| Oriental river prawn, Macrobrachium nipponense, extensively distributed everywhere in China. It is an important speciese for aquaculture in China with an annual cultured production of about 237,000 tons and an annual cultured output value of near 15 billions RMB￥ in 2012. Because of males individual of the oriental river prawn grow faster and expected to be larger size during harvest than females individual of the species. In addition, autumn propagation is a common phenomenon in the culture of oriental river prawn, leading to multigenerational together. Monosex culture will help increase social and economial value and control autumn propagation. However, it is still difficult to develop such effective technologies to implement large-scale cultivation due to unknown the molecular mechanism of sex determination and differentiation in the M. nipponense so far. As a result, it’s very important to understanding on the molecular mechanism of sex determination and differentiation in oriental river prawn. To understand the controlling mechanism of sex, a testis high-quality normalized cDNA library of orienial river prawn M. nipponense was constructed and ESTs were analyzed.12 genes related to sex determination and sex differentiation were discovered. cDNA of sex determination and sex differentiation related genes including transformer 2 (Mntra-2), sex lethal (Mnsxls), male-special lethal 3 (Mnmsl3) and extra sex combs (Mnesc) were obtained basis on expressed sequence tages, followed by analysis of characterization and spation-temporal expression. Moreover, RNA interference technology was also tentatively researched in M. nipponense. These data provide theoretical basis and guidance for illustrating the molecular mechanism of sex determination and differentiation and developing effective technologies on sex control in M. nipponense. In this paper, several aspects of researches were carried out as follows:1. Gonadal sex differntiation in M. nipponense and its relation to shrimp size and dayIn the present study, we systematically studied gonadal sex differentiation of the M. nipponense by histology section method and its relation to shirmp size and age from 1 to 35 days after metamorphosis using a fast-growing batch (FGB) and a slow-growing batch (SGB) of shrimp. The results indicated that the gonadal differentiation in M. nipponense was more related to day than to body size. The gonadal primordium was presented in M. nipponense 15th day after metamorphosis, the ovarian differentiation was initated in the 18th day after metamorphosis, and the ovarian cavity formed in 30th day. The testis began to different in 20th day after metamorphosis with the character of forming seminiferous tuble, which was filled with germ cells in the 25th day after metamorphosis. Ovary and testis differentiation were finished in the 30th day after metamorphosis.These findings indicate that the time that the ovarian differention was earlier than that of testis. Based on our results, we suggest that the critical period of sex differentiation in M. nipponense occurs between 15-20 day after metamorphosis.2. Screening, cloning and temporal-spatial expression of sex determination-related genes from M.nipponenseIn this study, a testis cDNA library of oriental river prawn is constructed, using the combination of SMART (Switch mechanisms at the 5’end of RNA transcript) and DSN (duplex-specific nuclease) techniques to reduce the abundance and discover functional genes at high level of transcripts.12 genes were identified to be sex-determination by GO classification and sequence comparability analysis with other publications, such as fruitless, transformer-2, sex-lethal, male sex-lethal 3, and so on.Four sex determination -related genes including Mntra-2, Mnsxl 1, Mnsxl 2 and Mnmsl 3 were cloned using rapid amplification of cDNA ends (RACE). Two of them such as Mntra-2 and Mnmsl 3 cDNAs isolated from M. nipponense testis with the size of 1,724 bp and 1378 bp respectively. The open reading frams (ORFs) of them are 579 and 1092 bp, which encode 192aa and 363aa respectively. We discovered that there two transcripts of Sxl gene, designated as Mnsxl 1 and Mnsxl 2. Mnsxl 1 and Mnsxl 2 with the size of of 1138bp and 1214bp, encode 308aa and 241aa respectively. There are 67aa residues absent in the C-terminal in the Mnsx1 2. The deduced amino acid sequences of Mnsxl 1 and Mnsxl 2 showed high sequence homology to the insect Sx1 and contained conserved domains in two RNA-binding motifs. The encoded protein (Mnmsl 3) has a Chromatin organization modifier domain (CBD) at the N-end and a MRG domain at the C-end. Poylogenetic analysis showed that Mntra-2, Mnsxl 1, Mnsxl 2 and Mnmsl 3 had a closer phylogentic relationship with Hymenoptera.To compare with the relative amount of mRNA for Mntra-2, Mnsxl 1, Mnsxl 2 and Mnmsl 3 between development stages (embryogenesis, larvae, post-larvae) and adult tissues, all samples were detected by qRT-PCR using beta-actin as an internal refence. qRT-PCR analysis results showed that the Mntra-2, Mnsxl 1, Mnsxl 2 and Mnmsl 3 genes were expressed in all investigated tissues, but expression levels of them were different between testis and ovary. The highest expression level of Mntra-2, Mnsxls and Mnmsl 3 is in the muscle and intestine, intestine and liver, testis respectively. The expression profile of them during the embryogenesis and post-larvae are similar. The levels of Mntra-2、Mnsxl 1^ Mnsxl 2 and Mnmsl 3 are the highest at the nauplius stage, the 1st～5th and the 15th～20th day post-larvae after the metamorphosis, which suggested that them may play an important role in embryogenesis and sex differentiation of M.nipponense.PCR was performed to amplify genomic DNA fragment of Mntra-2 using primers, which were designed on the full-length of Mntra-2 cDNA and the intronic sequences cloned. Spliced the isolated the DNA fragment sequences, the Mntra-2 DNA sequences cloned being comprised of 5 exons and 4 introns. Comparison of the cDNA and genomic sequences for Mntra-2 enabled the identification of an upstream 5’non-coding exon.3. Molecular cloning, expression and RNAi of Esc gene in M.nipponens.We have isolated the full-length cDNA of an Esc gene from the testis of oriental river prawn(M.nipponense) according to the established expressed sequence tags (ESTs) information using Rapid Amplification of the cDNA Ends (RACE) technique, designated as Mnesc. The full-length cDNA of Mnesc is 1,461 bp in size and has an open reading frame (ORF) of 1,068 bp, encoding a 355-amino acid protein.The protein have five WD40 repeats domains and a potential nuclear localization signal (NLS) in the N-terminal region. In addition, it has casein kinase Ⅱ phosphorylation site, protein kinase C phosphorylation site, an amidation site, a myristyl N-myristoylation sites and N-glycosylation site too. qRT-PCR analysis showed that mainly expression in the brain and ovary of the Mnesc transcript and little expression in the testis, which indicated that Mnesc has important role in oogenesis, its biological function may be controlled by central nervous system. Further qRT-PCR analysis during the developmental stages of embryo and postembryonic showed that Mnesc was mainly expression in the blastula stage of embryonic and the fourth larvae. These data indicate that the Mnesc may play important roles in the embryogenesis, oogenesis and morphological differentiation of the early larva, and may be used as a molecular marker for future studies of M. nipponense embryonic development. In vivo silencing of the gene, the dsRNA of Mnesc caused a significantly decrease in target gene expression level. RNA interference technology will provide an effective molecular biological approach for the identification of gene function and antiviral research in M. nipponense. |
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