| Penaeus monodon is the member of crustacean, decapod, natantia, penaeidae, and shrimp genus. It is the largest species of shrimp, with its high economic and natural nutritional value [1]. However, during its breeding process, broodstock breeding technology has been a very important limiting factor [2, 3]. With the development of molecular biology techniques, there are growing concerns about the function of cell cycle genes of crustaceans, especially shrimp [4]. Recently, molecular mechanisms of cell cycle regulatory proteins have been researched [5]. In the study, cell cycle-related protein family genes relating to ovarian development also become a hot topic [6, 7].In this study, on the molecular level and protein level, Penaeus monodon(Pm-cyclin Y) gene were studied to explore the relevance of Pm-cyclin Y and black tiger shrimp ovarian development, to the molecular mechanisms regulating ovarian development monodon enrich theory. In addition, based on the existing research of our laboratory Penaeus monodon cyclin E(Pm-cyclin E), Pm-cyclin E were further explored in order to lay the foundation for protein interaction studies of Pm-cyclin E. The main contents of the study are summarized as follows: firstly, high-throughput sequencing of Penaeus monodon established EST library, screened cyclin Y EST sequences associated with ovarian development monodon; secondly, at the gene level: primers were designed using the SMART-RACE technique cloned c DNA sequences of full-length gene and then bioinformatic analysis; and specific primers were designed using the genome walking technique to get the whole genome sequence of the gene of interest, and finally, bioinformatics analysis of all the results were made. The expression of target gene in 10 organizations of Penaeus monodon, including ovary, liver, eyes, etc., tongether with ovaries at different developmental stages were studied by using real-time PCR technology, then analysed experimental results by using statistical methods. All the study explored the relationship between the target gene and ovarian development monodon at the m RNA level. At the protein level, constructed the recombinant plasmid Pm-cyclin Y carrying p ET21 a, and found the optimized prokaryotic expression conditions, and then induced recombinant protein; purified protein by nickel affinity chromatography, detected until it reaches a predetermined concentration, then sent company to make antibody; antibody was used for the immunoblot and immunohistochemistry experiments during the black tiger shrimp ovarian development phases, so as to achieve the target of finding the relationship between ovarian development the target gene on protein level. In addition, for Pm-cyclin E, combined with existing researches and our laboratory studies, we added the authentication of the m RNA levels in the black tiger shrimp ovarian development, and at the protein level, we did prokaryotic expression and immunohistochemistry, with pull-down technique, we made a preliminary exploration in Pm-cyclin E protein interactionsThe results are summarized as follows:1. Pm-cyclin Y gene c DNA full length was 1576 bp, which contains 108 bp of the 5 ’untranslated region(UTR) and 439 bp of the 3’ untranslated region(UTR) and 1029 bp open reading frame(ORF), and it can encode 342 amino acids. Bioinformatics analysis showed that the amino acid sequence encoded by a conserved cyclin box(cyclin box) homology domain(172-257aa), the predicted molecular weight of about 38.7k D, theoretical isoelectric point 6.64. Pm-cyclin Y and Daphnia pulex(Daphnia pulex) cyclin Y has a high degree of homology(67% similarity). Blastx analysis, The predicted Pm-cyclin Y amino acid sequences has a very high homology with other known cyclin Y; Signal P 3.0 program analysis, Pm-cyclin Y does not contain a signal peptide sequence; Net NGlyc1.0 Server program analysis, Pm-cyclin Y glycosylation sites(N-Glycosylation) does not exist; Net Phos2.0 prediction results show: Pm-cyclin Y contains a total of 24 potential phosphorylation sites, including 17 loci Ser, Thr 2 sites and five Tyr sites. The prediction Pm-cyclin Y secondary structure encoded protein showed: protein Pm-cyclin Y encoding 13 α helix, 54% of the total structure; 1 β-sheet, 1% of the total structure, and 47% random coil structure. Clustalx1.83 software and Bio Edit software, Pm-cyclin Y multiple sequence alignments showed, Pm-cyclin Y and Hornet, and Daphnia pulex high degree of similarity, similarity were 72%, and 67%. MAGA6.0 software phylogenetic results show, Pm-cyclin Y encoded protein P.monodon and Hornet, Daphnia pulex and Drosophila clade, and cyclin Y cluster encoding protein species like mammals, fish, reptiles and other representatives.2. Real-time quantitative PCR showed that Pm-cyclin Y m RNA was significantly higher than other tissues(P <0.05) in the expression of ovarian tissue; and during the ovarian five different developmental stages, the highest expression in stage III.3. According to the known EST sequences, designed Pm-cyclin Y prokaryotic expression primers and then cloned cyclin Y open reading frame(ORF) successfully from Penaeus monodon ovary c DNA, and connected with the p ET-21 a vector, transforming E. coli BL21, then the recombinant protein was induced by IPTG and purified successfully, after BCA kit test protein concentration standard, the recombinant protein as antigen was sent to company to made antibody(designated R-CYCY).4. Prepare for paraffin sections of P.monodon ovarian development organization. And R-CYCY antibodies for immunohistochemistry experiments showed phase I(oogonia stage) and Phase II(nuclear chromatin period) the signal,occurred early in yolk oocytes,III period(around the nucleus Jen cytoplasmic phase) and stage IV(yolk sac) showed a a strong signal in primary oocytes, while the nucleus also had a weak signal, V period(maturity) signals mainly in the cytoplasm of the oocyte in stick cortex and nucleus signal is not detected.5. For Pm-cyclin E,researches were successfully studied the m RNA and protein levels during ovarian development stages of Penaeus monodon, the results showed that on the m RNA level, Pm-cyclin E expression was highest in stage III ovarian, and significantly higher than others; at the protein level, Pm-cyclin E was successfully carried the plasmid p RSET and by inducing expression, protein purification and antibody preparation and other experiments, finally we got Pm-cyclin E antibody.And the immunohistochemistry results showed that: Phase I and Phase II of vitellogenesis oocyte cytoplasm early in the nucleus signal, III and IV primary oocytes in a strong signal, no signal in the cytoplasm, V phase signal in oocytes cytoplasm and nucleus are present, but there is no signal in the cortex rod.The results were different from Pm-cyclin Y, which may be related to their phase, suggesting that they are cell cycle-related proteins. |
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