| Scophthalmus maximus belongs to the order Pleuronectiformes,family Bothidae,also known as turbot.It is a rare species of low-temperature commercial fish,native to Europe.China has now become the largest country breeding for turbot breeding worldwide.To keep fish alive without water,slow-cooling method is used to make fish remain in a dormant state.In this way,fish can be oxygenated and packed in a water-free sealed environment after leaving water environment and stored in an ice-temperature environment.After a period of time,the fish is placed in suitable low-temperature water environment for gradient warming to make it slowly get into normal living conditions.S.maximus is a cold water fish species that is an ideal material for the studies of waterless keep-alive technology.But the effect of various factors on the organism in the whole process is not yet clear.Therefore,the technologies such as biochemistry,proteomics,histology and transcriptomics are used in this study to explore the mechanisms of action of different oxygen concentrations on the organism of S.maximus during the process of slow cooling,cold acclimation and keeping alive.On this basis,the waterless keep-alive transport container for S.maximus is studied and developed.The main research results are as follows:1.After cold acclimation,the blood glucose,ATP,cortisol,and CAT,lysozyme,SOD,AKP,and ACP activity of S.maximus showed a significant rising trend,while the GOT and GPT activity decreased significantly.The two-dimensional protein electrophoresis analysis was adopted to screen out a total of 16 differentially expressed proteins related to the physiological function of S.maximus,including 7 up-regulated proteins such as complement component c3b,tandem duplicate 1 precursor,claudin-10A and pericentrin,and 9 down-regulated proteins such as brain creatine kinase,serine/threonin protein kinase ARAF,actin,alpha and cardiac muscle 1a.Based on the real-time qPCR detection analysis,it can be found that the amount of gene expression of 3 proteins,namely,pericentrin,beta-enolase and Enolase 1(alpha),showed a significant up-regulation trend;the amount of gene expression of 4 proteins,namely,complement component c3b,tandem duplicate 1 precursor,claudin-10A,phosphoglycerate mutase 2 and brain creatine kinase,showed a significant down-regulation trend.This was consistent with the analysis results of proteomics,which verified the accuracy and reliability of the results.2.The gill filaments were histologically observed at 6 h and 12 h after treatment with different oxygen concentrations in the waterless keep-alive process of S.maximus.The results showed that the gill lamellae among gill filaments in the air treatment group(group A)showed obvious morphologic changes,together with shrinking and puckering phenomena.At the same time,the thickness,length,perimeter and cross-sectional area of base of gill lamellae were measured.Compared with the control group(CK),the oxygen treatment group(group O)showed a significant increase(P<0.01)in the perimeter at 6 h after waterless keep-alive treatment,reached(356.29±32.76)?m,and returned to the normal level at 12 h.The thickness,perimeter and cross-sectional area of base in the group A showed a significant difference(P<0.05),and the length showed no significant difference(P>0.05).The group A decreased to(2.96±0.048)?mol/g at 12 h after waterless keep-alive treatment,which was significantly lower than that of the control group(P<0.05).For the group A,the concentration of glucose,cortisol and urea nitrogen in the serum of S.maximus increased obviously at 6 h and 12 h after waterless keep-alive treatment,with extremely significant difference(P<0.01).The serum CHE(cholinesterase)concentration of S.maximus in the two treatment groups had no significant difference in the whole waterless keep-alive process.3.For the purpose of systematically understanding the transcriptome characteristics of effect of oxygen concentration on gill tissue of S.maximus in the waterless keep-alive process,the gills of the control group(slowly cooled to 20C),group A and group O were selected in this study.A total of 9 cDNA sequencing libraries were constructed for RNA-seq sequencing and bioinformatics analysis.A total of 59 Gb data were obtained by sequencing,with a total length of 168,973,791bp,average length of 1,61 lbp,N50 of 3,127bp and GC content of 49.41%.After the removal of gill lamellae and quality screening,104,886 unigenes were assembled from high-quality sequences,among which the maximum unigene was 23,178bp and the minimum one was 201 bp.Blast aligning was conducted between the obtained 104,886 unigenes and Nr of NCBI.Among them,65,000(62.3%)unigenes in NR,78,000(74.9%)unigenes in NT,59,000(55.9%)unigenes in Swissprote protein sequence database,28,000(26.7%)unigenes in COG,58,000(54.4%)unigenes in KEGG,6,000(6.0%)unigenes in GO and 48,000(45.9%)Interpro were functionally annotated.After comparing the seven databases,a total of 81,186 unigenes were annotated,accounting for 77.40%of the total number.To further understand the integrity of transcriptome data,the obtained 104,886 unigenes were annotated with COG function.The 104,886 unigenes were annotated into 25 families with different functions.It's found by comparison that there were at most 10,219 unigenes related to general function prediction only function,4,771 unigenes related to transcription function,and 4,725 unigenes related to replication recombination and repair function.In order to further describe the unigenes,the obtained unigenes were annotated with GO.104,886 unigenes were annotated into 3 major types and 57 categories of cell components,biological processes and molecular functions in the GO database.Among them,biological processes accounted for 25 categories,cell components accounted for 18 categories,and molecular functions accounted for 14 categories.There were at most 3,225 cellular process unigenes in the biological process,accounting for 3.07%.There were 2,445 unigenes related to cell functions in cell components,accounting for 2.33%.There were at most 2,964 unigenes in the bing category of molecular functions,accounting for 2.83%.The significant difference of RPKA value(P<0.05)was used to calculate the different amounts of gene expression for the different groups,and 2-fold method was used for data statistics and analysis of differential gene among different groups.A total of 647 differentially expressed genes(DEGs)were obtained from the air and oxygen groups,among which 435 genes were significantly up-regulated while 212 genes were significantly down-regulated.A total of 662 DEGs were obtained from the control and air groups,among which 322 were significantly up-regulated and 340 were significantly down-regulated.A total of 441 DEGs were obtained from the control and oxygen groups,among which 244 were significantly up-regulated and 197 were significantly down-regulated.The number of DEGs(441)between the control and oxygen groups was far less than that(662)between the control and air groups,and also less than that(647)between the air and oxygen groups,suggesting that oxygen was more conducive to the keep-alive transportation of S.maximus than air.4.In order to put the waterless keep-alive transportation technology for S.maximus into practical application,the waterless keep-alive transport container for S.maximus has been designed and developed based on the early technology exploration.The container is mainly composed of hardware equipment and software monitoring system.The hardware equipment includes diesel generating set,refrigerating unit,oxygen concentration detector,carbon dioxide concentration detector,air solenoid valve,pressure probe,temperature detector,humidity detector,goods shelf,tray,and container body.The software monitoring system includes programmable logic controller(PLC),temperature monitoring system,oxygen concentration monitoring system,carbon dioxide concentration monitoring system,pressure and humidity detection system.The touch screen is used to monitor and control the temperature,oxygen concentrations,carbon dioxide concentrations,humidity and pressure in the keep-alive transport container. |
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