半滑舌鳎trim36基因cDNA克隆及表达分析  

Cloning and Expression Analysis of trim36 Gene in Half-smooth Tongue Sole(Cynoglossus semilaevis)

作　　者：王文文[1,2] 朱颖[1,2] 位战飞 郑卫卫 董忠典[1,2] 高峰涛[1,2] 邵长伟[2] 温海深[1] 陈松林[2,3] 

机构地区：[1]中国海洋大学水产学院,青岛266003 [2]中国水产科学研究院黄海水产研究所,青岛266071 [3]海洋科学与技术国家实验室/海洋渔业科学与食物产出功能实验室,青岛266237

出　　处：《农业生物技术学报》2016年第7期968-979,共12页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金重点项目(No.31130057);山东省泰山学者攀登计划专项

摘　　要：三重基序蛋白(the tripartite motif-containing protein,TRIM)家族是一类结构保守、进化快速的E3泛素蛋白连接酶,参与诸多重要的生命过程。TRIM36是TRIM家族的一员,trim36编码E3泛素蛋白连接酶。本研究在已完成的半滑舌鳎(Cynoglossus semilaevis)全基因组测序及转录组测序的基础上,利用RACE技术克隆得到半滑舌鳎trim36基因(Gen Bank登录号:KU244470),序列全长为2 899 bp,其中ORF为2 214 bp,编码737个氨基酸,5'非翻译区(untranslated regions,UTR)为289 bp,3'UTR为396 bp。生物信息学分析显示,半滑舌鳎TRIM36蛋白包含1个锌指结构,2个B-BOX类型锌指蛋白结构域(B-Box-type zinc finger,BBOX),1个卷曲螺旋结构,1个羧基末端的纤连蛋白Ⅲ(fibronectin typeⅢ,FN3)结构域和1个双特异性蛋白激酶spla和兰尼碱受体(spla kinase and ryanodine receptor,SPRY)结构域。此外,在不同脊椎动物中,trim36基因与其邻近的基因具有保守的共线性关系。半定量RT-PCR及q RT-PCR结果显示,在半滑舌鳎不同组织中,trim36基因表达存在差异。trim36在卵巢中表达量最高,其次为雌、雄鱼的脑中;不同发育时期中,trim36基因在120日龄半滑舌鳎性腺中开始表达,且卵巢表达量显著高于精巢(P<0.05)。原位杂交结果显示,trim36基因杂交信号主要出现在Ⅰ期和Ⅱ期卵母细胞。trim36时空表达特征表明,其在卵巢腔的形成及Ⅰ期、Ⅱ期卵母细胞的形成中起重要作用。结果表明,trim36在性别发生方面有一定的作用,这为进一步研究鱼类trim36基因功能及TRIM家族基因功能提供线索和参考。The tripartite motif-containing protein（TRIM） family belongs to a E3 ubiquitin protein ligase with conserved structure and rapid evolution. They are involved in a variety of biological processes, such as cell regulation, cell differentiation, metabolism and innate immunity. TRIM36 is one of members of TRIM family.In this study, trim36 gene（Gen Bank No. KU244470） was RACE cloned in the half- smooth tongue sole（Cynoglossus semilaevis） based on the entire genome and transcriptome sequence. trim36 gene contained 2 899bp, and the ORF was 2 214 bp, which encoded 737 amino acids, 289 bp of 5＇-untranslated regions（UTR） and396 bp of 3＇- UTR. The bioinformatics analysis showed that the amino acid sequences contained a RING finger, 2 B-Box-type zinc finger（BBOX）, a coiled-coil motif, a fibronectin type Ⅲ（FN3） and spla kinase and ryanodine receptor（SPRY） domains. In addition, trim36 and its adjacent genes had conservative genomic synteny in different vertebrates. The upstream of the trim36 gene appeared geranylgeranyl transferase type-1subunit beta（pggt1b）, coiled-coil domain-containing protein 112（ccdc112） and Fem-1 homolog C（fem1c） in sequence. According to the alignment result of TRIM 36 amino acid sequence of different species, the TRIM36 protein sequence of Cynoglossus semilaevis presented the highest consistency with that of Larimichthys crocea, and the homology was 89%, followed by Stegastes partitus and Oryzias latipes（88%）.Moreover, the consistency between Gorilla gorilla and Macaca mulatta was the lowest of 63%. The results of RT- PCR and q RT- PCR showed that trim36 mainly expressed in ovaries and brains, which was extremely higher than that of other tissues（P〈0.05）. The results of gonad in different periods by RT-PCR and q RT-PCR showed that trim36 gene began to rise at 120 d, which was consistent with the time of ovarian cavity formation, and the ovary expression was significantly higher than that in testis（P〈0.05）. The in situ hybridization results d

关 键 词：半滑舌鳎 三重基序蛋白基因(trim36) 克隆 QRT-PCR 原位杂交 

分 类 号：Q752[生物学—分子生物学]