CDNA Cloning And Expression Of The Genes Involving In Oogenesis And Embryo Development In Macrobrachium Nipponense

Sexual gametes combine to form fertilized eggs, which develop to adult through embryonic development. Oocytes play an important role in the process because they carry the genetic information and nutritious material. Oocytes are formed in the ovary. So ovary and embryonic development is the core of developmental biology research. Because large-scale shrimps and crabs have high economic and nutritional value, their developmental research has naturally been concerned by many scientists. However, researchers tend to focus on the morphology and physiology in recent years, and the molecular mechanisms on development has been little studied. In this study, five genes related to the development of oogenesis and embryos were cloned from M. nipponense. At the same time their molecular characterization and expression patterning in the process of ovary and embryo development were analyzed. We further discussed their role on oogenesis and embryonic development of M. nipponense. This study provides a useful reference for molecular mechanism of development of M. nipponense as well as crustaceans. This study includes the four parts as follows:1. Molecular cloning, characterization and expression of Mago nashi and Tsunagi in M. nipponenseMago nashi and Tsunagi genes, named as MnMago and MnTsu, were cloned from M. nipponense according to homogenous cloning and the established EST information using rapid amplification of cDNA ends (RACE). Two transcripts of Mago nashi were isolated from M. nipponense with the size of 746bp and 683bp respectively. It was found that the same open reading frame (ORF) and 3'-untranslated region (3'-UTR) but different 5'-UTR in the two cDNA sequences encoding 147 amino acids. The ORF of MnTsu cDNA was 486bp, encoding a protein of 161 amino acids. The Mago domain and RNA recognition motif (RRM) were detected in MnMago and MnTsu proteins respectively based on the motif scan at http://hits.isb-sib.ch/cgi-bin/motifscan and Blast p at http://blast.ncbi.nlm.nih.gov/Blast.cgi. Three-dimensional structure prediction of interaction between MnMago and MnTsu proteins showed that they apparently constituted a Mago-Tsunagi complex, which indicated that they may play a biological role in M. nipponense as in other model organisms. Real-time quantitative PCR (RT-QPCR) analyses revealed that the expression patterns of MnMago and MnTsu gene in the process of embryo and ovary development were consistent completely. In the early phase of embryo, their expression level kept steady, and increased substantially in the later, which suggested that MnMago and MnTsu were related to morphogenesis of later embryonic development. In the development of ovary, the expression level of MnMago and MnTsu gradually increased from the perinucleolus (PN) stage to the yolk granule (YG), and then decreased suddenly in the maturation stage (MA). However, the expression patterns of two genes in mature tissues were a little uniform. The maximum level of MnMago and MnTsu occurred in muscle tissue which indicated that the muscle cell may be in active proliferation. Interestingly, their expression levels in testis were both very low. These suggested that MnMago and MnTsu not only played critical roles in oocyte maturation but also performed multiple biological functions in oriental river prawn.2. Cloning, characterization and expression of gustavus in the development of M. nipponenseThe protein of the gustavus (GUS) gene contains a SPRY domain and a SOCS box. The SPRY domain is thought to mediate Ca2+release from the sarcoplasmic reticulum, and the SOCS box belongs to the suppressor of cytokines signaling family (SOCS) and functions on proteasomal degradation. In this study, the GUS gene was firstly indentified and termed as MnGus in oriental river prawn M. nipponense. Bioinformatics analyses showed that this gene encodes a protein of 261 amino acids with a predicted molecular mass of 29.70 kDa. Phylogenetic analysis showed that MnGus protein was clustered into the clade of SSB-1 in other species. Real-time quantitative PCR (RT-QPCR) analyses revealed that the expression level varied significantly in the process of embryo and ovary development and changed substantially in other tissues. In embryos, the expression level of MnGus was slightly higher in the cleavage stage (CS) than in the blastula stage (BS). Following an increase in the blastula stage (BS), the MnGus reached a maximum in the zoea stage (ZS). In the ovary, the minimum expression of MnGus occurred in the perinucleolus (PN) stage, while the maximum was in the oil globule (OG) stage. The MnGus gradually decreased from the yolk granule (YG) stage to the paracmasis (PM) stage. The expression level of MnGus in muscles was much higher than that in other tissues in a mature prawn. The differential expressions of MnGus in the embryo, ovary and other tissues suggest that the gustavus gene performs multiple biological functions in oriental river prawn.3. cDNA cloning and expression of Ubc9 in the developing embryo and ovary of M. nipponenseUbiquitin-mediated protein degradation pathway is the main regulator of protein function and the terminator. In the same way the small ubiquitin-like modifier (SUMO) pathway in eukaryotes is an essential biological process involving cellular processes, development and organelle biogenesis. In a sequential enzymatic action, Ubc9 is an important conjunction enzyme in the SUMO pathway. Although the Ubc9 has been found in vertebrates, its expression in crustaceans is little known. In this study, the Ubc9 was identified in the embryo and ovary of a freshwater prawn M. nipponense for the first time and it was denoted as MnUbc9. Bioinformatics analyses showed that this gene encodes a protein of 160 amino acids with predicted molecular mass of 18.32 kDa. Real-time quantitative PCR analyses demonstrated that the expression levels varied significantly in the developing embryo and ovary. In the embryo, the expression level of MnUbc9 was higher at the cleavage stage (CS) than at the blastula stage (BS), and reached even higher levels at the protozoea stage (PS) and the zoea stage (ZS). In the ovary, the MuUbc9 expression was low at the early stage, but reached the highest at the yolk granule stage (YG), and then abruptly declined at the maturation stage (MA). The differential expressions of MnUbc9 in the embryo and ovary suggest that MnUbc9 may play an important role in embryogenesis and oogenesis of M. nipponense.4. Molecular cloning, characterization and expression of MnvWD-Kazal gene in M. nipponenseA new gene, named as MnvWD-Kazal, containing the von Willebrand factor D (vWD) and Kazal-type domain was cloned from the ovary of M. nipponense. The full length of MnvWD-Kazal cDNA was 2713bp with the ORF of 2574bp encoding a protein of 857 amino acids. Bioinformatics analyses showed that there were a vWD domain at amino terminal and three Kazal domains at carboxyl terminal of MnvWD-Kazal protein. Real-time quantitative PCR (RT-QPCR) analysis revealed that the expression level of MnvWD-Kazal was very low in the process of embryo, but a regular expression in ovary development. The level of MnvWD-Kazal gene increased gradually from perinucleolus stage to yolk granule stage, and decreased suddenly at maturation. This suggested that MnvWD-Kazal may participate in the process of oocyte maturation of M. nipponense. RT-QPCR analysis of mature tissues showed that the high level MnvWD-Kazal transcript occurred in these tissues, such as intestines, thoracicganglia, yolk granule and heart, especially in intestines. The high expression level in intestines may be related to Kazal domain because the proteinase inhibitor with Kazal domain can restrain prematurity of insulin-activated enzyme zymogen.This is the first report that five genes related to oogenesis and embryo development were cloned from M. nipponense, and their spatio-temporal expression patterns were studied in the development of embryo and ovary by RT-QPCR technique. The two genes of MnGus and MnUbc9 participated in the whole process of embryonic development, while the other two genes of MnMago and MnTsu were extremely relevant to late morphogenesis of embryo. The five genes were all involved in formation and proliferation of oocytes. However the new gene, MnvWD-Kazal, was little to do with the embryo development of M. nipponense. In conclusion, this research not only provided clues to molecular mechanism of oogenesis and a theoretical basis for seedling quality improvement from M. nipponense, but also enriched the developmental biological resources of crustacean.