| Ubiquitin, composed of76amino acid residuesis, is a highly conserved small protein with thermal stability, widespread in the animal body to participate in approximately80%of intracellular protein degradation. Studies have proven that in the normal fertilization process of the same species or two genetic relationship closer species, the sperm mitochondria degraded by the oocytes ubiquitin. Compared with the normal fertilization, the fate of mitochondria of the cloned animals showed large differences from different species. Many scholars believe that cloned animals exist reprogramming and cytoplasmic mechanisms, but the lack of specific theoretical support. This research construct sheep-bovine species cloned embryos through somatic cell nuclear transfer, and observed under a fluorescence microscope before the nuclear transfer fluorescent dye-labeled goat for nuclear mitochondrial overexpression of the bovine receptor oocytes reconstructed embryos ubiquitin-protein interactions, aimed at the detection of species cloned embryos to the existence of heterogeneous mitochondrial ubiquitination degradation.This study has achieved the following results:(1) Constructed pVenus-Ub recombinant plasmid and completed the detection of ubiquitin gene expression in cellular and molecular levels. Synthetic ubiquitin gene by the sequence published by Genbank(GenBank accession number M17524.1). Recombinant prokaryotic expression vector pVenus-Ub is constructed by insert artificial synthesis ubiquitin gene into pVenus between BamH â… and Hindâ…¢. Then transfecting pVenus-Ub into Hela cells by Using Lipofectamine2000Reagent. Fluorescence microscope and PCR results showed that pVenus-Ub was expressed in Hela cells. Design primers for PCR amplification of cDNA from the transfected cells, the PCR result of264bp target band to prove that the recombinant vector pVenus-Ub transfected into Hela cells and expressed.(2) Achieve normal expression of ubiquitin genes in bovine oocytes and parthenogenetic embryos by in vitro transcription and cRNA microinjection. Recombinant prokaryotic expression vector pVenus-Ub linearized by the endonuclease EcoR â… digestion, then inject into the oocyte cytoplasm. Parthenogenetic activate mature oocytes by ionomycin, observe the expression and localization of oocytes under fluorescence microscope. And in the first polar body and oocyte nucleus, cell membrane, significantly brighter than the cytoplasm. The results showed that pVenus-Ub mRNA fusion protein was highly expressed in the bovine oocyte maturation process and parthenogenetic embryos, expression efficiency in the nucleus and the cortex is higher than in cytoplasm.(3) Observed the nuclear mitochondria in the early stages of sheep-bovine reconstructed embryos development was not degraded, and distribution to achieve the co-localization with ubiquitin. Selected high expression of ubiquitin mature bovine oocytes, blind suction to remove nucleus, observe mitochondrial distribution by staining oocyte mitochondria with JC-1fluorescence dye. The mitochondrial fluorescence intensity for Goat cells is enhanced with the early embryonic division, co-located with the ubiquitin fusion protein in the cytoplasm, and high intensity in the nucleus and cell membrane. This proved that mitochondria from goat donor cells are not degraded by ubiquitination and co-localization with ubiquitin.(4) Further evidence of mitochondria co-localization with ubiquitin is found by staining mitochondria of the various stages of bovine parthenogenetic embryo. Ubiquitin-protein expression in various stages of the cattle early parthenogenetic embryos showed extensive distribution of green fluorescence throughout the cytoplasm, nucleus and membrane is higher than in cytoplasm. JC-1stained embryos of each period show the mitochondrial distribution consistent with ubiquitin protein distribution, mainly concentrated in the nucleus and the cortex, distribute slightly in the cytoplasm. This proved that pVenus-Ub mRNA fusion protein was highly expressed in parthenogenetic embryos in the nucleus and the cortex, and mitochondria co-localization with ubiquitin.This study established eukaryotic, oocyte and embryo expression system of ubiquitin gene, built a sheep-bovine species cloned embryos by somatic cell nuclear transfer methods, and proved the species cloned embryos heterogeneous mitochondrial receptor package quality pan-factors of degradation, and associated with the ubiquitin expression. This study has provided reliable references for the further research in the genetic phenomena of mitochondrial nuclear transfer, and the mechanism of nuclear transplantation. |
PDF Full Text Request