Functional Study Of The DMRT Protein Family In Sexual Development Of The Brine Shrimp Artemia Franciscana

Artemia franciscana,also known as brine shrimp,is a type of planktonic crustacean that inhabits salt lakes and salt flats,and possesses a high commercial value in the fishing industry.In modern fish farming,brine shrimp is regarded as the preferred starter feed for fish cultivation.Due to its short life cycle,ease of experimental manipulation and verification,and ease of sexual differentiation,brine shrimp has gradually become an ideal model for studying the sexual development of crustaceans.The study of the sexual development mechanism of brine shrimp is not only conducive to promoting the development of mono-gender breeding techniques and improving the production efficiency of brine shrimp cysts,but can also be extended to the breeding of other economically important crustaceans.To study the sexual development of brine shrimp,we first conducted transcriptome sequencing and analysis on female and male brine shrimp samples.The results revealed6056 female-specific expressed genes and 18409 male-specific expressed genes.Subsequently,we identified five candidate genes from these gender-differentiated expressed genes that may play a role in sexual development,and belong to the Dmrt(Doublesex and mab-3 related transcription factor)gene family.Based on their position in the phylogenetic tree,they were named Afr Dsx,Afr Dmrt11E,Afr Dmrt93B,Afr Dmrt99B,and Afr Dmrt Un.Previous studies have shown that the Doublesex(Dsx)gene acts as a"molecular switch"that regulates the sexual development and differentiation of arthropods.Therefore,the Afr Dsx gene of brine shrimp may play a crucial role in sexual differentiation and development.Through the validation of RT-PCR,the results revealed a transcript variant called Afr Dsx~M,which was preferentially expressed in male brine shrimp,and another variant called Afr Dsx~F,which was expressed specifically in female brine shrimp.Through the analysis of the gene structure,promoter prediction,and polyadenylation site(polyA)prediction,we found that Afr Dsx~M and Afr Dsx~F were derived from the same gene loci,and the production of the two transcripts is regulated by a combination of selective promoter,splicing,and poly A sites.We used RT-q PCR to compare the expression of Afr Dsx~M and Afr Dsx~F in different tissues,organs,and developmental stages of male and female brine shrimp.The results showed that Afr Dsx~M had the highest expression in the male gonads,while Afr Dsx~F had a lower expression level in the ovaries than in other tissues.Moreover,the expression levels of Afr Dsx~M and Afr Dsx~F increased during the development from nauplii to adulthood,indicating the involvement of Afr Dsx in sex development in the early stages and plays a regulatory role throughout the brine shrimp’s life cycle.To investigate the functions of the two Afr Dsx transcripts in sexual regulation,we used RNA interference to knock down Afr Dsx~F and Afr Dsx~M in female and male brine shrimp,respectively.The results showed that knocking down Afr Dsx~M led to feminization of the male gonads,with over 60%of male brine shrimp showing female reproductive gland characteristics and containing a large number of yolk granules in the ovary-like structure.However,knocking down Afr Dsx~F in the female brine shrimp did not cause any significant changes.Through the RT-q PCR,immunoblotting,and immunohistochemistry experiments,we found that knocking down Afr Dsx~M induced the expression of the AfrVtg gene,which normally appears only in females,in the male brine shrimp,while knocking down Afr Dsx~F did not significantly affect the expression of AfrVtg.These results suggest that Afr Dsx~M plays a crucial role in maintaining the development of male brine shrimp’s reproductive organs while inhibiting the formation of female reproductive gland characteristics.To investigate whether AfrDSX protein directly interacts with the AfrVtg gene promoter region,we first synthesized a double-stranded DNA probe carrying the predicted AfrVtg promoter DSX binding site with a digoxigenin(DIG)label at the 3’end.Next,we used a prokaryotic expression system to express and purify the Afr DSX GST-DM recombinant protein.The results of electrophoretic mobility shift assay(EMSA)showed that the GST-DM recombinant protein formed a protein-DNA complex with the DNA probe.In the cold probe competition experiment,the addition of a 100-fold excess of unlabeled wild-type probe effectively competed with the DIG-labeled probe,resulting in the disappearance of the protein-DNA complex band.However,a 100-fold excess of mutated probes(with AA replaced by GG in the core region of the DSX binding site)could not compete.These results further illustrate the specificity of the binding of Afr DSX protein to the AfrVtg promoter.In summary,this study identified the DMRT protein family through the transcriptome analysis of sexually dimorphic brine shrimp.Our findings demonstrate that Afr DSXM acts as a transcriptional suppressor,selectively recognizing and binding to downstream target genes,including AfrVtg,and inhibiting the expression of genes associated with female reproductive gland development.This study provides an important theoretical basis for understanding the sexual development mechanism of crustaceans and developing mono-gender breeding techniques.