Cloning, characterization and manipulation expression of Follicle Stimulating Hormone gene β subunit from Striped Catfish Pangasianodon hypopthalmus for gonadal maturation

Abstract

Induced maturation of fish by hormonal plays important role to get mature broodstock at out of spawning season. In this study, striped catfish FSH β subunit was cloned, characterized, and analyzed its expression after PMSG and AD induction. Cloning of FSH is a step to construct recombinant protein expression vector. The aimed of this research were isolation and characterization of FSH β subunit and evaluation of FSH β subunit expression after hormonal induction. Isolation of total RNA was done by TRIzol invitrogen method, then synthesis cDNA by reverse transciptase. Then, the PCR product were ligated to pGEM Teasy (GEM-FSH) and transformed into cloning vector. The successful of transformation would be evaluated by blue white colony screening. Similarity and alignment analysis used NCBI site and MEGA 5 for phylogenetic tree. Signal peptide and mature peptide were examined by signal 4.1 site. Then, manipulation FSH β subunit expression by inducing PMSG and antidopamine was conducted. PMSG and AD was injected every week for three weeks to evaluate FSH endogenous activity and physiology respond. After inducing during three weeks, the analysis expression FSH was amplified as molecular analysis and physiological respond including gonadal somatic index, gonad histology, and fecundity. The sequence of FSH β subunit fragment was 404 bp with 132 amino acid residue and consisted of signal peptide on 1-16 and mature peptide on 17-132. Alignment amino acid was done to make phylogenetic tree between other species. Based on phylogenetic tree, FSH gene β subunit divided for two clade; scales and no scales fishes. Genetic distance FSH gene β subunit has 0.08 with scales fish while 0.01-0.015 no scales fish. related to evolution, 0.08 with FSH gene β subunit for scale fish. The amino acid residue alignment showed 79-99% showed that similarity of structur amino acid on no scales fishes namely catfish and sea eel. Expression of FSH β subunit got influence after inducing with PMSG, AD, PMSG+AD and physiological solution. Inducing PMSG+AD showed expression FSH β subunit was higher than other one. Gonad histology showed that eeg developed to be late vitelogenic stage. Injected of combination PMSG and AD also increase aromatase expression. Therefore, reproduction cycle on striped catfish can be induced in out of season spawning.