| Neurokinin B(NKB), encoded by Tac3 gene, belonged to the family of tachykinins, and widely distribited in the central nervous ststem and non-nervous tissues. NKB played a very important role in the physiological and nervous system activities. In recent years, it received attention from research scholars that NKB effected on reproductive endocrine regulation, and it was considered to be another factor which had an important role in the reproductive endocrine regulation, in addition to kissppetin. Currently, the research on the function regulation of NKB in reproductive activities has become a hot-spot at home and abroad.Platichthys stellatus was considered as one of the most promising farmed fish, and will become a new industry, following the Paralichthys and Scophthalmusmaximus. In this pager, P. stellatus was used as experimental material to clone the full-length c DNA of Tac3 gene, using rapid amplification of c DNA ends(RACE), proceeded bioinformatics analysis and prokaryotic expression, and detected Tac3 gene expression patterns and function. These results were as follows:1. A full-length tachykinin3(Tac3) c DNA was isolated from brain tissue of P. stellatus by rapid amplification of c DNA ends(RACE) PCR with special primers(GSP1 and GSP2). The Tac3 c DNA sequence was 710 bp, and contained an open reading frame of 366_base pairs encoding 121 amino acid, 5’-untranslation region of 72_base pairs, and 3’-untranslation region of 272_base pairs, Gen Bank Accession number was KP742474.2. Bioinformatics analysis revealed that molecular weight was 14.18 k Da and aisoelectric point was 10.31,a signal peptide and no transmembrane regon, The secondary structure prediction showed that α-helices was 41.32%, random coil was 36.36%, extended strand was 19.01%, and bate turn was 3.31%. Homology analysis showed that amino acid sequence of Tac3 had a high similarity with Sebastes rastrelliger(88%), and results from phylogenetic tree analysis also showed that P. stellatus formed a cluster with Sebastes rastrelliger, and they had a closer relationship.3.The prokaryotic expression vector p ET-21a/NKB was successfully constructed by connecting the open reading frame of NKB and the expression vector p ET-21 a.Then it was transformed into E.coli BL21, and was induced by IPTG. The SDS-PAGE showed that there was a bright band between 14.3KDa and 20.1KDa. This indicatesd that the fusion protein was successfully expressed in E.coli.BL21.4. This study analyzed the expression patterns of Tac3 gene by RT-PCR. Tissue distribution indicated that Tac3 m RNA was expressed in brain, liver, gonad, intestine, kidney, spleen and heart, and the highest Tac3 m RNA level was in brain tissue, followed by liver and gonad. In different periods of reproductive cycle, the highest Tac3 m RNA level was in mature stage, and it began to decrease in decline stage5. By way of injecting NKB mature peptide into mature female P. stellatus in vivo, the resulta showed that NKB can significantly regulate the expression level of Gn RH, FSH, LH gene. At dosages of 10ug/kg, NKB effective significantly enhance the expression level of Gn RH and LH gene(P<0.05), and decreased FSH expression level. Similarly, in vitro cell experiment, NKB had a significant impact on the expression level of Gn RH, FSH and LH gene in brain tissue.6. The effects of 17β-estradiol on the expression of the Tac3 in the P. stellatus brain tissue were performed by in vivo of intraperitoneal injection, the results showed that 17β-estradiol improved the Tac3 and LH m RNA expression in brain significantly(P<0.05), and it presented time-dose dependency relation. These results suggested that sex steroid hormones had a positive feedback regulation on Tac3 and LH expression in brain of sexually immature P. stellatus.
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