【作者】 李伟；

【导师】 刘少军；

【作者基本信息】 湖南师范大学 ， 发育生物学， 2008， 硕士

【摘要】 分子遗传标记和直接测序一直是对不同生物种类的遗传特性和它们之间遗传关系进行分析的两种主要方法。然而,目前的各种分子标记方法都存在各自的局限性,要么操作复杂,要么稳定性和重复性较差,有的还需要使用同位素等,而全基因组测序的方法又存在成本高、费时长等不足。在本研究中,我们利用一对特殊的Sox(SRY-box)基因简并引物对不同鱼类等动物的基因组进行PCR(PolymeraseChain Reaction)扩增和克隆实验,获得了一种新型的简单有效的分子遗传标记方法,也是一种研究鱼类等动物的分子遗传特性和遗传关系的新方法。Sox基因家族是一个具有高度保守的HMG盒(high mobilitygroup-box)编码转录因子的超基因家族。该基因家族中的HMG盒序列与与哺乳动物睾丸决定因子SRY的HMG盒具有60%以上的同源性,其编码的蛋白质可以和DNA序列特异性结合。从果蝇到人类,Sox基因家族在HMG-box区都呈现出高度保守性。我们根据多种动物Sox基因的HMG-box序列设计特殊的简并引物,在47个不同动物样品(包括19种自然鱼类、16种人工制备的杂交鱼类、4种两栖类、2种爬行类和4种鸟类,以及2种无脊椎动物)中进行Sox基因的扩增,在不同样品中得到了大小和数目不同的Sox基因的DNA片段,由此建立了不同动物的分子遗传标记。我们还对其中21个样品中的42个DNA片段进行了测序,特别是PCR扩增后显示出相似DNA片段的不同样品,进一步比较分析其核苷酸序列。本研究方法具有既快捷、方便,又准确有效的优点,是一种新型的鉴别和分析不同动物的分子遗传特征和分子遗传关系的方法。作为一种新的分子遗传标记方法,此方法既适用于简单快速的区分不同鱼类及其他亲源关系相近的动物,也可广泛应用到对鱼类等动物的遗传变异分析、动物的遗传育种以及种质资源调查等方面。更多还原

【Abstract】 Currently, molecular genetic markers and DNA sequencing are mainly adopted for the analysis on genetic characteristics and the genetic relationship in different species. However, the methods mentioned above are either of taxing operation, or of low stability and repeatability, even in need of isotopes, while whole genome sequencing is undeveloped considering its high expenses and time-consuming. In this study, we employed a pair of degenerate primers of Sox (SRY-box) genes, via PCR (Polymerase Chain Reaction) and clone in the whole genome of different fish and other animals, we got a new molecular genetic marker which is simple and effective. It’s also to be a novel method for studying on the genetics characteristics and relationship in different fishes and other animals.The Sox (SRY-box) gene family is a super gene family of HMG-box-containing genes whose predicted peptide sequence of the box shows >60% similarity to that of the SRY box. Each of the Sox genes encode a high mobility group HMG-box transcription factor, the amino acids motif is known to bind to specific target sequences in DNA. From Drosophila to mankind, the HMG-box of Sox genes were all presented highly conservation. We designed a pair of degenerate primers against the conserved region HMG-box in Sox genes, by PCR amplification, we obtained DNA fragments with definite sizes and numbers belonging to different Sox genes in many animals, including 19 natural fish species, 11 artificial diploid and polyploid hybrids, 4 amphibians, 2 crawlers, 4 Aves and 2 hexapods, from which we established molecular genetic markers for different animals. Furthermore, we sequenced 48 pieces of DNA fragments in 21 kinds of samples, especially those different samples which had similar DNA fragments appeared, in order to make a embedded analysis on their nucleic acid sequences. This novel method is simple, accurate and effective, applicable for study the genetic characteristics and genetic relationship of different animals.To be a new molecular genetic marker, the method can not only be adopted to identify different fish and other relative animals, but also be devoted to genetic variation analysis of animals, biological genetic breeding and animal resource investigation, and so on.更多还原