Polyclonal Antibody Preparation Of Cyp19a1a Gene In The Nile Tilapia Oreochromis Niloticus And The Influence By High Temperature Induced

Aromatase participate in mammalian central nervous system development,adjust the neuroendocrine， impact reproducing and sex, and it can control themammalian gonadal differentiation, impact sex differentiation and sex determinationof many fish。Aromatase is encodinged by cyp19a1aromatase gene. There are twokinds of aromatase gene in Nile tilapia, the cerebral aromatase gene (cyp19a1b) andthe gonads aromatase gene (cyp19a1a), they are encoded the cerebral aromatase(P450aromB) and the gonads aromatase (P450aromA). The gonad aromatase mainlyexpressed in the gonads, little or no expression in any other organizations. In order toinvestigate the molecular mechanisms of the gene expression related to sexdifferentiation in Nile tilapia which was induced by high temperature, obtainedcyp19a1a ORF area by RT-PCR technique from Nile tilapia gonads, expression andpurification of Nile tilapia cyp19a1a protein, then immune in mice to obtainpolyclonal antibody. Nile tilapia were treatment by36℃high temperature, Analysisthe change of cyp19a1a protein expression in gonad before and after high temperatureinduced Nile tilapia. Nile tilapia ovarian cells were cultured using tissue explant.qRT-PCR was used to analyze the expression level of hsp70and cyp19a1a genes inNile tilapia ovary which was induced by high temperature at37℃.1、RNA was extracted from Nile tilapia ovary and then was reverse transcribedto cDNA. The1200bp ORF region of cyp19a1a was amplified and ligated intopET-28a(+) expression vector to construct the prokaryotic expression vector ofpET-28a-cyp19a1a. Verified by enzyme digestion and DNA sequencing,pET-28a-cyp19a1a was transformed into E.coli BL21. In addition, IPTG inductionconcentration and induction time were optimized. The results displayed thatpET-28a-cyp19a1a recombinant protein abundantly expressed in0.5mM/L IPTGinduction after8h and form inclusion bodies. By Ni2+-NTA agarose gelchromatography column, expected fragment size expressed proteins was purified. Thepurified protein was determined by western blotting using His-tag antibody. UsingWestern blotting was given to verify the effectiveness of the polyclonal antibody.After high temperature induction, it was discovered that the expression of cyp19a1a quantity significantly reduced by Western blotting using antiserum.2、Nile tilapia ovarian cells were cultured using tissue explant. The results showthat the ovarian cells can grow healthily and continuous extend in L-15brothcontaining15%FBS and5%fish serum under29℃.Then, the qRT-PCR was used toanalyze the expression level of hsp70and cyp19a1a genes in Nile tilapia ovary whichwas induced by high temperature at37℃. Nile tilapia ovary which has no treatment,ovarian cells cultured in vitro at nomal temperature and ovarian cells cultured in vitroat37℃have also been done by qRT-PCR to analyze the expression level of hsp70and cyp19a1a genes. The results show that the expression of hsp70gene in Niletilapia ovary was significantly increased by high temperature, however, theexpression of hsp70gene in ovarian cells in vitro culture have a rising trend but notsignificant, indicating that Nile tilapia ovary cells in vitro is sensitive to heat stress.High temperature significantly decrease the cyp19a1a gene expression level in vitro;however, Nile tilapia ovarian cells cyp19a1a expression level was not significanteffect by high temperature, which suggested that the effect of high temperature andthe expression level of cyp19a1a may require the synergy of other protein orgenes(foxl2、sox9、dnmt1and so on).