【作者】 李林；

【导师】 邹桂伟；

【作者基本信息】 华中农业大学 ， 水产养殖， 2011， 硕士

【摘要】 DMRT基因家族是目前已知控制生物发育的基因家族之一,其编码产物均含有一个具有DNA结合能力的高度保守基序,即DM(果蝇的double-sex基因和线虫的mab-3基因共有的DNA盒基序)结构域,能以锌指结构与特定的DNA序列相结合,调节目的基因转录,从而调控下游基因表达,在性别决定与分化过程中具有重要作用。DMRT1 (double-sex and mab3-relatated transcription factorl)是DMRT基因家族的典型代表,广泛参与脊椎与无脊椎动物的性别发育过程,被认为是继SRY与Sox9之后发现的与性别决定相关的又一重要基因。为了阐明DMRT1在黄颡鱼性别决定及分化过程中的作用,给黄颡鱼的单性育种、雌雄生长差异研究以及鱼类的性别决定与性别分化机制研究提供理论基础,本文利用RACE-SMART及RT-PCR技术从黄颡鱼精巢中分离了DMRT1全长cDNA序列(GenBank Accession No. HM245921),对其在胚胎发育阶段、胚后发育阶段、性腺发育阶段和成体阶段的mRNA表达水平及DMRT1多态性与主要生长性状的相关性进行了研究,主要结论如下：1.黄颡鱼DMRT1基因cDNA全长分离及同源性分析黄颡鱼DMRT1 cDNA全长1381bp,其中5’端非翻译区30 bp,3’端非翻译区454 bp[不包括poly (A)],开放阅读框885bp,编码294个氨基酸。黄颡鱼DMRT1序列与其它物种DMRT1进行同源性比较,结果显示,黄颡鱼DMRT1与鲶形目鱼类胡子鲶、尖齿胡鲶及大口鲶DMRT1氨基酸序列的相似性分别为85%、82%和85%,而保守区DM-domian的相似性高达97.2%-100%,与其它鱼类奥利亚罗非鱼、尼罗罗非鱼、青鳝、斑马鱼及虹鳟的同源性分别为60%、60%、53%、64%和60%；与小鼠及人的同源性较低,分别为43%和45%。2.黄颡鱼DMRT1基因mRNA表达水平分析采用RT-PCR和Real-time RT-PCR技术对DMRT1在黄颡鱼不同发育阶段及雌雄成体不同组织的表达进行了分析。结果表明,在胚胎发育阶段,整个发育过程都有表达,原肠前期(早期、中期)表达水平显著高于其余各期；在胚后发育阶段,前期1-35天表达水平相对较高,31天其表达量达到最高峰,后期36-51天维持在一个相对较低的水平；性腺发育阶段,雄性精巢发育各期均有表达,Ⅳ期表达量最高,而卵巢发育各期均无表达；雌雄成体阶段,仅在雄性精巢组织特异性表达。3.黄颡鱼DMRT1基因多态性与生长性状的相关性分析利用PCR-SSCP方法对黄颡鱼DMRT1与其主要生长性状的关联性进行了分析,结果显示,在其外显子区域SS1位点检测出一个G→A突变位点,导致其编码的氨基酸由甘氨酸(G)突变成了丝氨酸(S)。该位点由一对等位基因(A和B)控制,表现出3种基因型(AA、BB及AB),不同基因型在群体中与主要生长性状的差异分析表明,雌性群体中,除头长、尾柄长及尾柄高外,AA基因型在全长、体长、体高、体厚及体重上均高于BB与AB基因型个体；雄性群体中,在全长、体高、头长及体重性状上,AA基因型同样均高于BB与AB基因型个体,但两个分析群体均没有达到显著水平(P>0.05)；整个群体中,AA基因型个体在全长、体长、尾柄高及体重性状上,显著高于BB基因型(P<0.05),极显著高于AB基因型(P<0.01)；体厚与尾柄长上,AA基因型显著高于BB与AB基因型(P<0.05)；体高与头长上,AA型极显著高于BB与AB基因型(P<0.01),BB与AB基因型之间差异均不显著(P>0.05),该位点可以考虑作为今后黄颡鱼分子标记辅助育种的候选标记,在选育群体中应尽可能地增加AA基因型个体。更多还原

【Abstract】 DMRT gene family is one of the known control biological development gene families. Its code products with a highly conservative sequences which can combine with the DNA, named DM domain region (the DNA-binding motif shared between files double-sex gene and nematode mab-3 gene). It was able to combine with particular sequences of DNA by the zinc finger structure, adjust the transcription of purpose gene, and control gene expression in gender downstream decision. It plays an important role in the process of sex determination and differentiation. DMRT1 (double-sex and mab3-relatated transcription factorl) is the typical representative of DMRT gene families, which participates widely the sex development of vertebrates and invertebrates, is considered another important gene related to sex-determinating after the discovery of SRY gene and Sox9 gene.To elucidate the role of DMRT1 in the sex determination and differentiation in Pelteobagrus fulvidraco, and provide the theoretical basis for researching of unisexual breeding, growth difference between male and female and sex differentiation mechanism, The full-length DMRT1 cDNA sequences from Pelteobagrus fulvidraco was isolated by RACE SMART and RT-PCR. Its mRNA expression level during the stages of embryonic development, post embryonic development, gonad development and adult was analyzed. Otherwise, the relationship between polymorphism of DMRT1 and growth traits was discussed. The main conclusions are as follows:1. Full-length of DMRT1 cDNA isolation and homology analysisThe full-length of DMRT1 cDNA was 1381 bp, including 30 bp of the 5’ untranslated region,454 bp of the 3’untranslated region and 885 bp of the open reading frame, encoding 294 amino acids. Deduced amino acids of Pelteobagrus fulvidraco DMRT1 was compared to Clarias batrachus, Clarias gariepinus and Silurus meridionalis, the similarity were 85%,82% and 85% respectively, while the conserved DM-domain similarity up to 97.2-100%. Other fishes Oreochromis niloticus, Nile tilapia, medaka, zebra fish and rainbow trout shared lower similarity of 60%,60%,53%,64% and 60% respectively, identity of 43% and 45% were also found when compared to mouse and human.2. The level of mRNA expression analysis of DMRT1 geneExpression of DMRT1 in different development stages and adult different tissues of Pelteobagrus fulvidraco were analyzed by RT-PCR and Real-time RT-PCT technologies. The results showed that it expressed throughout the developmental process, expression level of gastrulation period (early, middle) was significantly higher than the rest of the period during embryonic development stage. Expression of the early of post embryonic development (1-35 days after hatching, dah 1-35) was relatively high, its expression reached the highest level at dah 31, and maintained a relatively low level during dah 36-51. DMRT1 mRNA was detected during all development periods of testis with a highest level at periodⅣ, while none throughout ovarian development. In adult male and female, it only expressed in testis. 3. Relationship analysis between DMRT1 polymorphism and the growth traitsUsing PCR-SSCP method analyzed the relationship between DMRT1 polymorphism and growth traits in Pelteobagrus fulvidraco, the results showed that a G→>A mutation was detected in SS1 site of the extron, which leaded to the amino acid glycine (G) mutation into serine (S). The site was controlled by a pair of alleles (A and B), showing 3 genotypes (AA, BB and AB). Difference analysis between different genotypes and the main growth traits indicated that AA genotype in total length, body length, body height, body thickness and body weight were higher than BB and AB individuals in the female population, in addition to head length, caudal peduncle length and caudal peduncle height. For the male population, in the traits of total length, body height, head length and body weight, AA genotype were the same higher than the BB and AB. But the two analyzed groups did not reach significant level (P>0.05). In the entire population, AA genotype in the full length, body length, caudal peduncle height and body weight were significantly higher than that of BB (P<0.05), extremely significantly higher than AB (P<0.01), significantly higher than BB and AB in body thickness and caudal peduncle length (P<0.05), extremely significantly higher than BB and AB in body height and head length(P<0.01), while BB and AB genotype had no significant differences (P>0.05).The site can be considered as one of candidate markers in the process of marker-assisted breeding in Pelteobagrus fulvidraco, as far as possible increase the AA genotype in the breeding population.更多还原