Mouse 18s Rrna Gene In Ovarian And Oocyte Expression And Rnai Vector

Objective To determine whether 18S rRNA is expressed in ovaries and single immature oocytes in germinal vesicle (GV) and metaphaseⅠ(MⅠ) or not and reveal the relationship between 18S rRNA and maturation of oocytes. Based on bioimformatic analyses on 18S rRNA, the RNAi vector targeted in 18S rRNA was designed and constructed, which provide a tool for further studies on roles of 18S rRNA in embryonic developmentMethods According to the conservative sequence coding for 18S rRNA of mus musculus (363 bp), two pair primers used in RT-PCR were designed by DNAclub software. Subsequently, expression of 18S rRNA were determinted in ovaries and single immature oocytes in GV and MⅠstage by RT-PCR. After being amplified from immature oocytes in MⅠstage, the targeted gene fragment was inserted into pTG19-T vector, transformed into E. coli, then the positive recombinant plasmids were sequenced. The sequenced results was paid for bioinformatic analyses by RNAdraw, VECTOR NIT 9.0, ClutalX1.81, Treeview and other softwares. In the end, siRNA expression plasmid for interference 18S rRNA gene of mus musculus was constructed.Results (1) The gene fragment coding for 18S rRNA presented in ovaries tissues and single oocyte in GV and MⅠ, which was higher in MⅠstage than that in GV in immature oocytes. (2) The RT-PCR product was inserted into pTG19-T vector and transformed into E. coli successfully. By blastn, the sequence results of Kunming mus musculus were in complete accordance with the conservative sequence of Genbank NR₀03278 (791bp-1153bp). By Blastn in NCBI, the sequence with little difference among animals was confirmed to be conservative. After Blastn, fourteen complete CDS coding for different animals were chosen. According to VECTOR NIT 9.0 software, the similarities between Kunming mus musculus and bos taurus, homo sapiens, erinaceus europaeus, cricetulus griseus, sus scrofa, dasypus novemcinctus, rattus norvegicus, rabbit, equus caballus, macaca fascicularis, didelphis virginiana, monodelphis domestica and vombatus ursinus was 67%, 100%, 100%, 36%, 100%, 100%, 67%, 100%, 100%, 92%, 99%, 99% and 99% . In the phylogenetic tress constructed with the forteen 18S rRNA by Treeview, the Kunming mus musculus clustered with cricetulus griseus, sus scrofa and rabbit, which was nearer to cricetulus griseus and was most far away from macaca fascicularis. (3) After sencodary structure analyses of 18S rRNA of mus musculus, an oligonucleotide fragment for RNAi was designed and synthesized, which was transformed into plasmid, and restriction enzyme analyses and sequencing results should the expression plasmid pGPH1/ GFP/Neo-mouse-sh 18S rRNA were constructed for RNAi successfully.Conclusion (1) 18S rRNA presented in ovaries and immature oocytes in mus musculus, and expression of 18S rRNA was higher in M I stage than that in GV in oocytes, which showed that oocytes need protein synthesis in its development and its maturation should be associated with 18S rRNA expression. (2) The sequenced results were in complete accordance with the conservative sequence of 18S rRNA in Genbank, which suggested the conservative sequence of 18S rRNA to be little different. (3) Phylogentic analyses showed that mus musculus be nearest to cricetulus griseus, nearer to sus scrofa and rabbit and be most far away from macaca fascicularis. (4) Based on the conservative sequence of 18S rRNA, an oligonucleotide fragment for RNAi was designed and synthesized, the corresponding RNAi vector was designed and constructed, which provide a basic for further studies on function of 18S rRNA and its roles in oocytes maturation and early embryonic development.