【作者】 王家庆；

【导师】 侯林；

【作者基本信息】 辽宁师范大学 ， 海洋生物学， 2007， 硕士

【摘要】 大菱鲆是我国在上世纪90年代引进的名贵海水养殖鱼类,养殖规模较大。在苗种生产过程中常出现大规模的白化现象,白化大菱鲆商品价值大大降低。鲆鲽鱼类白化的分子机制一直是国内外未能解决的热点问题,因此,克隆鱼类白化相关基因具有重要意义。本研究应用mRNA差异显示技术,对正常与白化大菱鲆有眼侧皮肤组织进行了对比研究,克隆差异表达基因,共分离到22条大菱鲆白化相关基因片段,片段长度100～1200 bp左右。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:糖蛋白葡萄糖基转移酶(UGGT)基因、与挪威鼠类似尿苷二磷酸-葡萄糖:神经酰氨葡萄糖基转移酶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。这些结果也同样得到了BLASTX结果的证实。同源性较低的片段可能为未知新基因,对这些序列信息进行进一步研究将有助于获得完整基因和了解大菱鲆白化的分子机制。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,其表达量为:N1>N2≈A2>A1。电子延伸将DEN-2延伸为497 bp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。更多还原

【Abstract】 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. 22 DNA fragments related to albinism were isolated. The size of these fragments was 100-1200 bp in length. 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 Ugcgl1 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. The BLASTX results were consistent with the Blast results. The fragments showed no high degree of similarity to previously reported gene may be a novel gene. The further research on these sequence information are very useful to obtain the complete novel gene and to know the mechanism of albinism of turbot.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 byβ-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.更多还原