| Owing to the rich nutrition and good taste, Takifugu rubripes produces high economic efficiency.The tetrodotoxin (TTX) produced from genital gland and liver of Takifugu rubripes.Because of itsunique function of nerve block,the study of TXX becoming more meaningful.However, thanks tothe complicated and specific biological characteristics,the incidence rate of disease becomehigher.Immune system defend the living body from pathogenic bacteria,and it consists of immuneorgans, immune cells and many immune molecules. IL-7,IL-15,IL-15L and IRF2are the importantimmune molecules in the fish immune system.(1) The IRF2gene from Takifugu rubripes head kidney was cloned by rapid amplincation ofcDNA ends technique. Its full-length cDNA was1552bp, containing a936bp open reading frame(ORF), a187bp5’-untranslated region (UTR) and a429bp3’-UTR.The deduced amino acidsequence of IRF2consisted of311amino acid residues with a predicted molecular weight of35.5kDa, and the estimated isoelectric point was6.56. Maybe IRF2of Takifugu rubripes was thehydrophilic protein. IRF2had no signal peptide and transmembrane structure, it had aDNA-binding domain (DBD) and a interferon association domain (IAD2) at N terminus, atranscription repression domain (TRD) at C terminus. Takifugu rubripes IRF2had a highestsimilarity with S.chuatsi (79%). That supplied technical support for the gene function research inTakifugu rubripes.(2) According to the published IL-7,IL-15,IL-15L gene sequence on line and cloned IRF2genesequence in this study, pET32a-IL-7, pET32a-IL-15, pET32a-IL-15L, pET32a-IRF2andpET28a-IL-7recombinant plasmids were structured.They were transfered into E. coli BL21(DE3)and Rosetta (DE3) respectively and were over expressed when induced by IPIG. SDS-PAGEinitially indicated these genes were successfully expressed. The recombinant proteins werepurified by affinity chromatography on Ni-NTA resin. In addition, the optimum bacterial strain,IPTG concentration, induced time and induced temperature of each gene expression conditionwere found. And those offer technical support for mass production. The purified fusion proteinscan use to determine the biological activity and produce the vaccine for Takifugu rubripes.(3) Genomic sequences and EST sequences were acquired from NCBI.Then find theirmicrosatellite core sequence by SSR-Hunter software. Characteristics of35EST-SSR loci wereinvestigated using57Takifugu rubripes individuals. The number of alleles per locus ranged from2to7, The average number of effective alleles was3.3675.The observed heterozygosity rangedfrom0.2281to0.7368, average value was0.4924. while the expected heterozygosity ranged from0.4986to0.8235, average value was0.6859. The PIC values ranged from0.3712to0.7904,average value was0.6264.15loci conformed to the Hardy–Weinberg equilibrium,16lociproduced deviation. It possibly because that Takifugu rubripes group used in this study havegenetic relationship between the individual.(4) The correlation between microsatellite loci and growth trait were analyzed. Then thecorrelation between different genotypes of JX448611, JX448594, JX448602, AL837058,CA590514, CA332570, CA331902,CA331902gene loci and growth trait were analyzed.So the growth related genes maybe located in those loci. Growth related genes of Takifugu rubripeswould be obtained by analyzing the nearby gene sequence. And it can offer assist for molecularassisted selection in Takifugu rubripes. |
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