| In the teleosts,P450 aromatase plays a particularly important role in the process of sex differentiation and gonadal development.Aromatase can transform testosterone into estradiol,regulate gonadal differentiation and maintain ovarian development.The P450 aromatase of teleost fish is mainly composed of two kinds:gonad and brain type,respectively encoded by two genes of Cypl9a1a and Cypl9a1b.For hermaphrodite sex reversal fish,the activity of aromatase gene determines the direction of gonad development.The promoter is the main regulator of the aromatase gene expression,and the functional components on the promoter can be combined with a specific transcription factor to start and regulate the expression of Cypl9ala genes.In this study,a typical hermaphroditic fish fishes were selected as the research object.The response regions and response components of the androgen and estrogen on the Cyp19a1a gene promoter of the bellows were studied.The main results of this paper are as follows:1.Using Genome Walking technology,we successfully cloned the Cheilinus undulatus Cypl9a1a gene promoter by 1444 bp.It was found that the sequence contained five half-ARE sites and four half-ERE sites.2.The promoter regions were inserted into luciferase reporter gene vectors and transfected into HEK-293T,COS-7,L?T,CHO-K2,U251-MG and TM4 cells.The results showed that HEK-293T cell lines had the best effect in expressing Cyp19a1a promoter activity.In the HEK-293T cell line,when the concentration range is within 10-9M-10-5M,testosterone can significantly increase the activity of Cyp19a1a promoter through AR;when the concentration range is within 10-9 M-10-5 M,the estradiol can up regulate the activity of Cypl9a1a promoter,and the activity of Cypl9ala promoter is the highest at the concentration of 10-6 M.3.The deletion vector of Cyp19a1a promoter was constructed by promoter deletion technique.According to the location of ARE and ERE components,5 Testosterone deletion vectors were constructed and 3 estrogen deleted vectors were deleted,and the activity changes of promoters in HEK-293T cells were investigated by sex hormone stimulation respectively.The experimental results showed that when the first ARE was deleted from the promoter,the activity of testosterone was reduced sharply,and the remaining AREs was deleted in turn,and the activity could still be maintained.All deletion of ARE leads to promoter's loss of activity.In the estradiol induction experiment,the activity of the promoter decreased to the control level when the first ERE was deleted.Deletion of the remaining ERE has no effect on promoter activity.4.The 2 1/2ARE loci(1/2ARE#1,1/2ARE#2)and 1/2 site on the Cypl9ala promoter of PCR were mutated by fusion technology.The results showed that in the testosterone induction experiment,the mutation of 2 1/2ARE sites resulted in a significant decrease in promoter activity.The mutation of 1/2ERE#1 site leads to the loss of promoter activity on estradiol regulation.This study demonstrated that testosterone and estradiol can regulate the aromatase gene Cyp19a1a promoter of Cheilinus undulatus.There are 2 ARE loci that possess testosterone regulatory activity,while only ERE#1 components have estradiol regulatory activity.This study provides theoretical support for the reveal of Cheilinus undulatus P450 aromatase gene expression regulation. |
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