摘要
应用mRNA差异显示技术,对比研究正常与白化大菱鲆有眼侧皮肤组织,克隆差异表达基因,经测序,RT-PCR验证,差异表达基因片段DEN-1(GenBank登录号:DQ886390)与DEN-2(GenBank登录号:DQ886391)均在白化大菱鲆有眼侧皮肤组织中下调表达;通过BLAST检索发现,DEN-1与GenBank中的斑马鱼和牛的类似尿苷二磷酸-葡萄糖:糖蛋白葡糖基转移酶(UGGT)基因、与挪威鼠类似尿苷二磷酸-葡萄糖:神经酰氨葡糖基转移酶1(Ugcgl1)基因有较高同源性;DEN-2与原鸡、斑马鱼、人、挪威鼠、家鼠和狗的眼缺乏同源框4(eya4)基因的同源性均较高。采用相对定量RT-PCR对正常鱼有眼侧皮肤(N1)、白化鱼有眼侧皮肤(A1)、正常鱼无眼侧皮肤(N2)、白化鱼无眼侧皮肤(A2)进行表达谱分析,以内参β-actin基因分别对DEN-1和DEN-2表达量进行标准化的光密度扫描显示:DEN-1和DEN-2在4种皮肤组织中有相似的表达模式,即表达量:N1>N2≈A2>A1。电子延伸将DEN-2延伸为497bp的片段,该延伸片段与多种动物的eya4基因在核酸和蛋白水平均有很高同源性。本研究对DEN-1和DEN-2的不同组织器官和不同发育阶段的电子表达谱分析,将对其进一步研究提供有价值参考。
Two gene fragments of DEN-1 （GenBank accession number DQ886390） and DEN-2 （GenBank accession number DQ886391）, differentially expressed between the ocular skin tissues of normal and albino turbot, were isolated by mRNA differential display and sequenced. It was confirmed by RT-PCR method that the expression levels of both DEN-1 and DEN-2 were down - regulated in the ocular skin tissue of albino turbot. The sequence homology search of DEN-1 revealed high sequence homologies with the UGGT genes of zebrafish and cattle, and the Ugcgll gene of Norway rat. High sequence homologies of DEN- 2 were observed with the eya4 gene （the eye absent homolog 4） from red jungle fowl, zebrafish, human, Norway rat, mouse and dog. Expression pattern analysis was performed by relative quantitative RT-PCR to analyze the expression levels of DEN-1 and DEN-2 in four kinds of skin tissues： ocular side of normal turbot （termed N1 skin）, ocular side of albino turbot （termed A1 skin）, blind side of normal turbot （termed N2 skin） and blind side of albino turbot （ termed A2 skin）. The optical densities of the signals of DEN-1 and DEN-2 normalized by β-actin gene show that DEN-1 and DEN-2 have the similar expression pattern： N1 〉 N2 ≈ A2 〉 A1. A 497 bp sequence which had high homologies with the eya4 gene of many animals were produced by the in silico cloning. Electronic expression profile analysis of DEN-1 and DEN-2 in the different tissues, organs and development stages will provide valuable references for the further studies.
出处
《动物学报》
SCIE
CAS
CSCD
北大核心
2007年第1期159-166,共8页
ACTA ZOOLOGICA SINICA
基金
辽宁省科技基金资助项目(No.2050819)~~
关键词
大菱鲆
白化相关基因
MRNA差异显示
Turbot, Scophthalmus maximus, Albinism-related gene, mRNA differential display
