Cloning And Spatial-Temporal Expression Of Female Sterile Homeotic Gene (FSH) In Macrobrachium Nipponense

Oriental river prawn, Macrobrachium nipponense, is an important shrimp species in freshwater aquaculture. However, sexual precocity is a common phenomenon with the extension of farming, leading to multigenerational together, high amount of feed and falling commodity rate, and heavily autumn propagation. These negative problems have seriously hampered the further development of oriental river prawn farming. Therefore, the researches to carry out the genes related with gonadal development in oriental river prawn will not only provide information for molecular assisted breeding, but also has important application value to control phenomena such as autumn propagation in practice.The maternal-effect gene female sterile homeotic (fsh) has been reported in Drosophila melanogaster, related to female reproduction and growth. According to the previous studies, zygotic mutations of fsh cause either lethality or female sterility, whereas maternal mutations cause segmental deletions and thoracic homeotic transformations. However, fsh was not studied and reported in crustaceans. In this study, a highly homologous fragment of fsh was identified from the testis of orinental river prawn Macrobrachium nipponense, named as Mnfsh and the full-length cDNA sequence was obtained for the first time by the combination of reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The full-length cDNA sequence of fsh is2029bp, containing an open reading frame of1452bp,361bp of5’-untranslated region (UTR),462bp of3’-UTR with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail. The gene encodes a polypeptide of483amino acids with an estimated molecular mass of52.641kD and a theoretical pI of8.78. The deduced amino acid sequence analysis showed that Mnfsh contains the Bromodomain region, whereas the gene does not have signal peptide and transmembrane region, implying it is the nuclear proteins. The amino acids of Mnfsh shares including89%,90%and63%similarity to Harpegnathos saltator, Camponotus floridanus and Drosophila melanogaster, respectively, through the comparison with the amino acids of fsh in other insect. In addition, the spatial-temporal expression pattern of Mnfsh was detected by Quantitative real-time PCR (qRT-PCR) for the first time. According to the analysis results, the expression level reached the peak in matural ovum(O). The expression was slightly increased in zoea stage (ZS) during embryogenesis, the larvae VII (LVII) in the larvae and the1st day of the post-larvae (PL1). Based on the expression pattern in adult tissues, the highest expression level was observed in ovary, followed by eyestalk, muscle, testis, abdominal ganglion and heart.It is the first time to clone the full length cDNA sequence of Mnfsh gene and analysed the structure of Mnfsh gene in this research. Besides, qRT-PCR technique was firstly used to detect the spatial-temporal expression pattern of Mnfsh gene during embryogenesis, larvae, post-larvae and expression pattern of adult tissues. The results strongly support that Mnfsh is the maternal-effect gene, revealing that Mnfsh is an arthropods fsh homologue and probably also played important roles in embryogenesis, organogenesis and morphological differentiation of oriental river prawn. This research could provide basic information for the function of Mnfsh gene and references for further studies in molecular assisted breeding, related to the gonadal development process of oriental river prawn, as well as in other crustacean species.