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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。
The E3 family of ubiquitin ligases plays an important role in vertebrate innate immunity and many members of the tripartite motif (TRIM) protein family are reported to have ubiquitin ligase activity due to the presence of the RING finger domain. In order to study the role of TRIM16 and TRIM25 in the immune process of Oreochromis niloticus, cDNA and genomic sequences of TRIM16 and TRIM25 genes were obtained by reverse transcription PCR and RACE. Genomic structure and protein secondary structure analysis, qRT-PCR detection of gene tissue distribution, expression changes during embryonic development and its response to Streptococcus agalactiae infection. The results showed that the cDNA of TRIM16 gene (GenBank accession number: KY746714) was 2 314 bp in length and 1 677 bp in ORF encoding 558 amino acids. The cDNA of TRIM25 gene (GenBank accession number KY968697) was 2 748 bp in length and ORF1 677 bp , Encoding 558 amino acids, both genomic sequences without introns. Amino acid sequence analysis showed that both TRIM16 and TRIM25 possess the conserved structure of RING finger domain and B-box domain of TRIM family. Tissue analysis showed that the TRIM16 and TRIM25 genes were expressed in 11 tissues and organs tested, the highest expression in blood and the lowest expression in liver were 60.46 and 274.07 times higher than those in liver (control group) . Both of these genes are expressed during the embryonic development of Nile tilapia. After artificial infection of Streptococcus agalactiae, the expression levels of TRIM16 and TRIM25 genes in all the five tissues and organs tested were elevated, and the highest expression levels of TRIM16 in intestine, spleen, gill, kidney and blood The highest expression levels of TRIM25 in the intestine, spleen, gill, kidney and blood were 1.13, 1.22, 1.26, 61.41 and 77.80, respectively, of the control group, which were 1.30,2.09,1.61,7.81 and 6.05 times higher than that of the control group Times The above results indicate that the TRIM16 and TRIM25 genes play an important role in the infection of S. niloticus against S. agalactiae. The results provide a theoretical basis for further understanding of the anti-infective mechanism of tilapia and exploring new ways of disease control.