Cloning,Expression And Regulation Of Fruitless Gene In Gonadal Development Of Mud Crab(Scylla Paramamosain)

The mud crab,Scylla paramamosain,is an important economic marine crab resource.The mud crab is known because of its rapid growth,large size,nourishing and sweet meat,especially the mature female crab has been called"sea ginseng".Sexually mature female crab has higher economic value because of full crab paste and unique flavor.However,the crab farming industry is facing many problems,such as the shortage of artificial crab seedlings,the fighting and killing of crabs,and the difficulty of gender control.The reason is that the lack of research on the molecular mechanism of sex differentiation and development,same-sex struggle and courtship and mating greatly limits the development of crab breeding and larvae breeding.In this study,Spfru1-a,Spfru1-b and Spfru2 were identified and named by analyzing the transcriptome data of mud crab.Spfru1-a and Spfru1-b were alternative splicing isomers.The expression levels of Spfru1-a,Spfru1-b and Spfru2 in different tissues,gonads and mating states of mud crab were detected by RT-PCR.The tissue localization was performed by FISH.Finally,the miRNA expression analysis and silencing and overexpression experiments were carried out to explore the expression characteristics of novel＿miRNA-35 in different developmental stages of mud crab,and preliminarily studied the regulatory relationship between novel＿miRNA-35 and its target gene Spfru2.In this study,by cloning and analyzing the fruitless genes,we identified the fruitless genes:Spfru1-a,Spfru1-b and Spfru2,and preliminarily explored the role of fruitless genes in the sex regulation pathway of mud crab.The main results and conclusions are as follows:1.The Spfru1-a has full length of 3784bp for cDNA with 878 amino acids.The Spfru1-b has full length of 3178bp for cDNA with 475 amino acids.Spfru2 has full length of 4487bp for cDNA with 131bp 543 amino acids.After sequence alignment,it was found that the first 1747bp sequence of Spfru1-a was different from the first 1141bp sequence of Spfru1-b gene,but the last2037bp sequence of Spfru1-a and Spfru1-b was identical,and the sequence identity of the two alternative splicing isomers was 64.98%.The former 517aa sequence of Spfru1-a protein sequence is different from the former 114aa sequence of Spfru1-b protein sequence,but the latter361aa sequence of protein sequence of Spfru1-a and Spfru1-b are identical.The protein sequence consistency of the two alternative splicing isomers is 42.03%.The results of domain prediction show that Spfru1-a,Spfru1-b and Spfru2 all contain conserved BTB domains.The alternative splicing isomers Spfru1-a and Spfru1-b both contain C₂H₂-Zn F domains.Spfru1-a contains two C₂H₂-Zn F domains,while Spfru1-b contains three C₂H₂-Zn F domains.Spfru2 lacks C₂H₂-Zn F domains and contains a BEN domain.The phylogenetic tree constructed by BTB domain sequence showed that the frus of Insecta and Crustacea were clustered into 2 branches.It was obvious that Crustacea and Insecta were divided into two branches.Spfru1-a and Spfru1-b were the closest to Daphnia magna,and Spfru2 was the closest to Eriocheir sinensis.2.RT-PCR results showed that Spfru1-a,Spfru1-b and Spfru2 were expressed in different tissues of mud crab.The expression of Spfru1-a and Spfru2 in ovary was significantly higher than other tissues and testis.The expression of Spfru1-a in female ganglia,intestine and muscle was significantly higher than that in male.The expression of Spfru1-b in testis was significantly higher than other tissues and ovary（P<0.05）.The expression of Spfru1-a was higher in ovary stage I to III,and began to decline in ovary stage IV and V,and was relatively low in the three stages of testis;the expression of Spfru1-b was higher in ovary stage I to ovary stage IV,and significantly decreased in ovary stage V,while the expression of Spfru1-b was higher in testis stage I,testis stage II and testis stage III.The expression of Spfru1-b in testis increased rapidly from the stage II to the stage III,and exceeded ovary at the stage III.The expression of Spfru2was higher in ovary stage I to stage IV,but lower in testis.Therefore,it is speculated that Spfru1-a,Spfru1-b and Spfru2 may have a significant impact on the ovary development and maturation of mud crab,Spfru1-a may also participate in the development of nerve cells,and Spfru2 may also have a great impact on the testis development and maturation of mud crab.The expression of Spfru1-a and Spfru1-b in the gonads of mud crab in different mating states showed that the expression of Spfru1-b increased rapidly（P<0.05）during the period from unmating to premating in male mud crab and the expression of Spfru1-a and Spfru1-b decreased to close to unmated after mating,.Therefore,it is speculated that Spfru1-a and Spfru1-b may be involved in the regulation of mating behavior of male mud crab,while Spfru2 may not be involved in the regulation of mating behavior of male mud crab.In female mud crab,the expression of Spfru1-a gene showed an upward trend,especially from 1 day after mating to 3 days after mating,the expression of Spfru1-a increased sharply;the expression of Spfru1-b showed a downward trend from unmating to premating（P<0.05）,and the expression of Spfru1-b increased to the same level as unmating after mating.There was no significant difference in the expression level of spfru2 between unmating and premating,but the expression level increased from premating to 3days after mating（P<0.05）;On the one hand,because the cells of ovary development of stage I is relatively slow and static,the ovary cells of female mud crab（Scylla paramamosain）begin to enter a rapid development stage after molting and mating.Therefore,it is speculated that spfru1-a and spfru2 may play an important role in the process for rapid development and maturation of ovary.On the other hand,fruitless gene may also be involved in the regulation of female crabs’receptive behavior and post mating behavior,spfru1-b gene may be involved in the regulation of female crabs’courtship behavior.The results of FISH showed that the m RNA of spfru1-a and spfru2 were located in spermatid and spermatogenic epithelia,and spfru1-b was located in spermatid.In ovary,the m RNA of spfru1-a,spfru1-b and spfru2 were localized in the cytoplasm of oocytes.3.Expression of miRNA showed that the expression of novel＿miRNA-35 in ovary and testis at different developmental stages was significantly different.The expression of Spfru2 and novel＿miRNA-35 showed the opposite trend in the ovary and testis of mud crab.The results of overexpression and silencing of novel＿miRNA-35 showed that the expression changes of Spfru2and of novel＿miRNA-35 showed an opposite trend in the silencing and overexpression.Therefore,it is speculated that the target gene of novel＿miRNA-35 is Spfru2,and novel＿miRNA-35 may plays a negative regulatory role through complementary combination of seed sequence with 3’UTR of Spfru2.In conclusion,Spfru1-a,Spfru1-b and Spfru2 may play an important role in the sex determination pathway and gonadal development and maturation.Moreover,the expression of alternative splicing isomers Spfru1-a and Spfru1-b in mud crab has obvious gender specificity,suggesting that the alternative splicing patterns of fruitless gene in mud crab may similar to Drosophila melanogaster.The novel＿miRNA-35 may play an important role in sex determination,differentiation and maturation of mud crab by negatively regulating the target gene spfru2.