Association between AA-NAT gene polymorphism and reproductive performance in sheep

Arylalkylamine N-acetyltransferase (AA-NAT) is critical enzyme in Melatonin (MLT) biosynthesis for MLT regulating the animal seasonal breeding. In this study, DNA sequencing methods were applied to detect the polymorphisms of the AA-NAT gene in 179 Chinese sheep belonging to two non-seasonal reproduction breeds and two seasonal reproduction breeds. One mutation at exon 3 (NM_001009461:c.486A > G) was firstly described at the sheep AA-NAT locus. Hence, we described the SmaI PCR-RFLP method for detecting EX3 486A > G mutation, frequencies of the AA-NAT-G allele varied from 0.871 to 0.908 in two non-seasonal reproduction breeds and 0.517 to 0.578 in two seasonal reproduction breeds. The associations of SmaI polymorphism with estrus traits was analyzed in non-seasonal reproduction breeds sheep and seasonal reproduction breeds sheep, the significant statistical results were found between them, the GG genotype frequencies was higher in non-seasonal reproduction breeds (p < 0.001), while, the GA genotype frequencies was higher in seasonal reproduction breeds (p < 0.05). Hence, the EX3 486A > G mutation could facilitate association analysis and serve as a genetic marker for Chinese sheep breeding and genetics.

In vitro effects of 5-hydroxytryptophan, indoleamines and leptin on arylalkylamine N-acetyltransferase (AA-NAT) activity in pineal organ of the fish, Clarias gariepinus (Burchell, 1822) during different phases of the breeding cycle.

Arylalkylamine N-acetyltransferase (AA-NAT) is the rate-limiting enzyme of melatonin biosynthetic pathway. In vitro effects of 5-hydroxytryptophan (5-HTP) and indoleamines (serotonin, N-acetylserotonin and melatonin) were studied on AA-NAT activity in the pineal organ of the fish, C. gariepinus during different phases of its annual breeding cycle. Further, in vitro effects of leptin on AA-NAT activity in the pineal organ were studied in fed and fasted fishes during summer and winter seasons. Treatments with 5-HTP and indoleamines invariably stimulated pineal AA-NAT activity in a dose-dependent manner during all the phases. However, leptin increased AA-NAT activity in a dose-dependent manner only in the pineal organ of the fed fishes, but not of the fasted fishes irrespective of the seasons.

CONSTRUCTION,EXPRESSION AND DETECTION OF THE EUKARYOTIC EXPRESSION VECTOR OF SEROTONIN N-ACETYLTRANSFERASE GENE / 解剖学报

Objective To investigate the possibility that the construction and expression of a eukaryotic expression vector system of rat NN-NAT gene. Methods The full-length cDNA fragment of rat AA-NAT gene was amplified by RT-PCR method.After retrieving the PCR products,ligating it with pTARGET~(TM) vector,transformating ligation reaction to JM109 huge efficiency competent cells and identifying the recombinant plasmid,the recombinant eukaryotic expression vector pTARGET~(TM)-AANAT was transfected into rat L6 myoblasts with lipofectamine.Accordingly,engineered cells selected by antibiotic G418 were detected by the methods of RT-PCR and Westem blotting. Results It was revealed that,amplified AA-NAT cDNA confirmed by agarose gel electrophoresis could ligate with pTARGET~(TM) vector and subcloned into JM109 cells.L6 cells transfected with pTARGET~(TM)-AA-NAT survived well after G418 selection and expressed AA-NAT protein. Conclusion Our results suggest that we have prepared rat AA-NAT stable eukaryotic expression system successfully although it was just a primary result.This system can be used for the transfection of L6 myoblast.