| Macrobrachium nipponense, commonly known as shrimp, belongs to Crustacea,Decaplda, Palaemonidae, Macrobrachium,which is widely distributed in our country's fresh waters. Macrobrachium nipponense is one of the important aquaculture species of China's freshwater shrimp. Growing disease problem in shrimp farming,become an important problem restricting the develpment of aquaculture.This problem need to solveProphenoloxidase activating system--proPO, invertebrate innate immune system plays an important role in the immune system. prophenoloxidase-activating factor, PPAF is an important phenol oxide plasminogen activator and its cofactors serine homolog activated can directly affect the vitality and the body's immune defense mechanism phenol oxidase.These immune-related factors are how to resist the invasion of pathogenic microorganisms, how to participate in immunization activities of the body, has been widespread concern, this study first cloned Macrobrachium nipponense full length PPAF cDNA by RACE method; by semi-quantitative PCR, analyzes the gene expression in different tissues, the results are as follows:We present full-length PPAF cDNA. sequence length 1261 bp, including 35 bp of 5'UTR,893 bp ORF, 316 bp of 3'UTR.Coding region of 271 amino acids, the molecular weight of the protein prediction 30.389.86 kD.Isoelectric point is 9.727,Which contains 34 basic amino acids(K, R), 18 acidic amino acids(D, E), 94 hydrophobic amino acids(A, I, L, F, W, V), 75 polar amino acids(N, C, Q, S, T, Y).Analysis of the composition of the open reading frame found that G, C content bases(51.09%) higher than the A, T content bases(48.91%).Analysis of the amino acid sequence of the gene Macrobrachium nipponense PPAF domains, found in a region of about 84-277 trypsin domain, there is a family of trypsin-like serine protein domains in the 77-278 region.Phylogenetic tree analysis showed that PPAF Gene of the Macrobrachium nipponensehave the highest homology with the Fenneropenaeus chinensis,lowest homologywith silkworm and bollworm.By semi-quantitative PCR analysis the different tissues of healthy Macrobrachium nipponense, PPAF gene expression levels were significantly different(P<0.05), higher expression of immune-related organizations hepatopancreas, gills, blood,lower expression in muscle tissue, testis, ovary.After challenged with A.hydrophila, the expression level of hepatopancreas, gills and blood changes in different periods, with the?-actin gene as a control group standards, detecting the expression variation of different periods.After challenged with A.hydrophila 72 h later, mRNA gene expression level of PPAF in hepatopancreas, gills, blood tissue were significantly different(P <0.05).The group which challenged with A.hydrophila 3 hours changed significantly stimulation, the PO activity increased 4.23 times, comparing with the saline injection group were significantly improved.Significant changes A.hydrophila bacteria PO activity after injection showed that bacteria can trigger prophenoloxidase activation system into PO. Macrobrachium nipponense Prophenoloxidase system can resist the invasion of external bacteria, it explains Prophenoloxidase system plays an important role in the immune system of shrimp.PPAF is a key factor of the proPO. And it plays an important role in Macrobrachium nipponense immune system.This study of the gene expression pattern in immune PPAF tissue infections A.hydrophila after the revelation, for further study nipponense against bacterial diseases of the immune defense mechanisms of the foundation.The study of the PPAF has a great significance and theoretical value in Immune Mechanism of the Macrobrachium nipponense. |
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