尼罗罗非鱼TRIM16和TRIM25基因的克隆及表达分析

发布时间：2019-07-19 13:27

【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,ORF1 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。
[Abstract]:Ubiquitin ligase E3 family plays an important role in innate immunity of vertebrates. Due to the existence of RING finger domain, many members of the triple motif (tripartite motif,TRIM) protein family have been reported to have ubiquitin ligase activity. In order to study the role of TRIM16 and TRIM25 genes in (Oreochromis niloticus) immunization of Nile tilapia, the cDNA and genomic sequences of TRIM16 and TRIM25 genes of Nile tilapia were obtained by reverse transcription PCR and RACE. The gene structure and protein secondary structure were analyzed. QRT-PCR was used to detect the tissue distribution of the genes, the expression changes during embryonic development and their response to Streptococcus lactis (Streptococcus agalactiae) infection. The results showed that the full length of TRIM16 gene was 2314 bp,ORF1 677 bp, encoding 558 amino acids, and the full length of TRIM25 gene was 2748 bp,ORF1 677 bp, encoding 558 amino acids, both of which had no introns. Amino acid sequence analysis showed that both TRIM16 and TRIM25 had conserved structures such as RING finger domain and B-box domain of TRIM family. Tissue analysis showed that TRIM16 and TRIM25 genes were expressed in 11 tissues and organs, the highest expression was found in blood, and the lowest expression was found in liver. The expression level in blood was 60.46 and 274.07 times higher than that in liver (control group), respectively. These two genes were expressed during embryonic development of Nile tilapia. After artificial infection with Streptococcus lactis, the expression of TRIM16 and TRIM25 genes in the five tissues and organs detected was increased. The highest expression of TRIM16 gene in intestine, spleen, Gill, kidney and blood was 1.30, 2.09, 1.61, 7.81 and 6.05 times of that in intestine, spleen, Gill, kidney and blood, respectively. TRIM25 gene was in intestine, spleen and Gill. The highest expression levels in kidney and blood were 11.13, 1.22, 1.26, 61.41 and 77.80 times higher than those in control group, respectively. These results suggest that TRIM16 and TRIM25 genes play an important role in the prevention of Streptococcus lactis infection in Nile tilapia. The results provide a theoretical basis for further understanding the anti-infection immune mechanism of tilapia and exploring a new way of disease control.
【作者单位】：中国水产科学研究院珠江水产研究所/农业部热带亚热带水产资源利用与养殖重点实验室;上海海洋大学水产与生命学院;
【基金】：现代农业产业技术体系专项(CARS-49) 广东省创新载体建设项目(No.2013B090700004) 广州市科技计划项目产学研协同创新重大专项(No.201508020046)
【分类号】：S917.4

【相似文献】