| As an important freshwater shrimp culture species,Macrobrachium rosenbergii is also one of the main shrimp cultures in China.Whether it is live shrimp or shrimp meat,it is a common daily food.The research on white tail disease of Macrobrachium rosenbergii is not only related to the shrimp farming industry,but also closely related to the food safety of consumers.In this study,a convenient detection method for on-site rapid detection was developed from the existing diagnostic techniques on the basis of the pathogenicity of Macrobrachium rosenbergii nodavirus(MrNV).A practical high-sensitivity rapid diagnostic kit was then designed.At the same time,the reverse genetics techniques were employed to construct the reverse genetic vector of MrNV and extra small virus(XSV).The mechanism of replication mechanism,pathogenicity of XSV and MrNV were analyzed by RNA interference technique in cell or vivo,so as to elucidate the pathogenic mechanism of MrNV and to lay the foundation for immune antigen molecule and drug treatment.The deferentially expressed genes and proteins responding to the MrNV/XSV stress were searched,screened and identified by transcriptome technique and differential proteomics technique;a high-throughput screening and evaluating method was constructed in vitro to choose prawn immune enhancers from fungi,bacteria,animals and plants.Through the above research,we promoted the early diagnosis of MrNV and XSV before its entering the market.This research will provide solid data for effectively avoiding the outbreak of white-tailed disease of Macrobrachium rosenbergii,lowering the culture risk,reducing the economic loss of prawn farmers and preventing the diseased prawn entering into the food market.The specific research contents are as follows:(1)Construction of the rapid diagnosis technology system for MrNV/XSVA rapid detection method for white-tailed disease in the Macrobrachium rosenbergii was established by loop-mediated isothermal amplification(LAMP)and lateral flow dipstick(LFD).The results showed that the detection of a single sample using the duplex RT-LAMP-LFD method for MrNV/XSV can be completed within 1 hour,and the sensitivity can reach about 1.0pg,which is 100 times that of conventional RT-PCR.On the other hand,nucleic acid sequence-based amplification(NASBA)reaction system of MrNV was established and optimized by using a nucleic acid test paper labeled with a biotin probe to detect NASBA reaction products.While the detection of a single sample using the NASBA-LFD method can be completed within2 hours,and the sensitivity can reach about 1.0 fg,which is 10~4 times that of conventional RT-PCR.Compared with the LAMP method,NASBA method costs more than twice as high,but has lower false positive and secondary pollution.At the same time,a diagnostic kit was designed and assembled for convenient on-site detection.Two diagnostic kits were developed according to the two detection technologies,which can be selected to use according to different demand.(2)Prototypical expression and antibody preparation of capsid protein of MrNV.According to the whole genome sequence analysis results of MrNV,full-length genomic cDNA of MrNV capsid protein CP43 gene was obtained by RT-PCR,then cloned to the appropriate vector to get the target protein by inducing expression and purification.The prepared polyclonal antiserum of the CP43 gene had strong specificity testified by Western blot,which was then used for immunofluorescence localization in sf9 cells.(3)Transcription and expression of MrNV and XSV in Macrobrachium rosenbergii of different agesTranscription expression levels of MrNV and XSV were compared at the cellular and in vivo levels.It was found that the expression level of XSV was significantly higher than that of MrNV at the early stage of infection.The interest gene can be detected in various tissues of the adult shrimp,but the expression level is relatively high in hemolymph,hepatopancreas and muscle tissues.Histopathological and ultrathin histological study showed that virus particles existed in tissues in the form of inclusion bodies,damaging to cells and fibrous tissues of the body and causing pathological changes.(4)Study on transcriptome of Macrobrachium rosenbergii larvae infected with MrNV/XSV-chin.Total RNA was extracted from Macrobrachium rosenbergii larvae before and after the MrNV/xsv-chin infection for transcriptome sequencing analysis.40.67 Gb Clean Data was gained from two groups of samples with three biologically repetitive in each group after sequencing.The Clean Data of each sample reached 6.16 Gb,and the base percentage was92.56%and above.A total of 97,770 Unigene were obtained after assembly.Among them,there are 16,990 Unigene with a length of more than 1 kb.A total of 17,428 Unigene annotation results were obtained by performing functional annotation on Unigene,including comparison with NR,swiss-prot,KEGG,COG,KOG,GO and Pfam databases.A total of 14,854 SSR markers were obtained from the Unigene library.161 deferentially expressed genes were found after the gene expression level analysis in each sample,among which 30 were up-regulated and131 were down-regulated.According to bioinformatics annotation of deferentially expressed genes,50 genes that may be involved in various metabolic and immune pathways were selected for fluorescence quantitative PCR verification,of which only 9 were up-regulated and 41 were down-regulated,and the relative expression level was basically consistent with the prediction.(5)Proteomics study on MrNV/XSV-chin infection of the Macrobrachium rosenbergii larvaeTwo-dimensional electrophoresis-mass spectrometry(MrNV)was used to compare and analyze the protein of susceptible larvae and normal larvae,so as to identify the existence of differential expressed proteins.By using the traditional bidirectional electrophoresis technique,35 significantly deferentially expressed proteins were screened from the symptomatic Macrobrachium rosenbergii seedlings at 7 days after infection and their negative controls.These protein spots were selected for mass spectrometry analysis,and 19 protein spots were successfully identified.13 primers could be designed according to the amino acid sequence of the protein database and the mass spectra.Among the 13 protein sequences that were compared in the NCBI protein database,the amino acid coverage was very low,in which the highest was33%,while the lowest was only 2%.The results of fluorescence quantiative PCR verified the significant differences in expression levels of the 13 gene.10 genes were significantly up-regulated after challenge,and only 3 genes were significantly down-regulated.(6)Screening and evaluation of anti-disease immune enhancer for Macrobrachium rosenbergiiThe prawn immune enhancers were screened in vitro.Dozens of immune enhancers from different plants,fungi,bacteria and so on were screened.After optimization,enhancers was added to the feed for feeding Macrobrachium rosenbergii.Immune preparations were screened out after the detection of phenol oxidase,alkaline phosphatase,peroxidase,superoxide dismutase and blood cell phagocytosis from the hemolymph collected at different time.Through comparison of non-specific immune indicators before and after infection,two groups of samples were selected for high-throughput measurement of small RNA to obtain miRNA with significant differences in expression.The immune effect of immune enhancers was preliminarily evaluated by fluorescence quantitative PCR using miRNA with significant expression.3 microRNA molecules(bta-mir-2478,mu-mir-3968 and has-mir-7975)were successfully obtained for the evaluation of immune effect. |
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