Sequence Characteristics And Expression Analysis Of FTZ-F1、CYP17A1 And CYP17A2 Genes In Chlamys Farreri

FTZ-F1,a member of the orphan nuclear receptor family,is an important transcriptional regulator gene involved in biohormone production and gonadal differentiation.Cytochrome P450 family members CYP17A1 and CYP17A2 genes are the key rate-limiting enzymes in the steroid hormone synthesis pathway.The FTZ-F1 gene and CYP17A1 and CYP17A2 genes have been studied extensively in vertebrates,but there are relatively few studies in molluscs.Chlamys farreri is an important economical shellfish species.This study used Chlamys farreri as the research object.The three gene sequences characteristics,multiple sequence alignment and phylogenetic analysis of FTZ-F1,CYP17A1 and CYP17A2 were analyzed,the three-dimensional structure was also predicted.The expression of the above three genes in different tissues of the female and male individuals of Chlamys farreri were detected,and in the gonadal development cycle as well.The expression rules and characteristics were explored,and the function of the genes was inferred.The main research is as follows:1.The CDS of the FTZ-F1 gene is 1668 bp,encoding 555 amino acids.Amino acid sequence analysis showed that it contains the conserved regions of DBD and LBD(the LBD region contains an activation domain AF-2).The three-dimensional protein structure prediction also confirmed the existence of the above two conserved regions.The alignment results of multiple amino acid sequences showed that Chlamys farreri FTZ-F1 is similar to the conservative regions of other species.Phylogenetic analysis showed that the Chlamys farreri FTZ-F1 protein phylogenetic relationship is basically consistent with the taxonomic status of the species.The genes of this family can be named FTZ-F1 or NR5 A subfamily,or SF-1 subfamily,combined with the sequence and expression pattern of Chlamys farreri FTZ-F1.This gene was named FTZ-F1 ccording to the classification and naming methods of other different species FTZ-F1 families in the literature.FTZ-F1 expression in tissues were detected by semi-quantitative PCR,in female individuals,relatively stronger expression were derected in the adductor muscle kidney and gill,weaker expression in other different tissues.In male individuals,the stronger expression were in shell muscle,kidney and gill tissues,and weaker expression in other tissues,which indicated that this gene is expressed extensive in all tissues.The expression characteristics in male and female individuals are differences.Using quantitative real-time PCR(q PCR),the FTZ-F1 expression was compared between the ovary and the testis during the reproductive cycle,FTZ-F1 is mainly expressed in the mature testis,and the expression level is significantly higher than in others gonads of different stages.It is speculated that the highest expression is a concern in significantly increased testosterone content in the mature testis.A certain relationship confirms that FTZ-F1 participates in the development of the testis of Chlamys farreri and plays an important role in the mature stage of the testis.2.The CYP17A1 and CYP17A2 genes are members of the CYP17 family and contain three conserved regions: Ono sequence,Ozols’ tridecapeptide region and heme binding region.Ono sequence conserved region differents CYP17 from other p450 genes.This study firstly compared CYP17A1 and CYP17A2 Ono sequence conserved regions of different species,the unique sites of CYP17A1 and CYP17A2 were analysised,two similar genes in Chlamys farreri were classified.The CYP17A1 gene is 1522 bp,encodes 428 amino acids,and contains three conserved regions of the CYP17 family.The alignment results of multiple amino acid sequences showed that the conserved regions of Chlamys farreri CYP17A1 are consistent with other species.The construction of a phylogenetic tree showed that the Chlamys farreri CYP17A1 protein first clustered with the Mizuhopecten yessoensis CYP17A1,then clustered with the Crassostrea gigas CYP17A1 and Aplysia californica CYP17A1,and finally clustered with the CYP17A1 protein of other species,showing the phylogenetic evolution relationship is basically the same as the taxonomic status of species.Semi-quantitative PCR technology detected the expression of CYP17A1 gene in different tissues.In females,the gene is expressed stronger in the kidney and hepatopancreas,while the expression in other tissues was weaker.In males,the expression is stronger in hepatopancreas,and weaker in others tissues,indicating that the CYP17A1 gene is widely expressed in different tissues of Chlamys farreri,and may have physiological functions;QPCR detection of CYP17A1 expression in the gonadal cycle were used,CYP17A1 expression is the highest at growth stage during ovarian development,we considered the gene may play a role in oocytes growth.It may be due to the production of estradiol which affects the growth of oocytes;during the development of testis,the expression of CYP17A1 increases with the testis development.Based on the literature,this gene may be involved in the production of testosterone,it may play a role in the development of male gonads by participating in testosterone production.The CYP17A2 gene is 3600 bp long,encodes 503 amino acids,and also contains three conserved regions of the CYP17 family.The expression of CYP17A2 was detected by semi-quantitative PCR.In females,higher expressions were detected in the kidney and gill,and weaker in others tissues.In males,the expression was higher in the adductor muscle,kidney,gill and testis,weaker in others tissues,the expression research of this gene is currently only seen in fish,its function in different tissues requires more experimental data.QPCR results show that the CYP17A2 expression is the highest at mature stage of ovary,which is significantly higher than at proliferative stage and growth stage,it is speculated that this gene may be involved in 17α,20β-DP(17α,20β-dihydroxy-4-pregnen-3-one,DHP,a kind of progesterone)producing to play a role in the maturation of oocytes.During the development of the testis,the CYP17A2 expression is highest at mature stage,which is significantly higher than other stage,it is believed that this gene may affect sperm excretion and sperm motility by participating in the production of progesterone.