, respectively. TRIM16 and TRIM25 genes were all without introns. The results of putative protein indicated that the TRIM16 and TRIM25 had conserved structures of the TRIM family, such as the RING finger domain and the B-box domain. The putative protein of TRIM16 had 29.0%~92.6% identities with other teleosts and mammals. The putative protein of TRIM25 had 20.0%~88.1% identities with other teleosts and mammals. The putative protein of TRIM16 and TRIM25 exhibited the highest identity with Maylandia zebra (92.6% and 88.1%, respectively). Tissue distribution analysis revealed that TRIM16 and TRIM25 genes expressed in all tested tissues with the highest expression level in blood and the lowest expression level in liver. The expression levels of TRIM16 and TRIM25 in the blood were 60.46 and 274.07 folds compared with that in the liver, respectively. Both TRIM16 and TRIM25 genes expressed during the embryonic development of Nile tilapia, which suggested that they played an important role in the immune process of Nile tilapia in the early stage of embryonic development. Upon stimulation with intraperitoneal injection with Streptococcus agalactiae, the expression level of TRIM16 and TRIM25 genes were up-regulated in all test tissues (intestine, spleen, gill, kidney and blood) at the same times. The highest expression levels of TRIM16 gene in intestinal, spleen, gill, kidney and blood were 1.30, 2.09, 1.61, 7.81 and 6.05 folds compared with 0 h (control group), respectively. The highest expression levels of TRIM25 gene in the intestine, spleen, gill, kidney and blood were 11.13, 1.22, 1.26, 61.41 and 77.80 folds compared with 0 h (control group), respectively. The results showed that TRIM16 and TRIM25 genes played an important role in the immunoreaction of Nile tilapia against S.agalactia infection. This study provides a theoretical basis for further understanding of the anti-infective immune mechanism of Nile tilapia and exploring new ways of disease prevention and control.
Key wordsNile tilapia    Tripartite motif (TRIM) protein family    Gene cloning    Streptococcus agalactiae
收稿日期: 2017-01-09      出版日期: 2017-06-01
ZTFLH:  S917.4  
基金资助:现代农业产业技术体系专项资金资助;广东省创新载体建设项目;广州市科技计划项目产学研协同创新重大专项
通讯作者: 高风英     E-mail: fengyinggao2011@163.com
引用本文:   
郑建美 高风英 卢迈新 刘志刚 曹建萌 可小丽 王淼. 尼罗罗非鱼TRIM16和TRIM25基因的克隆及表达分析[J]. , 2017, 25(6): 861-873.
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http://journal05.magtech.org.cn/Jwk_ny/CN/     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2017/V25/I6/861
 
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