| Sex determination and differentiation is the most common physiological phenomenon in the entire animal kingdom.Therefore,the study of gender regulation mechanism is one of the hottest research areas in life sciences.The evolutionary status of crustaceans is relatively low.The features of sex determination mechanism of crustaceans are aboriginality,diversity and flexibility.The growth process of crustaceans is sexual dimorphic.There are significant differences in the size of males and females.Hence,the study on the sex determination mechanism of crustaceans can not only provide a theoretical basis not only for reproductive reproduction,but also provide a theoretical basis for sex reversal and unisexual breeding technology.Till now,only Insulin-like androgenic gland hormone(IAG)secreted by androgenic gland plays an important role in male sex differentiation and functional maintenance.There are few reports on other key factors,especially ovarian development-related genes and their mechanisms of action,which need to be further studied.Cherax quadricarinatus,commonly known as freshwater blue dragon from Australia,is one of the important economic freshwater shrimps.There are obvious differences between male and female C.quadricarinatus in growth rate and individual size.Moreover,compared with the spawning quantity of Fenneropenaeus chinensis with tens of thousands eggs,C.quadricarinatus has low fecundity and the rate of ovum holding.The prices of redclaw crayfish are high due to the lack of larves.This is also one of the main factors affecting the development of the industry chain.Therefore,more female shrimp are needed in production to meet the demand of seedlings.The above situation shows that unisexual culture is with great significance to improve the yield and specification of redclaw crayfish and provide more parents for the breeding of the larvae lacking in females.In this study,the transcriptome of gonadal tissues of redclaw crayfish was obtained and analyzed through RNA-Seq.A large number of functional genes involved in gonadal development were screened according to previous reports,including Tra2 and Foxl2.The full-length sequences of these two genes were obtained by RACE.The homology and phylogeny analysis were carried out by biological information.The profile detect expression of Tra2 and Foxl2 was analyzed based on q RT-PCR.Finally,RNAi technique was used to explore the role of Tra2 and Foxl2 in the early sex differentiation of redclaw crayfish.The main results are showed as follows:1)High-throughput sequencing technology,Illumina Hi Seq 2000,was used to construct the gonadal transcriptome database of redclaw crayfish.A total of 189456923high-quality original sequences were obtained.Seven public databases such as GO,NR and KEGG were applied to annotate the Unigenes sequence from the gonadal tissue of redclaw crayfish.And a total of 126368 Unigenes were obtained.A total of 64138 differentially expressed genes were found in the comparative transcriptional library of testis and ovary,of which 32822 Unigens were highly expressed in ovary and 31316 Unigenes were highly expressed in testis.According to the reported literature,a large number of functional genes related to gonadal development were screened,including vitellogenin,IAG,cdc2,Dsx,Tra2,Fem1 and Foxl2.2)The Tra2 gene was isolated and characterized by RACE technique.Three splice variants were identified,designated as Cq Tra-2A,Cq Tra-2B,and Cq Tra-2C,and sequence analysis showed that they had a highly conserved RRM domain.Phylogenetic analysis was performed by the NJ method,and the results revealed that the Tra2 protein of the redclaw crayfish was very closely related to those of Macrobrachium rosenbergii,Fenneropenaeus chinensis and Macrobrachium nipponense.Real-time PCR analysis showed that the three isoforms were predominantly expressed in the ovary.Additionally,the expression pattern of CqTra2 at different developmental stages was analyzed by q PCR and revealed that the phase of having a body length of 3 cm may be the key period for the sex differentiation of C.quadricarinatus.The results of RNAi-targeting gene silencing showed that the transcription level of CqTra2 gene decreased by about85%.And the expression level of Cq Dsx gene was down-regulated,which further confirmed the function of CqTra2 in sexual differentiation in redclaw crayfish.3)The transcript of CqFoxl2 was annotated according to the data of the transcriptome database.And the full length of Foxl2 was obtained by molecular cloning technique.The coding length is 1695 bp and encodes 564 amino acids.The amino acid sequences of CqFoxl2 gene was multiply aligned with those of 18 species,such as fish,mammals and crustaceans.It was found that redclaw crayfish had high homology with shrimp and crabs.Phylogenetic analysis showed that redclaw crayfish clustered with crustaceans and was closely related to Procambarus clarkii.In order to study the effect of Foxl2 on sexual differentiation of redclaw crayfish,q PCR technique was used to detect its expression in different tissues.The results showed that Foxl2 had the highest m RNA amount in the ovary.Furthemore,the expression of Foxl2 in different developmental stages of ovarian was measured by Real time-PCR.It was indicated that the expression of Foxl2 was higher in I-V stage of ovarian development.These results suggested that Foxl2 may be an important gene for ovarian function and maintenance of feminization.Finally,the expression of CqFoxl2 gene was interfered by using RNAi technology.The results showed that the m RNA level of sex regulation gene Doublesex decreased significantly.The gonadal index of female redclaw crayfish injected with ds Foxl2 was significantly lower than that of the control group.These results indicated that transcription factor Foxl2 has an important regulatory effect on ovarian development.In this study,the gonadal transcriptome library of C.quadricarinatus was constructed for the first time.A large number of genes related to gonadal development were screened by functional annotation.Secondly,CqFoxl2 and CqTra2 genes were isolated and characterized in redclaw crayfish.Their structures were characterized.By using q PCR and RNAi,the function of Tra2 and Foxl2 were preliminarily analyzed.It was proved that they were involved in the development of ovary.These results will help to better understand the molecular mechanism of sex determination in crustaceans and provide a strong theoretical basis for the development of unisexual breeding techniques. |
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