Analysis Of Ovary ESTs And CDNA Cloning And Expression Profile Of Reproduction-related Genes From Macrobrachium Nipponense

Ontogenesis of germ cells is an important subject in developmental biology.The animal ovary is functionally important in reproduction and secretion of hormones for growth and development regulation.Unfortunately,the regulative mechanism for ovarian maturation of decapods at the molecular level is quite limited compared with some model organism,as well as in freshwater prawn.For most prawns,however,the number of expressed sequence tags(ESTs)available is inadequate.There were only 11 ESTs of Macrobrachium in GenBank,and no any EST of oriental river prawn was found.Acquiring plenty of reproduction-related genes may prompt prawn genomics and developmental biology.Currently,some functionally conserved genes through evolution have been discovered,which might be useful for an extensive investigation on germ cells of non-model animals.In this thesis,an ovary cDNA library of oriental river prawn Macrobrachium nipponense was constructed and ESTs were analyzed.29 genes related to reproduction and development were discovered.cDNAs of reproduction-related genes including DExD-box family and Mn-cyclin B,Mn-cdc2, Mn-Peritrophin were gained using a homologous cloning strategy or basis on expressed sequence tags,followed by analysis of characterization and spatio-temporal expression.There were four major parts,listed as follow:1,Gene discovery from an ovary cDNA library of Macrobrachium nipponense by ESTs annotationA high-quality cDNA library of M.nipponense was constructed from the ovary tissue.A total of 3,294 successful sequencing reactions yielded 3,256 expressed sequence tags(ESTs)longer than 100bp.The cluster and assembly analyses yielded 1,514 unique sequences including 414 contigs and 1,168 singletons.About 719 (47.5%)unique sequences were identified as orthologs of genes from other organisms. By sequence comparability analysis,29 important genes including cathepsin B, chromobox protein,Cdc2,cyclin B,DEAD box protein and ADF/Cofilin protein were expressed.These genes may be involved in reproductive and developmental functions in prawn.Peritrophin consisting of cortical rods was also found in this species.2,Cloning and expression profile of reproduction-related DExD-box family genes from M.nipponenseFour genes of DExD-box family,including Mn-vasa,Mn-PL10,Mn-p68 and Putative Mn-DDX39 were cloned using rapid amplification of cDNA ends(RACE). Three of them such as Mn-vasa,Mn-p68 and Putative Mn-DDX39 cDNAs isolated from M.nipponense ovary with the size of 2391 bp,2174 bp,1774 bp respectively. The open reading frames(ORFs)of them are 1806 bp,1623 bp and 1299 bp,which encode 601aa,540 aa and 432 aa respectively.We first discovered that there were two alternative spliced isoforms of Mn-PL10(Mn-PL10A and Mn-PL10B).Mn-PL10A and Mn-PL10B with the size of 2247 bp and 2649 bp,encode 485 aa and 709 aa respectively.There is 224 aa residues absent in the N-terminal in Mn-PL10A isoform. Mn-vasa,Mn-PL10A,Mn-PL10B,Mn-p68 and Putative Mn-DDX39 proteins share nine conserved motifs and a GG doublets with other DExD-box members.The conserved phe is in 17 amino acid residues upper the Q-motif except Mn-PL10A.Mn-vasa contains a zinc-finger CCHC motif conserved among vasa-related proteins.There is a RGG repeat in Mn-vasa and three RGGs in Mn-PL10B,which only found in vasa-related and PL10-related proteins.All the conserved structures ensure the RNA helicase function.RT-PCR analysis of spatial expression revealed a specific expression of Mn-vasa in gonads,suggesting that Mn-vasa might be involved in M. nipponense gametogenesis.To compare with the relative amounts of mRNA for Mn-vasa between stages during oogenesis,ovary samples from development stages were subjected to quantitative Real-time RT-PCR analysis using beta-actin as an internal reference.The level of Mn-vasa transcript is the highest at perinucleolus stage, but drops at followed development stages and reaches the lowest point at paracmasis stage.The results indicated that Mn-vasa might play a role during the oogonium formations.In addition,RT-PCR results also indicated that mainly expression in ovary and hepatopancreas of Mn-PL10,Mn-p68 and Putative Mn-DDX39,little expression in other tissues.The expression profiles of the transcripts during oogenesis are similar.The levels of Mn-PL10,Mn-p68,Putative Mn-DDX39 are the highest at yolk granule stage and reach the lowest point at paracmasis stage,which suggested that Mn-PL10,Mn-p68,Putative Mn-DDX39 might be involved in prawn oocyte maturation.This is the first report on p68 and DDX39 in crustacean and the discovery of expression profile of Mn-DDX39 during oogenesis might prompt DDX39 function research.3,Cloning and expression profile of cyclin B and Cdc2 genes from M.nipponenseThe meiotic maturation of oocyte in animals is regulated by maturation promation factor(MPF),a complex of Cdc2 and cyclin B.In this study,full-length cDNAs of cyclin B and Cdc2 was cloned using RACE technique.The prawn cyclin B cDNA was 2318 bp containing a long 3' untranslation region(UTR)of 989 bp and an open reading frame encoding for a protein of 398 amino acids.There are two cytoplasmic polyadenylation signals,five cytoplasmic polyadenylation elements(CPEs),a translation control element(TCE)and a K-box motif in the 3'UTR of prawn cyclin B transcription,which suggested that the translation or post-transcription of Mn-cyclin B might be regulated cooperatively via all the present elements.The deduced amino acid sequence of Mn-cyclin B has high identity with the reported cyclin B sequences of other species in the GenBank,sharing 69%,68%,60%and 60%identity with Penaeus monodon,Marsupenaeus japonicus,Scylla serrata,Eriocheir Sinensis respectively. Several obvious sequence motifs or domains are present in the Mn-cyclin B:a cyclin family signature,the consensus sequence known as the destruction box and the amino acid residues of pkA site.Poylogenetic analysis showed that Mn-cyclin B was highly conserved through evolution.A Blast searching GenBank database revealed that the deduced amino acid sequence of the prawn Cdc2 kinase shared 92%identity with P. monodon,91%with S.serrata and 91%with E.sinensis.Mn-Cdc2 kinase include several signature domains,such as elements involved in ATP binding,catalytic domain PSTAIR,Serine/Threonine protein kinase active-site,conserved phosphorylation site Thr161,dephosphorylation sites Thr 14 and Thrl5.RT-PCR results indicated mainly expression in the ovary of Mn-cyclin B transcript,also expression in the testis,muscle,hepatopancreas,skin and hemolymph,little expression in the intestine.The expression of the prawn Cdc2 mRNA was detected mainly in the ovary,testis,hepatopancreas and skin.Quantitative Real-time RT-PCR analysis revealed that the levels of Mn-cyclin B transcript were cyclincal fluctuation during oogenesis.It has a relative high quantity at perinucleolus stage,and drops at fusion nucleolus stage,but a little increase at oil globule stage and reaches the highest point at yolk granule stage,then drops remarkably at paracmasis stage.The fluctuation characteristic is consistent with the role of the cyclin B in cell cycle regulation.The high level of Mn-cyclin B at maturation stage indicated that it is closely related to oocytes meiotic maturation in the prawn ovary.Quantitative Real-time RT-PCR analysis also revealed that the levels of Mn-Cdc2 mRNA showed no statistically significant difference during oogenesis,which is consistent with the mechanisms of Cdc2 kinase regulating cell cycle.4,Cloning and expression profile of Peritrophin genes from M.nipponensePeritrophin is one of the major components of cortical rods in the jelly layer of shrimp and is proposed to play a role in the protection of spawned eggs.At the end of vitellogenesis in penaeid shrimp,oocytes maturation was characterized by the appearance of rod-like bodies arranged radially around the periphery of the oocyte plasma membranes.When the oocytes are released into seawater,the contents of cortical rods are released,forming a jelly layer,a corona,around the egg. Nevertheless,in oriental river prawn,cortical rods were not found even in the fully matured oocytes.It is generally believed that this prawn species lacks cortical rod protein in the oocytes.Peritrophin was found only one EST homology in constructed ovary cDNA library.The full-length cDNA of Mn-Peritrophin was cloned using RACE method.It was 654 bp,including a 5'UTR of 192 bp,a 3'UTR of 314 bp and an open reading frame of 291 bp excoding a polypeptide of 96 amino acids.The protein has a transmembrane helice,a CBM₁4 domain and a cleavage site containing 19 amino acid signal peptide.In addition,it has a casein kinaseâ…¡phosphorylation site,a myristyl N-myristoylation site and a protein kinase C phosphorylation site too. Quantitative Real-time RT-PCR analysis revealed that mainly expression in the ovary and hepatopancreas of the Mn-Peritrophin transcript and little expression in the testis, which indicated that Mn-peritrophin has important role in oogenesis not in spermatogenesis.Further quantitative RT-PCR analysis during the oogenesis showed that the level of Mn-Peritrophin transcript was no difference at oil globule stage but was the highest at the maturation stage,which suggested Mn-Peritrophin might contribution to nutrient accumulation of the oocyte except for yolk proteins.Moreover, it occurs only at the later stage during the oogenesis.The result indicated that the ovaries might contain cortical rod protein or a homologous protein which was a chemical component of cortical rods in penaeid prawn even without the formation of cortical rods in these species.The expression profile in the tissues of the Mn-Peritrophin mRNA also indicated that hepatopancreas might be the main Peritrophin protein synthesis site.The high level of Mn-Peritrophin at maturation stage also suggested that Mn-Peritrophin played a critical role prompting oocyte maturation finally.This dissertation provides sufficient basic data for research on decapods developmental biology.The identification of EST sequences in M.nipponense would improve our understanding on the genes that regulate reproduction and development in prawn species.This study also lays the groundwork for development of molecular markers related to ovary development in other prawn species.Further study on these genes might prompt prawn genomics and developmental biology.It not only provided oriental river prawn gene sequences information for researchers who use the data from NCBI to perform character string searches of the annotations but also facilitated research in crustacean molecular genetics.