Cloning And Analysis Of Aromatase Gene Promoter In Cheilinus Undulatus

Cheilinus undulatus is one of the large coral reef fish who live in warm water.C.heilinus undulatus meat is rich in protein,unsaturated fatty acids as well as a variety of trace elements and what's more,it tastes delicious.All these traits prompt people to overfish Cheilinus underlatus and therefore some international organizations has listed it as an endangered species.In order to protect and develop this kind of endangered fish,China will breed C.heilinus undulatus artificially.However,one problem existing in the process of artificial breeding is the disproportionality of male and female fish;therefore,in order to solve this problem,the research mainly probe into the sex control mechanism of Cheilinus undulatus,with a purpose of laying the theoretical foundation of artificial breeding of Cheilinus undulatus.P450aromatase,a key enzyme in the conversion of testosterone to estradiol,is encoded by Cyp19al.Because the Gonads primordium of fish have the potential of developing into both gender,Aromatase can monitor the proportion of estrogen and androgen and thus regulating the sex of fish and maintaining gonadal function.However,little is known about the first "switch" genes and subsequent regulatory pathways of social sex reversal fish.Finding upstream regulatory factor of the aromatase gene Cyp 19al is a process revealing a feasible strategy.Therefore,this study will starts with the research about Cheilinus undulatus of Cyp 19a1 gene.There are two aromatase genes in teleost fish:gonads type Cyp19ala and brain type Cyp19alb.The level of Cyp19ala is directly related to sex control and gonad development,while Cypl9alb may affect sexual differentiation indirectly.Based on the findings of Cheilinus undulatus' cDNA we have got in our previous study,We intend to use the promoters(which is acquired by Genome walking technology),and possible sequence(which is calculated by related software)to analyze transcription binding sites.We will get the recombination plasmid by inserting the aim gene fragment into the dual luciferase expression vector on the basis of related software prediction and analyze the transcription binding sites.After the transfection of COS7 cell,Dual Luciferase Reporter Gene Assay System is used to detect the recombinant plasmid.Through the experiment,we get 5'flanking sequences of 1119bp and 1632bp respectively and find that there are many different kinds of transcription elements,for example,CREB,SRY,Sox5,SRF,TFIID and receptor binding sites,GR,AR,PAR,AhR,and PXR.It is predicted that the CpG methylation binding sites and estrogen receptor(ER)can be founded in Cyp 19alb promoter,while in Cyp 19ala can not.It also suggested that these transcription elements plays a key role in the regulation of P450.It is showed that different density of androgen could increase the expression of Cyp19ala promoter,while Cyp19alb has no significant change.Under different density of estrogen,the promoter activity of Cypl9alb is significantly different,which bear a close relationship with ER,but no significant relationship with cypl9ala.It is hoped that this study can help to clarify the expression and regulation mechanism of P450 and provide a theoretical basis and techniques of artificial reproduction of Cheilinus undulatus.