| The frequent outbreak of prawn viral disease has caused huge economic losses to prawn breeding industry all over the world. In vitro immortalized shrimp cell line can provide us a useful tool for the study of infection mechanism of shrimp virus as well as the preparation of antiviral medicines and vaccines. Transforming immortalization-related genes into shrimp primary cells is an efficient method for establishing continuous shrimp cell line. However, up to date, no immortalized shrimp cell line is available because of the absence of active mitosis and difficulty to be transformed in the in vitro cultured shrimp cells. Thus, efficient gene transferring technology for shrimp cells is in urgent need. Attempts have been made on the genetic transformation of both living penaeid shrimps and in vitro cultured shrimp cells using non-viral and retroviral vectors. Unfortunately, high lethality rate and low transformation efficiency are still bottleneck problems to deal with.Thus the present study has attempted (1) to clone the promoter region of shrimp translationally controlled tumor protein (TCTP) gene (Ptctp) and shrimp white spot syndrome virus (WSSV) early gene iel (Pie1). These two promoter regions were then inserted into viral or nonviral vectors to construct recombinant expression plasmids containing double promoters. And then their promoter activity in mammalian cells, fish cells and shrimp primary culture cells were examined and compared to seek for shrimp cell-specific active promoters; (2) to clone and insert SV40 enhancer into the reporter vectors constructed for the gene transfer of shrimp cells and assess its potential application in promoting foreign gene expression in nondividing shrimp cells by evaluating its activity of DNA nuclear targeting using mammalian cells and shrimp primary culture cells; (3) to clone the gene fragments of vp19 and vp28, two key envelope proteins of shrimp WSSV, and construct their corresponding eukaryotic expression vectors. These two envelop proteins were then both assembled into packaged virion particles by co-transfection method to increase the shrimp cell-tropism of the mammalian-derived retrovirus gene transferring system; (4) to predict potential microRNAs and target genes from the transcriptome sequences of shrimp early embryos by bioinformatic approaches. microRNAs related to shrimp embryo development and cell cycle were further explored and discussed.In the analysis of TCTP gene promoter (Ptctp) activity, two 612 bp TCTP promoters were successfully cloned from Metapenaeus ensis and Marsupenaeus japonicus and named as Pme-tctp and Pmj-tctp, respectively. The binding sites for transcription factors in these two promoters were analyzed by TFsearch and Match softwares. Meanwhile, we also successfully cloned the ORF sequence of enhanced green fluorescent protein (eGFP). Using retroviral reporter vector of pMCs-GFP as basic plasmid which has viral long terminal repeated elements (LTR) as promoter (PLTR), the above two promoters were directionally inserted into it at the site between 5’LTR and GFP/eGFP gene, or the GFP gene was replaced by eGFP gene through double endonuclease digestion method, and constructed five retroviral expression vectors as followed:pMCs-PLTR-eGFP, pMCs-PLTR-Pme-tctp-GFP, pMCs-PLTR-Pmj-tctp-GFP, pMCs-PLTR-Pme-tctp-eGFP and pMCs-PLTR-Pmj-tctp-eGFP. When these five expression vectors were transformed into mammalian cells of Plat-GP by lipofection method, obvious green fluorescence was observed in all the transformed cells under inverted fluorescence microscope. Of those vectors, pMCs-eGFP which contained single promoter of PLTR produced the stronger green fluorescence than the other four vectors which contained double promoters of PLTR-Pme-tctp/mj-tctp, indicating that LTR promoter has strong activity in mammalian cells and the insertion of Pme-tctp and Pmj-tctp pushed LTR promoter away from the initiation site (ATG) of downstream gene and thus inhibited the activity of LTR promoter. When the above five vectors were transferred into shrimp primary cells by liposome method, no green fluorescence was observed in the pMCs-eGFP-transformed shrimp cells, but obvious green fluorescent was observed in the other double-promoter vectors-transformed shrimp cells, indicating that the promoter Pme-tctp and Pmj-tctp could successfully drive foreign gene to be transcribed and translated in shrimp primary cells. The results of PCR and RT-PCR analysis confirmed the fluorescence observation results, that is, the vector plasmids could be introduced to shrimp cells by liposome method but only plasmids with double promoters could be transcribed into mRNA.In the improvement of the shrimp cell-tropism of the mammalian-derived retrovirus gene transferring system, the full-length ORF of vp19 and vp28 genes were successfully cloned from shrimp WSSV. The obtained ORF fragments of vpl9 and vp28 genes are 366 bp and 615 bp in size, encoding 121 and 204 amino acids, respectively. Then these two genes was directionally inserted into the eukaryotic expression vector of pcDNA3.1-V5/HisA flanking with BamHI and EcoRI to construct recombinant plasmids of pcDNA-VP19 and pcDNA-VP28. The contribution of VP19 and VP28 to the shrimp cell-tropism of the retrovirus gene transferring system was analyzed by co-transfecting the following different sets of viral plasmids into packaging cells, followed by retroviral packaging, virus titer measurement and infection of shrimp cells. The four co-transfection combinations were as followed:(1) pMCs-PLTR-eGFP and pCMV-VSV-G; (2) pMCs-PLTR-Pme-tctp-eGFP and pCMV-VSV-G; (3) pMCs-PLTR-eGFP, pCMV-VSV-G, pcDNA-VP19 and pcDNA-VP28; (4) pMCs-PLTR-Pme-tctp-eGFP, pCMV-VSV-G, pcDNA-VP19 and PcDNA-VP28. It was found that, obtained viral titer was calculated as (1.9-2.1)×108TU/ml. At 48 hours post-infection, no green fluorescence signals were detected in the shrimp cells infected by viral concentrates not co-packaged with VP19 and VP28 or the viral vectors not driven by Pme-tctp promoter. Only the viral concentrates packaged by combination of PCMV-VSV-G, pcDNA-VP19, pcDNA-VP28 and pMCs-PLTR-Pme-tctp-eGFP could successfully mediate the reporter gene expression in shrimp cells. Although the number of positive clones was scare and the fluorescent intensity was weak, it was clearly indicated that the introduction of VP19 and VP28 into the retroviral system obviously improved the shrimp cell-tropism of this retroviral gene transferring system.In order to investigate the mechanism of contribution of VP19 and VP28 to the shrimp cell-tropism of the retrovirus gene transferring system., the amino acid sequences of VP19 and VP28 were analyzed by softwares of DAS, Toppred2, Tmpred, and HMMTOP to identify the potential hydrophobic regions and transmembrane regions (TMDs). It was found that the residues from 36 to 55 and from 95 to 114 of VP19 protein, residues from 7 to 27 of VP28 protein were potential TMDs with high hydrophobility and enough length to span a lipid bilayer. To investigate the cell membrane localization of over-expressed VP19 and VP28 in packaging cells of Plat-GP, reporter genes of eGFP and mCherry were respectively fused to the C-terminal of stop codon TAA-deleted vpl9 and vp28 to construct two fusion protein expression vectors of pcDNA-VP 19-△TAA/eGFP (protein VP19 marked with eGFP) and pcDNA-VP28-△TAA/mCherry (protein VP28 marked with mCherry). When the above two fusion protein vectors were transformed into Plat-GP cells by lipofection method, fourty eight hours later, membrane-anchored location of VP19 and VP28 was observed under the inverted confocal fluorescence microscope, inferring the improved shrimp cell-tropism of this system was attributed to the introduction of WSSV envelop protein VP19 and Vp28.The promoter activity of Ptctp in shrimp cells was not high enough, thus more efficient promoter for shrimp cells were needed. For this, we further cloned the promoter of early gene iel from shrimp WSSV with 2000 bp in size, named as Pie1-2000, and then directionally inserted it into vector pcDNA3.1/V5-HisA (with CMV promoter of Pcmv) to construct two expression vectors of pcDNA-Pcmv-Pie1-2000-eGFP and pcDNA-APcmv-Pie1-2ooo-eGFP (Pcmv was deleted), respectively. The above two expression vectors were then transferred into mammalian cells of Plat-GP and Neuro2A, fish cells of FG and shrimp primary culture cells by lipofection method, respectively. The results showed that, plasmid pcDNA-Pcmv-Pie1-2000-eGFP mediated weak GFP expression in Plat-GP and Neuro2A, suggesting that the insertion of Pie1-2000 have caused an inhibitive effect on the activity of CMV promoter. Plasmid pcDNA-APcmv-Pie1-2000-eGFP, which has only one promoter of Pie1-2000, mediated similar weak expression of green fluorescent protein, indicating the activity of Pie1-2000 was also very low in mammalian cells. In FG cells, just like plasmid pcDNA-Pcmv-eGFP which has only one CMV promoter, the above two expression plasmids containing Pie1-2000 successfully mediated expression of reporter genes, inferring that both Pcmv and Pie1-2000 have a promoter activity in FG cells. However, green fluorescent signal failed to be detected in the shrimp primary cells transfected with pcDNA-Pcmv-Pie1-2000-eGFP or pcDNA-△Pcmv-Pie1-2000-eGFP, suggesting that Pie1-2000 have no or extremely low activity in shrimp cells. The results of PCR and RT-PCR analysis showed that although the expression vectors were introduced into shrimp cells by liposome method, mRNA transcription mediated by Pie1-2000 was in extremely low level which is consistent with those detected under microscopy.Due to the low promoter activity of Pie1-2000, we further analyzed the transcription factor binding sites of Pie1-2000 and attempted to investigate the activity of truncated iel gene promoter region with 504 bp in size (at positions from-1 to-504), named as Pie1-504.Then we cloned the Pie1-504 and ORF of red fluorescent protein(mCherry) gene and inserted them into pcDNA3.1-V5/HisA to construct two plasmids of pcDNA-Pcmv-mCherry and pcDNA-Pcmv-Pie1-504-mCherry. SV40 enhancer sequence with 72 bp in size was previously reported as DNA nuclear targeting sequence. It was also cloned and inserted into the plasmids of pcDNA-Pcmv-mCherry and pcDNA-Pcmv-Pie1-504-mCherry at the site located after mCherry gene and another two plasmids of pcDNA-Pcmv-mCherry-SV40 and pcDNA-Pcmv-Pie1-504-mCherry-SV40 were constructed. These four constructed expression vectors were then transferred into Plat-GP, Neuro2A, FG cells and shrimp primary culture cells by lipofection method to analyze the activity of Pie1-504 and SV40 enhancer. It was found that, robust expression of red fluorescence were observed in all the transfected Plat-GP, Neuro2A and FG cells, demonstrating that Pie1-504 did not cause obvious inhibition on the activity of CMV promoter in the double-promoter plasmids. However, in shrimp cells, except for the shrimp cells transfected with pcDNA-Pcmv-mCherry, sporadic red fluorescent signals were detected in others under microscopy.Compared with samples transfected with pcDNA-Pcmv-mCherry, sporadic red fluorescent signals detected in shrimp cells tranfected with pcDNA-Pcmv-mCherry-SV40 indicated SV40 enhancer can work as DTSs in shrimp cells and promote nuclear entry of foreign plasmids.Likewise, compared with samples transfected with pcDNA-Pcmv-mCherry, pcDNA-Pcmv-Pie1-504-mCherry also mediate bits of red fluorescent signals, indicating successfully transcription and expression of reporter gene mediated by Pie1-504 in shrimp cells but in low efficiency.The results of PCR and RT-PCR analysis also showed that expression plasmids can be introduced to shrimp cells by liposome method and plasmids with double promoters or SV40 enhancer can also be transcripted but not for pcDNA-Pcmv-mCherry just with Pcmv, which is consistent with those detected under microscopy. Moreover, the amplifying band derived from sample transfected with pcDNA-Pcmv-Pie1-504-mCherry without SV40 enhancer sequence was apparently weaker than SV40 enhancer-containning plasmids of pcDNA-Pcmv-Pie1-504-mCherry-SV40, suggesting that SV40 enhancer sequence have a certain DTS activity in shrimp cells.In order to verify the above conclusion, the fluorescence micrographs of Neuro2A cells transformed by above-mentioned three GFP plasmids and four mCherry plasmids were further analyzed using ImageJ software to investigate the positive signal rate (PSR) and average fluorescent intensity for each positive cell, which was presented with the average corrected total cell fluorescence (CTCF). Compared with plasmid pcDNA-Pcmv-eGFP, Pie1-2000 did generate a considerable space steric hindrance to CMV promoter, which led to significant decrease of PSR and CTCF values (t-test, p<0.01) for pcDNA-Pcmv-Pie1-2000-eGFP and pcDNA-△Pcmv-Pie1-2000-eGFP. Compared with pcDNA-Pcmv-m Cherry and pcDNA-Pcmv-mCherry-SV40, The CTCF values for pcDNA-Pcmv-Pie1-504-mCherry and pcDNA-Pcmv-Pie1-504-mCherry-SV40 indicated that Pie1-504 exerted certain space steric hindrance to Pcmv but it was obviously less than Pie1-2000 did. And also, the SV40 enhancer-containing plasmids of pcDNA-Pcmv-mCherry-SV40 and pcDNA-Pcmv-Pie1-504-mCherry-SV40 generated higher PSR value than pcDNA-Pcmv-mCherry and pcDNA-Pcmv-Pie1-504-mCherry did at 24 and 48 hours post-transfection, which confirmed the DTSs activity of SV40 enhancer again.For microRNA mining from shrimp early embryos, using transcriptome sequences generated by Illumina platform as reference dataset, we employed BLASTN and RNAfold to carry out homologous search and secondary structure analysis. Finally twenty-one conserved miRNA sequences were predicted, belonging to 19 families:mja-miR-14,44,242,287,317,927,966,969,1014,1175,2491,2731, 2774,2788,3338,3885,4961,6489 (6489-5p/3p) and 6493 (6493-5p/3p). In order to validate the existence of predicted miRNAs sequences and compare miRNAs expression patterns in different developmental periods, RT-qPCR was performed to investigate miRNAs expression profiles at gastrula and nauplius stages. The results showed that fifteen of them could be amplified and got positive bands through gel electrophoresis. Compared with expression profiles at gastrula stage, the miRNAs expression at nauplius stage presented a different profile. However, mja-miR-3885 was expressed in surprisingly high level at both gastrula and nauplius stages. The results suggested that mja-miR-3885 may play a key role in the shrimp development process.Using all unigene sequences as reference dataset, target genes for miRNAs were predicted by miRanda algorithm and another set of strict criteria and finally a total of 120 mRNA sequences were predicted as targets. All the predicted target genes were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) database for analysis. The results showed that all target genes were assigned to 11 categories at cellular component level,10 categories at molecular functions level and 18 categories at biological process level. KEGG pathway analysis was performed for pathway enrichment. The results showed that 19 targets were involved in 31 metabolic pathways. The main metabolic pathways involved included RNA degradation, Wnt signaling pathway, cell cycle and mineral absorption and so on, which are all crucial process during the development period. In the process of targets annotation, some genes were found to be closely related to developmental behavior and immune activities. A total of eleven sequences were collected from the targets pool to form this cluster. In this paper, seven of them were abstracted for RPKM analysis and RT-qPCR was performed to detect their expression abundancy. Taken together of the different expression profiles of miRNAs in different stages with the significant different expression level of target genes, we assumed that there must be a very complex miRNA:mRNA regulation network involved in the delicate control of the development process.Taken together, in this study, the activity of promoters of Ptctp from shrimp TCTP gene and Pie1 from shrimp WSSV iel gene as well as DTS activity of SV40 enhancer was explored in shrimp primary culture cells systematically. It is found that both promoters can mediate forgeign gene expression in shrimp cells, but the efficiency is low and SV40 enhancer can improve nuclear entry efficiency of vectors in shrimp and Neuro2A cells. Noteworthy, we have successfully established a shrimp cell-tropic retroviral expression system. The improved shrimp cell-tropism of this system was attributed to the introduction of WSSV envelop protein VP19 and Vp28. By bioinformatic approaches, twenty-one microRNAs and one hundred and twenty target genes were found from the transcriptome of shrimp early embryos.All the obtained data in the present study have laid a technical foundation for the future work on the technologies of foreign gene transfer and expression in shrimp cells and even transgenic live shrimps. |
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