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中华鳖(Pelodiscus sinensis)MITF基因的生物信息学及表达差异研究
田儒品1, 张海琪2, 许晓军3, 何中央3, 丁雪燕3, 苏胜齐1
1.西南大学动物科技学院 重庆 400715;2.浙江省淡水水产研究所 湖州 313001;3.浙江省水产引种育种中心 杭州 310012
摘要:
小眼畸形相关转录因子(microphthalmia-associated transcription factor,MITF)是一种黑色素细胞重要的转录因子。本研究通过中华鳖(Pelodiscus sinensis)的肝脏组织进行转录组测序,获得MITF基因的全长序列,生物信息学分析结果显示中华鳖MITF基因5’和3’非编码区分别为32bp和175bp,开放阅读框1512bp,编码503个氨基酸,预测蛋白分子质量为56kDa,理论等点电为6.06。中华鳖MITF蛋白二级结构预测显示,α-螺旋(Alpha helix)占25.37%,延伸链(Extended strand)占20.90%,β-转角(Beta-turn)占7.76%,无规则卷曲(Random coil)占45.97%。氨基酸序列多重比对分析显示,中华鳖MITF基因核苷酸序列与绿海龟、西部锦龟、美国短吻鳄、非洲爪蟾和人的同源性分别为93%、93%、85%、58%和76%。构建进化树分析表明,中华鳖与西部锦龟、绿海龟MITF基因分子进化距离最近,亲缘关系最密切。实时荧光定量PCR对中华鳖8个组织MITF基因表达差异性研究结果表明,该基因在中华鳖的肌肉表达量最高,心脏次之,二者存在显著性差异,肝脏、脾脏、肺、肾脏、眼、皮肤等组织表达量相对较少,显著低于肌肉和心脏。这些研究结果不仅为研究中华鳖MITF基因在黑色素合成过程中的重要作用提供理论基础,还有助于了解龟鳖类体色调控的内在机理,同时也可为中华鳖等爬行动物的遗传育种提供参考。
关键词:  中华鳖  小眼畸形相关转录因子(MITF)基因  体色  转录组  基因表达
DOI:10.11693/hyhz20171100300
分类号:Q789;S917
基金项目:浙江省农业(水产)新品种重大育种专项项目,2016C02055-4号。
BIOINFORMATICS AND EXPRESSION DIFFERENCES ANALYSIS OF MITF GENE IN PELODISCUS SINENSIS
TIAN Ru-Pin1, ZHANG Hai-Qi2, XU Xiao-Jun3, HE Zhong-Yang3, DING Xue-Yan3, SU Sheng-Qi1
1.College of Animal Science and Technology, Southwest University, Chongqing 400715, China;2.Zhejiang Institute of Freshwater Fisheries, Huzhou 313001, China;3.Zhejiang Fisheries Introduction and Breeding Center, Hangzhou 310012, China
Abstract:
To understand the molecular mechanism of MITF (microphthalmia-associated transcription factor) and the body color change of Chinese soft-shelled turtle Pelodiscus sinensis, we built the cDNA library of P. sinensis, obtained the full-length sequence of the MITF gene by transcriptome sequencing, and analyzed the tissues expression of the MITF. Bioinformatics analysis showed that the 5' and 3' non-encoding regions were 32bp and 175bp, with an open reading frame of 1512bp, encoding a putative 503aa. The deduced molecular weight of MITF protein was 56kDa and the theoretical isoelectric point is 6.06. The secondary structure showed that a-helix (Alpha helix) accounted for 25.37%, extension chain (Extended strand) for 20.90%, a beta angle (Beta-turn) for 7.76%, and a random coil (Random coil) for 45.97%. Alignment analysis indicated that the nucleotide sequence showed a high homology with the MITF sequences of Chrysemys picta bellii (93%), Chelonia mydas (93%), Alligator mississippiensis (85%), Xenopus laevis (58%), and Homo sapiens (76%). In the phylogenetic tree, the molecular evolutionary distance of MITF gene among P. sinensis, Chrysemys picta bellii and Chelonia mydas was the closest, and the genetic relationship was most closely related. Real-time PCR revealed the expression of MITF mRNA in the muscle of P. sinensis was significantly higher than in heart, and the expression of MITF mRNA in heart was significantly higher than in the liver, but there was no significant difference between liver, spleen, lung, kidney, eye, and skin. These results are helpful to provide a reference for genetic breeding of P. sinensis and other reptiles.
Key words:  Pelodiscus sinensis  MITF gene  body color  transcriptome  gene expression
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