橘色双冠丽鱼TYR基因的克隆及其发育时序和组织表达分析  

Cloning and Expression Analysis of the Developing Sequence and Tissue Expression of TYR Gene in Amphilophus citrinellus

作　　者：蒋燕玲[1,2] 宋红梅[1] 刘奕[1] 韦敏侠[1] 汪学杰[1] 胡隐昌[1] 罗建仁[1] 

机构地区：[1]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,广州510380 [2]上海海洋大学水产与生命学院,上海201306

出　　处：《农业生物技术学报》2016年第5期697-707,共11页Journal of Agricultural Biotechnology

基　　金：广东省自然科学基金(No.2015A030313700);广东省科技计划(No.2013B020401004);国家科技基础条件平台子项目(No.2015DKA30470)

摘　　要：酪氨酸酶(tyrosinase,TYR)是动物体内黑色素合成信号通路中起关键性作用的限速酶,黑色素生成的速度和种类取决于TYR基因的表达量和活性水平。为了解色素相关基因与体色变化的关系,采用cDNA末端快速克隆(rapid-amplification of cDNA ends,RACE)技术获得橘色双冠丽鱼(Amphilophus citrinellus)TYR基因cDNA全序列,并利用qRT-PCR分析了橘色双冠丽鱼体色变化不同时期和不同组织中TYR基因的表达差异。实验获得c DNA全长序列2 683 bp,其中阅读框1 620 bp,编码540个氨基酸,5'UTR区246 bp,3'UTR区817 bp。氨基酸序列和系统发育分析表明,相比与脊椎动物间的保守性,TYR蛋白序列在鱼类间的保守性更高。qRT-PCR结果表明,TYR基因在胚胎各期均有表达,受精期表达量最低,血液循环期开始急速上升;在"黑色-灰色-亮黄色"三个典型体色褪黑过程中,各组织TYR基因表达量均在黑色期呈现最高值,显著高于灰色和亮黄色期,灰色到亮黄色时期鳞片和尾鳍的TYR基因表达量逐渐降低,但差异不显著(P>0.05),而皮肤组织的TYR基因表达量在三个体色褪黑期均显著降低(P<0.05);亮黄色时期,TYR基因在心、肾、脑、鳔、性腺、尾鳍和眼等组织均有表达,其中眼部表达量最高,其次是鳔和尾鳍,皮肤和性腺表达量最低。橘色双冠丽鱼TYR基因在三个典型体色褪黑期表达逐渐降低,可能与红色素细胞和黄色素细胞逐渐增多以及黑色素细胞数量和分布比例发生变化相关。本研究通过了解体色变异的分子基础,为鱼类体色遗传和体色改良研究积累资料。Tyrosinase （TYR） is the one of the most important rate-limiting enzymes in animals within the signal path of melanin synthesis, which plays a crucial role in the formation of melanin. So the expression and activity level decide the speed and type of melanin. This study was conducted to reveal the relationship between genes related to pigment and body color. We obtained tyrosinase gene （TYR） sequence of Amphilophus citrinellus by rapid-amplification of cDNA ends （RACE） technique and also investigated the expression of TYR in different developmental stages and tissues using qRT-PCR. The total length of TYR was 2 683 bp which contained 246 bp 5＇-UTR, 817 bp 3＇-UTR, and 1 620 bp open reading frame （ORF） encoding 540 amino acids in A. citrinellus. Sequence analysis of amino acids and phylogenetic tree analysis indicated that conservation of TYR was higher in fish than that in other vertebrates, qRT-PCR analysis revealed that TYR expressed in embryonic periods was lower in the conception and began to sharply rise in the circulation of the blood period. In the process of body color changes （black to gray to bright yellow）, the expression of the TYR in black period of all tissues was significantly higher than that in grey and bright yellow period （P〈0.05）. TYR expression of tail fin and scale was gradually reduced in gray to bright yellow period, while there is no significant difference between these two periods （P〉0.05）. However, expression of TYR in skin significantly decreased during the body color developmental stages （P〈0.05）. In the bright yellow period, TYR expressed in all kinds of tissues （heart, kidney, brain, maw, gonad, tail fin and eye）, which expressed the highest amount in eyes, intermediate in swim bladder and tail fin and the lowest in skin and glands. Also, TYR expression gradually decreased among three periods of body color development. It may be related to the gradual increase of red and yellow pigment cells, as well as the changes of the number and distri

关 键 词：橘色双冠丽鱼 酪氨酸酶基因(TYR) QRT-PCR 黑色素细胞 

分 类 号：Q786[生物学—分子生物学]