【作者】 童英；

【导师】 罗琛；

【作者基本信息】 浙江大学 ， 发育生物学， 2010， 博士

【摘要】 随着越来越多物种全基因组序列的测定,基因组的结构、组织和进化也开始揭开其神秘的面纱。对目前已知的多个物种基因组全序列的研究表明,所有的脊椎动物,尽管它们在整体水平上是二倍体,但它们的基因组中却存在着大量的重复基因。最近的研究表明,转录调控元件和蛋白结构域的互补性丢失可以使重复基因拷贝被保留,这些结果都有力地支持了加倍—退化—互补模型(DDC model)。但翻译调控区的序列变化能否对重复基因的命运产生影响并不是很清楚。在本研究中,我们通过对还处于恢复二倍化过程中的金鱼的vsxl(visual system homeobox-1)基因5’侧翼序列的克隆和分析,确定了该基因的拷贝数目,进一步分析了不同拷贝的表达、功能和组织特异性翻译差异,以为基因组加倍后重复基因拷贝被保留的分子机制提供新的线索。本研究的主要研究结果和结论如下：1.克隆了金鱼vsxlA2的全长mRNA序列,确定了其基因组结构,证明了vsx1A2和vsxlAl都是在基因组加倍后通过亚功能化而保留下来的两个有功能的vsxl基因座位。2.将金鱼vsx1Al和vsx1A2的近端启动子区域与斑马鱼及人类vsxl基因对应的转录调控区域进行序列比对分析,发现在vsxl重复拷贝的转录调控区发生了调控元件的互补性丢失。基因表达分析结果显示vsxlAl和vsxlA2的表达时间既不相同,又有重叠。vsxlA2在原肠期胚胎有较高水平的转录,在体节期胚胎中的转录水平很低,而vsxlAl在胚胎发育早期的转录水平很低,在体节期胚胎中开始有较高水平的转录；在成体视网膜中vsxlAl和vsx1A2都能被转录,但vsxlAl的转录水平显著高于vsxlA2的转录水平。基因功能分析表明vsxlA2具有比vsxlAl更强的胚胎早期发育调控活性。3.将金鱼vsxlAl和vsxlA2的翻译调控区(3’UTR)与斑马鱼及人类vsxl基因对应的3’UTR区域进行序列比对分析证明在3’UTR区域也发生了翻译调控元件的互补性丢失。通过构建包含vsxlAl和vsxlA2翻译调控区(3’UTR)的绿色荧光蛋白载体,对绿色荧光蛋白在不同发育时期胚胎中的表达模式进行检测,我们发现在孵育期及其以后发育时期的金鱼胚胎中,vsxlAl 3’UTR能够特异性地介导绿色荧光蛋白在视网膜中翻译,而vsxlA2 3’UTR已经失去了这种组织特异性翻译介导能力,提示vsxlA1在神经视网膜的发育调节中起更重要的作用。这些研究结果说明：(1)vsx1基因在基因组加倍后通过亚功能化而使重复拷贝得以在基因组中保存；(2)亚功能化是通过转录调控区域和翻译调控区域调控元件的互补性丢失,造成vsxlAl和vsxlA2在表达时空上的区隔和互补而实现的；(3)在vsx1的亚功能化过程中,转录调控元件和翻译调控元件的互补性丢失,以及转录调控区和翻译调控区的协调变化都对重复基因拷贝通过亚功能化而保留和分享原vsxl基因的不同发育功能是必须的。我们的研究结果使DDC模型得到了更进一步的拓展和完善。更多还原

【Abstract】 With the sequencing of entire genomes from various model organisms, the mysteries of genome structure, organization and evolution are beginning to be unveiled. Studies on the available whole-genome sequences revealed that, all vertebrates, despite their generally diploid state, have a lot of duplicated genes in their genomes. Recent studies support the duplication, degeneration, complementation (DDC) model of genome evolution through whole genome duplication by providing evidences that both complementary loss of transcription regulatory elements and peptide domains of the ancestral gene lead to preservation of duplicate genes. However, whether sequence divergence in translation regulatory region has impact on the preservation of duplicate genes remains unclear. Here, we choose goldfish, an ancient polyploidy animal which might be in the process of diploidization, as our research object. By cloning and analyzing the 5’flanking sequence of goldfish vsxl, we identified two vsxl gene loci in the goldfish genome, which were named vsx1A1 and vsx1A2 respectively. We further studied the expression patterns, function and tissue-specific translation of the goldfish vsx1 duplicates. Our results might add new clues to the molecular mechanism underlying the diploidization of the duplicated genes after the genome duplication.The main results and conclusions of this research are as follows:1. We cloned the full-length mRNA of goldfish vsx1A2 and identified its genomic structure. We further proved that both vsx1A1 and vsx1A2 were functional loci, they were retained in the goldfish genome by subfunctionalization and they shared the two different regulatory functions of acient vsxl. 2. By comparing the proximal promoter regions of goldfish vsx1A1 and vsx1A2 with the cognate regions of zebrafish and human vsxl, we found that complementary loss of regulatory sequences occurred at the transcription regulatory regions of the duplicate vsxl loci. Expression analysis showed that vsx1A1 and vsx1A2 were transcribed at overlapping and distinct developmental stages, vsx1A2 initiates transcription at gastrula stage and its transcription level was very low in segmentation stage embryos, while the transcription level of vsx1A1 was very low at early developmental stages embryos and much higher than vsx1A2 at segmentation stage embryos. Both vsx1A1 and vsx1A2 could be transcriped in the adult retina and the transcription level of vsx1A1 was significantly higher than vsx1A2. Gene function analysis indicated that vsx1A2 had much higher regulatory activity than vsx1A1 during early developmental stages.3. By comparing the translation regulatory regions(3’UTR) of goldfish vsx1A1 and vsx1A2 with the cognate regions of zebrafish and human vsxl, we found that complementary loss of regulatory sequences also occurred at the translation regulatory regions of the duplicate vsxl loci. By constructing vsx1A1 or vsx1A2 3’UTR-1 inked Green Fluorescent Protein (GFP) reporter gene sensors and examining the GFP expression pattern in goldfish during different developmental stages, we demonstrated that vsx1A1 3’UTR can mediate retina-specific translation after hatching stage but vsx1A2 3’UTR has lost this translation mediating capability, suggesting that vsx1A1 might play a more important role in regulating retina development.These results indicate that (1) after genome duplication, the goldfish vsxl duplicates were retained by subfunctionalization;(2)the subfunctionalization of the goldfish vsxl duplicates were accomplished by complementary loss of both transcription and translation regulatory elements, which resulted in the different spatio-temporal expression patterns of vsxlAl and vsx1A2; (3) Both the complementary loss of transcription and translation regulatory elements and the intergenic synergetic divergences at transcription and translation regulatory regions of the duplicate vsxl genes are essential for the preserving of the vsxl duplicates and the partitioning of the developmental functions of ancestral vsxl.更多还原