| 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.
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