Proteome Analysis Of Gynagenetic Haploid And Normal Diploid Embryos Of Goldfish (Carassius Auratus)

It was found recently that in the gynogenetic haploid and diploid embryos of goldfish,which have exactly the same genome,the haploid condition results in obstruction of gene expression and abnormal development while the diploid embryos have normal gene expression and development.We used proteomics technology to analyze gynagenetic haploid and normal diploid embryos of goldfish(Carassius auratus).Part one:To better understand the molecular events underlying the processes occurring at the embryos,we conducted a proteome comparative analysis of the gynogenetic haploid and diploid embryos of goldfish in the neuralplate stages(18hrs after fertilization).We extracted the total protein of both the gynogenetic haploid and diploid embryos of goldfish.Proteins were separated by two-dimensional gel electrophoresis (2-DE)and analyzed by PDQUEST software.43 protein spots that were differentially expressed in haploid and diploid embryos respectively were identified by high capacity ion trap and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF-MS).Such as molecular chaperones,cytoskeleton proteins and enzymes were identified,and a large number of yolk protein to be identified.During the early development process,the yolk protein was the abundant class,but most of proteins havins dramatic expression change in the development,such as transcription factors and signaling proteins,may not expressed in high abundance and could not be clearly displayed on the 2-D gels.In order to identify transcription factors and signaling proteins with low abundance,we used the gynogenesis haploid embryos nucleoprotein(HEN)and normal diploid embryo nuclearprotein(DEN) as materials in our study.Differential in-gel electrophoresis(DIGE)was used to separate proteins.97 differential proteins were analyzed with the Decyder software.66 protein spots were identified by high capacity ion trap.It showed that gene expressions have significant difference between diploid and haploid during embryos development.These differences may cause the abnormal development of haploid embryos.Part two:Our previous work indicated that obstruction of some protein expression cause the abnormal development of haploid embryos, such as the protein Vsx1(Huang LY et al).Further studies on Vsx1 confirmed the diploid development regulatory mechanism of gene expression during embryonic morphogenesis of goldfish(Luo et al).The two genes of Vsx1 are expressed in gynogenetic haploid embryos with normal eyes of goldfish,while not with cupless eyes at all.To better understand the molecular events underlying the processes occurring at the embryos developing of gynogenetic haploid and elucidate the underlying molecular mechanism of abnormal development in haploid embryos,we performed a proteome comparative analysis of the gynogenetic haploid embryos with normal eyes and cupless eyes of goldfish in the same stage to disclose more differentially expressed proteins as Vsx1.The results indicated protein profiles of embryos with normal eyes and with cupless eyes exhibited some differences.Fifty-two protein spots were directly identified by mass spectrometry and mascot search.Seven protein spots were identified by sequence similarity search using mass spectrum driven BLAST(MS BLAST).Western blot analysis and immunohistochemistry were further carried out to verify the different expression of these four important proteins,which may offer important information for the study of embryos development between haploid embryos with normal eyes and with cupless eyes.