| Alburnus(Culter alburnus) is one kind of rare Chinese carps(Cyprinidae) freshwater fish, with high economic value. Xingkaihu alburnus own silvery white body, tender meat and delicious taste, its trait of excellent meat quality attracts widespread attention. However, the overfishing leads to natural resources dwindling and much tighter market demand. Therefore, alburnus predict a good prospect for artificial breeding. The quality, flavor and taste of fish meat are significant factors affecting the popularity of consumers. At present, there are more and more researches about inosine monophosphate(Inosine-5’-Monophosphate, IMP) content in muscle tissue from the molecular level. Many fish flavor candidate genes have been gradually uncovered, but few researches focus on variation of IMP content under different storage temperatures, IMP content during different growth stages and adenosine monophosphate deaminase(Adenosine 5’-monophosphate deaminase, AMPD1) gene related to IMP metabolism.In this study, by high performance liquid chromatography, under(20±1)℃, 4℃and-20℃ storage conditions, inosine monophosphate(IMP) content and its variation of age 2, 3, 4th instar Xingkaihu alburnus muscle tissue have been measured. According to Genbank known carps AMPD1 gene sequence, gene primers were designed. By using RT-PCR and RACE, partial c DNA sequence of AMPD1 was cloned AMPD1, and then we use the obtained sequence to design primers for amplifying other parts of c DNA sequence contained 3’UTR and 5’UTR, and the resulting sequences were spliced to obtain the complete sequence; by using quantitative PCR(Real-time PCR), we detected the expression of AMPD1 gene in Xingkaihu alburnus muscle tissue. Test results are as follows:(1) IMP content of 2-year-old age group was 3.4297mg/g, 2 and 3 years old age group were significantly higher(p <0.05). Under different three storage temperatures, IMP content of muscle tissue degraded faster under storage conditions of(20±1)℃; the IMP contents of specimens stored at-20℃ even maintained higher level and degraded slower than the fresh muscle tissues in specimens. Regardless of the influence of age and temperature change, IMP content also had relationship with storage time of muscle tissue. After reaching peak level, IMP content negatively correlated with storage time. It was recommended in low storage temperature and edible within 7 d.(2) Obtained the CDS region of alburnus AMPD1 gene and unknown area of the gene 3 ’end and the 5’ end by using RACE technology. Obtained 2592 bp c DNA sequence after sequence assembled, including encoding 717 amino acids and a termination codon of complete CDS sequences(2154bp), complete 3’UTR region(351bp) and 5’UTR region(87bp). Genbank accession number was KP735961.(3) Blast comparison displayed that the CDS region between alburnus AMPD1 gene and zebrafish(Danio rerio) showed a high homology of 89%. Amino acid homology comparison between AMPD1 gene and European flounder(Platichthys flesus) showed a high homology of 84%; with zebrafish(Danio rerio) showed a high homology of 93%, and the number of encoded amino acids was the same as zebra fish, both of them encoded 717 amino acids.(4) Analyzed the expression of AMPD1 gene in muscle tissues between 2, 3, and 4 age instar breeding Xingkaihu alburnus groups and 4 age instar wild groups. Real-time PCR results showed that the mRNA expression of 2 age alburnus AMPD1 gene was the highest, followed by the expression of the 3 age instar group, the former two were significantly higher than 4 age instar muscle tissues both the wild and breeding groups(p <0.05); between 4 age instar groups, the muscle tissue expression of wild groups was significantly higher than that of breeding groups(p <0.05). Consistent with existing research, AMPD1 gene expression decreased with age. AMPD1 expression quantity and IMP content were positively correlated. |
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