Early Development Observation Of Sipunculus Nudus And Study On Key Genes Of Oocyte Development

Sipunculus nudus has high economic and ecological value.The existing research on the reproductive biology of S.nudus is still weak,which restricts the improvement of artificial breeding techniques.The germ cells of S.nudus are freely developed in the coelomic fluid and the oocytes at different developmental stages are independent.In this study,ultrastructural observation combined with transcriptome sequencing was used to understand the molecular biological processes of the development of S.nudus oocytes,and key genes were screened for cloning and expression verification.The result couldprovide data support and theoretical guidance for the reproductive regulation and artificial breeding of S.nudus and alsoaccumulate basic data for the molecular mechanism of invertebrate oogenesis.The specific research results are as follows:1.The development process of S.nudus oocytes,embryos and larvae was observed by light microscopy and electron microscopy.The results showed that:(1)according to the ultrastructure of oocytes and the changes of existing sites,the development process can be divided into 6 stages.The Egg1-Egg4 developed in the coelomic fluid,which mainly synthesized and accumulated various substances in the oocyte growth stage;Egg5 developed in the nephridium,and germinal vesicle breakdown usually;Egg6 was emitted into the water.(2)the diameters of oocyte between Egg1-Egg4 were determined,thus the oocyte can be isolation by screen.(3)Embryonic and larval went through six development stages including cleavage,blastula,gastrula,trochophore,pelagospheric larva,and creeping larva.From cleavage to trochophore occurred within the egg envelope.The pelagospheric larva,feed on the benthic diatoms,was out of the membrane.2.A transcriptome library of oocytes Egg1-Egg6 was constructed,and 82,485 Unigenes were obtained.And functional annotations were performed.22,691 CDS,10,132 SSRs were detected,and 3,926 Unigenes encoding transcription factors were predicted.Differential expression analysis showed that Egg2 VS Egg3 and Egg4 VS Egg5 were the two most important turning points in oocytes development.KEGG and GO enrichment analysis found that Egg2 VS Egg3 involved in gene replication,transcription and translation process-related pathways were significantly enriched,and the related genes showed a significant upward trend,indicating that Egg3 has obvious DNA replication and protein synthesis process.The pathway about membrane change was significantly enriched in Egg4 VS Egg5.This result was consistent with that of ultrastructure observation which occurred the GVBD in Egg5.We selected out the vitellogenin,ferritin,Peritrophin,cyclin B,collagen,heat shock protein,growth arrestspecific protein 8,which involved in accumulation,cell cycle regulation,extracellular structure formation,immunity,protein folding and degradation,transcriptional regulation,and cytoskeletal remodeling.3.The stability of reference genes was evaluated by four methods.The results showed that TBP(1)can be used as the optimal single reference gene for qRT-PCR analysis of S.nudus.60S-L7 can be used as the optimal single internal reference gene in qRT-PCR analysis of oocytes or combined with 60S-L10 as the optimal double reference gene.4.The full-length cDNA sequences of five genes including Vg,Peritrophin,FoxL2,Ferritin,Hsp90 were obtained by RACE technology.We also performed bioinformatics analysis such asORF,amino acid sequence,protein domain,transmembrane domain,tertiary structure prediction and homology comparison.And qRT-PCR analysis showed that five genes were expressed in the different periods oocytes.Vg,Peritrophin,Ferritin,Hsp90 genes were highly expressed in Egg3 and Egg4,while FoxL2 genes were highly expressed in Egg5 and Egg6.The results of qRT-PCR were consistent to the transcriptome data.5.In the process of experimental animal breeding,some automated breeding facilities were designed,including cooling system used in little water,miniature frequency-adjusting air pump,particulate feed feeding device,and micro-feed mixing and distribution device.