Cloning And Expression Of Dmrt1 And Sox9 Genes In Discus Fish(Symphysodon Aequifasciatus)

Discus fish(Symphysodon aequifasciatus)is a tropical ornamental fish,native to Amazon River basin of South America and known as the "King of tropical ornamental fish".The breeding technology of discus fish has been popular,but it is hard to distinguish male and female of the early stage,like distinguishing sex from the appearance is unworkable.At present,the reproduction of discus fish is still carried out by natural pairing and natural spawning.Such a reproduction mode made by artificial simulation,not only spending a lot of time to pair,but lengthening the reproductive cycle.Moreover,the artificial spawning technology of the fish has not yet been solved with low fecundity,which greatly restricted the yield of seed breeding and limited the large scale farming of discus fish.Therefore,in order to promote the artificial breeding and industrialization development of discus fish,it is necessary to make a deep research on the genes about gonad differentiation and development.This research in the prophase research of discus fish gonad differentiated time further on the basis of using characteristics dmrt1 and sox9 gene markers in the process of its development in the characteristics of gene expression and its function to explore further experiment.In discus fish gonad differentiated with ovary and testis differentiation related in the process of biological information will help us to adopt molecular biology method further research may artificial oxytocin or gene engineering technology development and research to genetic potential function for discus fish propagation speed set up technical support,further promoting the industrialization of discus fish.The full-length cDNA of dmrt1 and sox9 gene was cloned and verified from discus fish gonad transcriptome data using RACE technology(Accession No.dmrt1: KX845007;sox9: MF285604).With the technique of Q-PCR the expression of dmrt1 gene and sox9 in gender differentiation period were analyzed.The probe was designed according to the cDNA sequence of dmrt1 and sox9 gene which can marked.In situ hybridization was performed on sections of fish,in order to observe the position of fuction in cells.The results are as the followings.The results showed that the full length cDNA of dmrt1 was 888 bp in length and 929 bp in length,encoding a polypeptide of 295 amino acids.The length of sox9 gene was 1390 bp and encoding a polypeptide of 334 amino acids.Dirichlet dmrt1 and sox9 genes are closely related to tilapia phylogenetically.The results showed that the evolutionary direction of dmrt1 and sox9 genes was in accordance with their evolutionary status.The expression of dmrt1 gene in S.aequifasciatus was specifically expressed in gonads,while no expression was detected in other tissues.sox9 gene was expressed in testis,brain,stomach,intestine and skin,but mainly expressed in testis and brain.During the process of juvenile fish development,the expression of dmrt1 firstly increased and reached the maximum at 55 d,then decreased to the lowest at 60 d,then continued to increase at 65 d,and then decreased at 70 d.The expression of sox9 first increased,Reaching the maximum at 45 days,then dropping to its lowest level at 50 days,continuing to increase at 55 days and then declining at 70 days,but remained at a relatively high level.dmrt1 mRNA was mainly expressed in the gonads and sox9 mRNA was mainly expressed in the testis and brain.During larval development,both dmrt1 and sox9 genes were in the 50-60 day after hatching occurred differentially expressed and involved in the development of the gonads may play a role in the regulation of the early sex differentiation of S.aequifasciatus...