Expression characteristics of ADSL gene in pectoral muscle tissue of Chishui black-bone chicken and the construction of ADSL gene interference vector

【Objective】This experiment was carried out to investigate the expression characteristics of adenylosuccinate lyase gene(ADSL) in the pectoral muscle tissue of Chishui black-bone chicken and to construct the interference vector of the target gene,.which provided a theoretical reference for an in-depth study of the expression pattern of inosine monophosphate acid(IMP) in the muscles of livestock tissues and the mechanism by which the ADSL gene affected the flavour of livestock and poultry. 【Method】Chishui black-bone chicken(half male and half female) of different ages(3, 4, 5 and 6 months old) were used as experimental materials. The expression of ADSL gene in the pectoral muscle tissue of Chishui black-bone chicken at different ages was detected by real-time fluorescence quantitative PCR. Meanwhile,four pairs of shorthairpinRNA(shRNA) interference sequences(sh1-ADSL,sh2-ADSL, sh3-ADSL and sh4-ADSL) and one pair of negative control sequences(sh-NC) were designed according to the mRNA sequence of chicken ADSL gene in GenBank(NM_205529.2). They were sequenced after ligated with pGPH1/GFP/Neo vector. The constructed ADSL gene interfering vectors were transfected into myoblasts, and the most efficient interference vector was detected and screened.【Result】The relative expression level of ADSL gene in the pectoral muscle tissue of Chishui black-bone chicken at different months age was in the order of 5 months>4 months>6 months>3 months. The relative expression level of ADSL gene in the pectoral muscle tissue of Chishui black-bone chicken was extremely significantly higher in 3-month-old females than in males(P<0.01, the same bellow). The relative expression level of ADSL gene was significantly higher in 4-monthold females than in males(P<0.05). The relative expression level of ADSL gene was extremely significant lower in 6-month-old females than in males. The sequencing results of the ADSL plasmid showed that the interference plasmid was successfully constructed and the green fluoresce was observed in the interference group and the negative control after plasmid transfection,and the blank control showed no fluoresce, indicating that each recombinant plasmid successfully transfected the myoblasts of Chishui black-bone chicken. The ADSL gene silencing results showed that sh2-ADSL silencing effect was the optimal, with an interference efficiency of 90.30%. 【Conclusion】The relative expression level of ADSL gene shows a trend of increasing and then decreasing with the increase of month age. The relative expression level of ADSL gene in the pectoral muscle tissue of females at 3, 4 and 5 months old higher than that of males. The relative expression level of ADSL genes increases with increasing month age, and the differences between males and females decrease.The experiment is successful in isolating and extracting myoblast from Chishui black-bone chicken, and screening out the sh2-ADSL vector with the best RNA interference effect. This study provides a reference for further research on the molecular mechanism of ADSL gene regulation of meat inosinate and flavor.