【作者】 刘季芳；

【导师】 刘少军；

【作者基本信息】 湖南师范大学 ， 动物学， 2004， 硕士

【摘要】 本文综合运用PCR亚克隆、RT-PCR以及Northern、Southern杂交等分子生物学技术，对异源四倍体鲫鲤性别决定相关基因Sox9进行了研究，主要结论如下： （1）用特异于HMG-box（high mobility group-box）保守区的简并引物扩增了异源四倍体鲫鲤及其原始亲本（红鲫和鲤鱼）的Sox基因。异源四倍体鲫鲤的扩增产物共有四条带，大小分别为200bp、600bp、900bp和1900bp左右，而在其原始母本红鲫和原始父本鲤鱼中均只有三条扩增带，大小分别为200bp、600bp和1900bp左右及200bp、500bp和900bp左右。这些结果表明在四倍体鱼及其原始亲本中存在SRY的同源基因，但在雌雄个体中均未发现性别特异扩增带。四倍体鱼不但兼有父母本的扩增带，而且也有变异，为四倍体鱼是异源四倍体而不是同源四倍体提供了分子生物学证据。目前四倍体鲫鲤已连续繁殖了12代(F₃-F₁₄)，形成了一个稳定的新的四倍体鱼繁殖群体。 （2）回收雄性四倍体鱼200bp，600bp和900bp左右扩增带，经亚克隆及测序分析，200bp片段属于HMG盒，600bp和900bp片段为2个新的编码71个氨基酸残基的基因Sox9a和Sox9b，通过计算机分析和RT-PCR方法确定，这2个基因均在HMG-box中含有内含子，其插入位点符合gt-ag规则。 （3）用地高辛标记的900bp片段做探针，斑点印迹结果显示，Sox9基因明显存在于四倍体鱼基因组中。为进一步研究该基因的组织表达特异性，抽提四倍体鱼精巢、卵巢、肝脏、脑、心脏等组织总RNA进行Northern杂交，结果表明Sox9只在精巢、脑和心脏表达，而不在卵巢表达，且在精巢宏量表达，说明该基因在异源四倍体鱼的精子发生及性别决定与分化中可能具有重要作用。而且，多组织RT-PCR及Southern印迹结果进一步证明Sox9在不同组织的差异表达。 （4）从Northern杂交结果观察到两个转录子，大小分别为1.7kb和2.1kb，这表明四倍体鱼中存在2个Sox9基因，但是否分别是Soxga和Soxgb，尚有待进一步研究。更多还原

【Abstract】 In the present study, a series of molecular biological techniques including subcloning of polymerase chain reaction(PCR) product, RT-PCR, Northern blot, Southern blot, were used to analyze Sox9 genes related to sex determination in allotetraploid fish. The main results were summarized as follows:(1) Using degenerate primers within the HMG-box (high mobility group-box) , the Sox gene of the male and female allotetraploid fish and their original parents (red crucian carp and common carp) was amplified by PCR. Four DNA bands with the length 200bp, 600bp, 900bp and 1900bp were observed in the PCR products of allotetraploid fish whereas only three bands existed in their original parents with the length 200bp, 600bp, 900bp and 200bp, 500bp, 900bp, respectively, indicating that there were homologous genes of SRY in allotetraploid fish and their original parents, but no sex-specific band was found in male and female individuals. Allotetraploid fish not only possessed sex-linking bands which almost covered all the bands their parents had, but also had some variation, providing further evidence that tetraploid fish was allotetraploid fish instead of autotetraploid fish. Since F3, the tetraploidy was maintained from one generation to another and to date FH allotetraploid hybrids were produced(F:!-FM)and a stable and new allotetraploid fish population was formed.(2) The excised 200bp, 600bp and 900bp fragments were extracted, subcloned and sequenced. The sequencing showed that an HMG-box with the length 200bp and two Sox9 genes (Sox9a and Sox9b) yielding a putative 71 amino acids protein separately were obtained. Both of them had an intron within the HMG-box, whose intron junctions were in accordance with the "gt-ag" rule by analyses of computer and RT-PCR.(3) Using the 900bp fragment labeled by DIG as probe, dot blotting analysis demonstrated Sox9 was obviously present in the allotetraploid fish genome. To futher the study on tissue-specific expression, Northern blot was performed with total RNA extracted from different tissues like testis, ovary, lung, brain and heart, indicating Sox9 was expressed in testis, brain and heart, but not in ovary. Morever, strong hybridization signals were observed in testis , which suggested Sox9 likely played an important role during spermatogenesis and sex determination and differentiation. In addition, it was confirmed by RT-PCR and Southern blot.(4) Two transcripts, approximately 1. 7kb and 2.1kb were found in Northern blot, suggesting two Sox9 genes existed in allotetraploid fish but that whether they were Sox9a and Sox9b respectively, relied on further study.更多还原