| P450 aromatase is an important sex hormone regulating factor in vertebrates.It plays an important role in sex differentiation and reproductive regulation.In most of the teleost fish,there are two kinds of aromatase,gonadotropin and brain type.These two enzymes are encoded by cyp19a1a and cyp19a1b genes,which play a different role in the sex development of fish.Cheilinus undulatus is a social sex reversed fish hermaphrodite,the sex hormone regulation mechanism is not clear.In this paper,the activity of brain aromatase gene promoter is studied,and the regulation mechanism of sex hormone on cypl9a1b promoter is revealed.1.Genome walking was used to further amplify the cyp19a1b gene promoter obtained in our laboratory,and the sequence of cyp19a1b gene promoter with a full length of about 2.3kb was obtained.The sequence was analyzed with the online software of the transcription factor binding site(TFBS).The results showed that there were a variety of transcriptional regulators on the cyp19a1b gene promoter.Parts,such as four AREs,one complete ERE,one 1/2ERE,including SP1,AP-1,cAMP response element(CRE),etc.2.cyp19a1b promoter and PGL-4 vector were digested and connected to construct a double luciferase reporter gene vector.Then,the eukaryotic expression vector of estrogen receptor(ER)and the eukaryotic expression vector of androgen receptor(AR)were co transfected to HEK-293T cell line respectively,and the different concentrations of estradiol(E2)and testosterone(T)were used to stimulate the cell line respectively.The dual luciferase detection system was used for detection.The results show that when the concentration of E2 is at 10-9M-10-6M,it can increase the activity of cyp19a1b promoter through ER,and the concentration of E2 is 10-6M,and the activity of cyp19a1b promoter is the highest.When T concentration is at 10-9M-10-5M,it can also increase the activity of cyp19a1b promoter,and the activity of promoter is the highest at the concentration of 10"5 M..3.In order to further study the area of estrogen,androgen and cyp19a1b promoter response,this study constructed a total of 2 estrogen deletion carriers and 4 androgen deletion vectors based on the location of ARE and ERE components,and studied their activity changes after estrogen and androgen stimulation.The results showed that when the ERE regulator on the cyp19a1b promoter was deleted,the activation reaction of the promoter disappeared,and the activity of the cyp19a1b promoter of the 1/2ERE element was deleted and the activity was not disappeared.This indicates that ERE gene significantly regulates cyp19a1b gene expression.When the ARE element is deleted on cyp19a1b promoter,the activity of promoter decreases.4.We use fusion PCR technology to carry out the mutation of ERE elements on the cyp19a1b promoter and 4 ARE elements(ARE#1,ARE#2,ARE#3,ARE#4)on the corrugated lip fish.The results showed that when the ERE element was mutated,the activity reaction of cyp19a1b promoter completely disappeared,and when the ARE#1,ARE#3 and ARE#4 components were mutated on the promoter,the promoter activity decreased in varying degrees,and the ARE#3 was abruptly changed,and the promoter activity of the ARE#4 site decreased significantly.The promoter activity of mutant ARE#2 element is not different from that of the non mutant promoter.These results indicate that ERE elements have significant effect on the activity of cypl9a1b promoter,and two regulatory sites of ARE#3 and ARE#4 play a major role in the regulation of cypl9a1b gene promoter.The results of this study proved that estrogen and androgen can regulate the aromatase gene cypl9alb promoter of Cheilinus undulatus.The effects of sex hormones on the expression of aromatase genes in the naked lip fish were revealed,which lay the foundation for further understanding the regulatory mechanism of aromatase expression. |
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