Molecular Cloning And Expression Study Of Brain Aromatase In Carassius Auratus

As a natural inducer of fish ovary development,estrogen plays an important role in fish sex determination and differentiation.Aromatase is an important enzyme in the process of estrogen synthesis.There are two different types of aromatase enzyme in teleosts,which are respectively encoded by cyp19a1 a mainly expressed in gonads and cyp19a1 b mainly expressed in brains.Currently,studies on aromatase genes are mainly focused on the expression and role of cyp19a1 a in gonads and cyp19a1 b in the brains.But there are relatively few studies on the expression of cyp19a1 b in gonads and its role in sex determination and differentiation of fish.As a unique species in Henan province,Qi river crucian carp(carassius auratus)can produce offsprings through gynogenesis.In this study,The full legth cDNA of cyp19a1 b of Qi river crucian carp was cloned and the Cyp19a1 b expression profile from RNA and protein level were detected respectively,then the expression in different development stages and tissues by real-time PCR were also detected.In addition,the gene expression changes in brains and gonads were also examined after the Letrozole induced sex reversal and intraperitoneal injection of hCG,the main results are as follows:1.In the present study,brain aromatase gene(cyp19a1b)was cloned by using reverse transcription-polymerase chain reaction(RT-PCR)and rapid amplification of cDNA 5? and 3? ends(RACE)in Qi river crucian carp.The results showed that the full-length cDNA of cyp19a1 b was 2894 bp including a 132 bp 5?-untranslated region(UTR),a 1319 bp 3?UTR,and a 1533 bp open reading frame(ORF)encoding a deduced protein with 510 amino acids.The predicated molecular mass and pI value of the deduced Cyp19a1 b protein were 58.11 kDa and 7.57 respectively,sequence analysis showed that Cyp19a1 b contained a transmembrane region and a I-helix region,Ozol's petide region,aromatase specific region and Heme-binding region respectively.Sequence analysis revealed that Cyp19a1 b from Qi river crucian carp showed the highest amino acid similarity with Cyprinidae fish,while has low similarity with other vertebrates include mammalian and reptiles.A phylogenetic tree was constructed to understand the phylogenetic relationships of the Cyp19a1 b proteins among the vertebrates.It showed that Cyp19a1 b was clustered into one clade with Cyp19a1 b from Cyprinidae fish,while fish Cyp19a1 a formed a distinct fish specific clade,demonstrating that the cloned gene was cyp19a1 b of the Qi river crucian carp.2.The expression level of cyp19a1 b in different development stages and different tissues were also be examined by real-time PCR.Tissue distribution by real-time PCR revealed that cyp19a1 b was abundantly expressed in the brain,while barely detectable in other tissues.The expression of cyp19a1 b was gradually upregulated from blastula stage,and peaked at neurula stage,then declined during the embryonic development stage.Ontogenic analysis demonstrated that cyp19a1 b expression level in the brain was gradually increases with the development of the individual and began to rise from 20 dah and peaked at 150 dah,but in the overy,cyp19a1 b was expressed highly in the 30~40 dah which was the critical period of sex determination and differentiation of Qi river crucian carp and continued to decline in the subsequent period of development.The above results indicate that this gene plays an important role in the formation of nervous system and neuroendocrine,sex determination and differentiation and the ovarian maintenance.3.The Unfertilized egg of Qi river crucian carp can be stimulated by heterologous sperm to produce whole female offspring.Letrozole was used to treat 5 dah Qi river crucian carp larvae for 60 d to cause sex reversal.The expression of cyp19a1 b in the brain was significantly decreased after sexual reversal,suggesting that this gene may play an important role in ovarian development and maintenance by controlling the synthesis of estrogen in the brain.Intraperitoneal injection of hCG can promote the expression of cyp19a1 b in the brain and indicating that the level of exogenous hormones has a great effect on the expression of cyp19a1 b in the brain.4 In order to detect the protein localization of the Cyp19a1 b,the polyclonal antibody of Cyp19a1 b was prepared based on the cDNA of cyp19a1 b we have cloned.The SDS-PAGE electrophoresis showed that Cyp19a1 b polyclonal antibody can specifically recognize the recombinant protein of Cyp19a1 b induced by IPTG with a molecular weight of about 44 kDa,and identify a single size around 44 kDa in the brain of Qi river crucian carp,which indicating that the Cyp19a1 b antibody has a good specificity.5.Furthermore,the antibody was used to investigate the expression profiles of Cyp19a1 b proteins in the brain of different development stages and different tissues of Qi river crucian carp.Western blot analysis showed that the expression of Cyp19a1 b in brains and gonads was significantly decreased after sex reversal induced by Letrozole treatment.By Immunohistochemistry,positive signals of Cyp19a1 b were detected in radioactive glial cells(RGCs)of brain from 20 dah and the somatic cells of the ovary from 30 dah,which are basically consistent with RT-PCR results and confirmed the role of brain aromatase in nervous system formation,gonad development and sex determination in Qi river crucian carp.To sum up,Cyp19a1 b is highly expressed in the brains of Qi river crucian carp both at mRNA and protein level,and significantly decreased with Letrozole induced sex reversal.RT-PCR results showed that the expression of cyp19a1 b in the brains was gradually increases with the development of the individualand,in the gonads,the expression of cyp19a1 b was significantly up-regulated in the critical period of sex determination(30~40 dah).Immunohistochemistry results showed that brain aromatase began to be detected in RGCs of brain at 20 dah and the somatic cells of the ovary at 30 dah,and also be detected in the Ledig cells of the sex revesal individuals.The above results all suggest that Cyp19a1 b played an important role in its nervous system formation,sex determination and differentiation and maintenance of gonadal development of Qi river crucian carp.