Molecular Cloning And Expression Analysis Of Five Genes Related With Sex Or Reproduction In Macrobrachium Nipponense

Oriental river prawn, Macrobrachium nipponense, distributes everywhere in east Asian countries including Chaina. It is an important species for aquaculture in China with an annual cultured production of about 250,000 tons and an annual output value of over 10 billion RMB. Male individuals of oriental river prawn grow faster and have a larger body size at harvest than female individuals of the species. However, it is difficult to develop an effective technology to perform large-scale cultivation of all male population due to unknown the molecular mechanism of sex control (including sex determination and sex gonad differentiation) in M. nipponense so far. As a result, it's very important to investigate the molecular mechanism of sex determination and sex gonad differentiation (reproduction)in oriental river prawn. In our laboratory androgenic gland transcriptome and gene expression profile of testis and ovarywere constructed in M. nipponense. These data provide theoretical guidance for screening of sex or reproduction related genes. In this study, 5 candidates of sex or reproduction related genes (Feminization-1 homolog b, Nitric oxide synthase, glycogen debranching enzyme and two Serine protease inhibitors) of M.nipponense were chosen to clone their cDNA sequence and analyze their temporal and spatial expression patterns in order to clarify whether those genes involve in sex control or not. We wish the work would promote the studies of molecular mechanism of sex control.Feminization-1 homolog b (Fem1b) is one of the genes essential for male development and play central roles in sex determination of Caenorhabditis elegans. In this study, we cloned and characterized the full-length Fem1b cDNA from Macrobrachium nipponense(MnFem1b). Real-time quantitative reverse polymerase chain reaction (qPCR) showed that MnFemlb gene was expressed in all investigated adult tissues. The expression levels in all investigated adult tissues showed a certain degree of sexual dimorphism, the expression levels in males was significantly higher than that in females (P < 0.05). Notably, the highest expression of MnFemlb was found in the testis. The results revealed that the MnFem1bgene might play roles in male reproduction. The qPCR also revealed that MnFemlb expression was significantly increased at 10 days after metamorphosis.According to the histology slice analysis, androgenic gland starts to develop at 10 days after metamorphosis in M. nipponense. That implies Femlb involves in the differentiation of androgenic gland.Nitric oxide synthase (NOS) produces nitric oxide (NO) by catalyzing the conversion of L-arginine to L-citrulline, with the concomitant oxidation of nicotinamide adenine dinucleotide posphate (NADPH). Recently, various studies verified the importance of NOS in vertebrates and invertebrates. However, knowledge about NOS gene family in Macrobrachium nipponense remains unknown. In this study, we cloned and characterized the full-length NOS cDNA from M. nipponense (MnNOS). Real-time quantitative reverse transcription-polymerase chain reaction (qPCR) showed that the MnNOS gene was expressed in all the investigated adult tissues, with the highest level of expression in the androgenic gland (P<0.05). The results revealed that the MnNOS gene may play a role in male sexual differentiation of M. nipponense. Moreover, qPCR revealed that MnNOS mRNA expression was significantly increased at 10 days after metamorphosis (P<0.05).Because androgenic gland starts to develop at 10 days after metamorphosis, Femlb also probably involves in the differentiation of androgenic gland.A complementary DNA (cDNA) that encodes the glycogen debranching enzyme (AGL)was cloned in Macrobrachium nipponense (MnAGL). The coding region consists of 5451 base pairs (bp) that encode a 1707 amino acid protein, with a predicted molecular mass of 189.3 kDa and pI of 5.93. The coding region is 327bp 5'-untranslated region (UTR) and a 256bp 3'-UTR. The deduced amino acid sequence has typically conserved domains, such as a Pfam: Hgde N domain, Pfam Alpha-amylase domain, hGDE_central domain and GDE_C domain. Quantitative real-time PCR (qPCR) indicated that MnAGL is highly expressed in the different tissues and at different developmental stages. Its functions need to investigate further.Serine protease inhibitors (Serpins) have been found in a diverse range of organisms and have many functions as a regulator of various biological processes such as coagulation of haemolymph, proPO activation and synthesis of antimicrobial peptides. Two genes from Serine protease inhibitor family designated as MnSERPIN1 and MnSERPIN2 were cloned from M. nipponense testis. The full length of MnSERPIN1 and MnSERPIN2 cDNA sequence was 1879 bp and 1788bp encoding 413 and 416 amino acid respectively.MnSERPIN1 displayed an estimated molecular mass of 45.57 kDa and theoretical pI:8.99.MnSERPIN2 displayed an estimated molecular mass of 45.75 KDa and theoretical pI:8.55.Adult tissues distribution showed MnSERPINl expressed in gill, abdominal ganglion and the highest expression level in testis, whereas MnSERPIN2 expressed only in testis among all tested adult tissues. The results indicated that these two genes may play important roles in male reproduction of M. nipponense.