Molecular Cloning And Expression Analysis Of Genes Of Kazal-Type Serine Proteinase Inhibitor And Sperm Gelatinase From Macrobrachium Nipponense

Oriental river prawn, Macrobrachium nipponense, subordinated to Decapoda, Palaemonidae, Macrobrachium, is an important aquaculture species in China due to its advantages, such as high nutrient value, fast growth rate, short reproductive cycle and less diseases. Precocious puberty is a major problem along with the expansion of prawn culture in recently years. To resolve problem of precocious puberty and to develope high-qualified varities of oriental river prawn, it is necessary to research the mechanism of reproductive development of oriental river prawn. Here, two genes of male production related genes were studied based on the testis cDNA library of oriental river prawn.First, a full-length KSPI (Kazal-type serine proteinase inhibitor) cDNA sequence was cloned from the testis of oriental river prawn (Macmbrachium nipponense) for the first time, using RACE technique. The full length KSPI cDNA was890bp, containing a80bp5’untranslated region (UTR), a546bp3’UTR and a246bp open reading frame (ORF) encoding87amino acids. There is a conserved Kazal-type domain, CⅠX5CⅡX(6)VCⅢX(5)TYXNXCⅣX6CⅤX12CⅥ, and its P1active site was threonine, in this gene. The phylogenetic tree based on15species KSPI proteins showed that KSPI from testis of M. nipponense shared the closest relationship with M. rosenbergii.and KSPI from hepatopancreas and hemocytes of other species formed other two main branches. The expression levels of KSPI in heart, ovary, testis, brain, liver and intestine were detected by Quantitative PCR (qRT-PCR). It was found that KSPI was rarely expressed in liver, brain and intestine, whereas relatively higher in ovary, heart and highest in testis. The differences level between testis and heart, testis and ovary were P<0.05and P<0.01, respectively. This implied that KSPI may play an important role in the male-reproductive process of oriental river prawn.Then, a full-length SG (sperm gelatinase) cDNA sequence was cloned from the testis of oriental river prawn (Macmbrachium nipponense) for the first time, using RACE technique. The full-length cDNA sequence of SG gene was711bp with a271bp3’-untranslated region, a56bp5’-untranslated region and a408bp open reading frame encoding127amino acids. A peptide signal, containing21amino acids, is known as Macrobrachium nipponense Sperm gelatinase (MnSG-Fu). Based on blast results of the nucleotides and proteins in the NCBI database, only one SG gene sequence which was homologous to this gene was found in M. rosenbergii with90%of identification. The expression levels of SG gene in different tissues of oriental river prawn, such as heart, testis, ovary, brain, liver and intestine were detected by qRT-PCR. The results showed that SG was highly expressed in the testis, while weakly expressed in heart. Moreover, SG cannot be detected in ovary, brain, liver and intestine of oriental river prawn, these results indicated that this gene was a male-specific gene in the reproductive system of M.nipponense and extremely expressed in male individual, suggesting that this gene may play an important role in the male reproductive process. However, the gene function needs further studies.Our results not only supplied basic information for the research on the function of Kazal-type serine proteinase inhibitor (KSPI) and M.nipponense sperm gelatinase (MnSG) genes of oriental river prawn, as well as providing reference for the research on the function and structure of reproduction-related genes in oriental river prawn, but also improves the researches on the reproduction-related genes in other crustacean species.