| The golden cuttlefish,Sepia esculenta Hoyle,1885,is an important economic species of cephalopod family.It was once an important fishing target in the Yellow Sea and Bohai Sea in China.However,since the 1980s,due to overfishing,habitat destruction and global changes,the golden cuttlefish resources have declined sharply.Golden cuttlefish has the characteristics of rich nutrition,rapid growth,large weights(700g),rapid replacement of short generations in the life cycle(about 1 year),and distinct migratory habits.To restore and utilize the resources of S.esculenta,researchers began to overcome the golden cuttlefish artificial breeding and proliferation and release technology,and achieved breakthroughs around 2010.In order to develop the reproductive regulation technology of golden cuttlefish,it was urgent to reveal the genetic basis and molecular mechanism of its growth,reproduction and death.Studies on the reproductive biology of golden cuttlefish showed that the amount of eggs of the sexually mature golden cuttlefish was about 2500,and the ovulation period lasted for about one month.But the number of spawns was only 1/3 of the eggs.In the later period of reproduction,the parents died successively,with a large number of mature eggs remaining in the dead female’s ovary.It can be seen that the reproductive capacity of the golden cuttlefish was restricted by certain factors.Can the ovulation period of golden cuttlefish be prolonged?What factors regulated the rapid death of golden cuttlefish after reproduction?In order to address these issues,the present study intended to use transcriptome sequencing technology to study the regulation of transcript expression and regulation of subpeduncle lobe/olfactory lobe-optic gland-gonadal axis at different periods of S.esculenta.Through the above research,it not only filled in the blank of the molecular mechanism research of the aging process of the golden cuttlefish,but also provided basic information for revealing the mechanism of the subpeduncle lobe/olfactory lobe-optic gland-gonadal axis regulating the rapid post-reproductive death of S.esculenta.The main results were as follow:1.Construction of full-length transcriptome of female Sepia esculenta and quality evaluationTotal RNA was extracted from brain,optic gland,ovary and nidamental gland samples at different periods of golden cuttlefish and cDNA libraries were constructed.The full-length transcript of the golden cuttlefish was obtained by using PacBio single-molecule real-time(SMRT)sequencing technology combined with Illumina RNA-Seq technology.A total of 14.16 Gb SMRT sequencing data were assembled into 94,635 transcripts.Meanwhile,35.15 Gb Illumina HiSeq data were assembled into 177,226 non-redundant transcripts.Then,we merged SMRT and Illumina assembled data to generate a more complete/full-length S.esculenta transcriptome with 177,951 high-quality transcripts.Based on the obtained transcriptome data,total 81,459 transcripts were annotated in at least one of seven functional databases(Nr,Nt,Swiss-Port,Interpro,GO,COG and KEGG)and 49,189 nucleotide sequences of coding regions were identified.Additionally,161,327 SSRs distributed in 64,933 transcripts were identified based on SSR analysis.The full-length transcript of S.esculenta would provided information for studying the post-reproductive death mechanism of golden cuttlefish in the subpeduncle lobe/olfactory lobe-optic gland-gonadal axis.2.Transcriptome analysis of the subpeduncle lobe/olfactory lobe and screening of the post-reproductive death regulatory signaling pathways in female Sepia esculentaCephalopods have complex brain structures that are most developed in invertebrates and even comparable to vertebrates.The subpeduncle lobe of the brain and its adjacent areas,located below the vertical lobe of the esophageal nerve mass of the central nervous system,inhibits the secretion of the optic gland,which is an important endocrine organ related to reproductive regulation and can regulate gonad development and maturation.The subpeduncle lobe/olfactory lobe is equivalent to the hypothalamus in vertebrates,which can secrete and produce many neuroendocrine factors.What are the changes in gene expression patterns in the subpeduncle lobe/olfactory lobe before and after reproduction?Which signaling pathways are involved in regulating the secretion of the optic gland?To address these issues,the transcriptome analysis was performed on the brain tissue of female S.esculenta in the three key periods:growth period(BG),spawning period(BS),and post-reproductive death period(BA).Finally,66.19 Gb clean data were obtained.A comparative analysis of the 3 periods showed 2,609,3,333,and 170 differentially expressed genes(DEGs)in BG-vs-BA,BG-vs-BA,and BS-vs-BA,respectively.The Gene Ontology(GO)enrichment analysis of DEGs revealed that the regulation of cyclin-dependent protein serine/threonine kinase activity,oxidative phosphorylation,and respiratory chain were significantly enriched.The significant enrichment analysis of the Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway identified pathways associated with the regulation of death,such as the mammalian target of rapamycin(mTOR)signaling pathway,AMPK signaling pathway,oxidative phosphorylation,and cell cycle.The above results revealed that the post-reproductive death of S.esculenta was a complex signaling pathway regulation network.The mTOR molecule acted as a nerve center-brain energy receptor and had a key role in the regulation of energy homeostasis.3.Transcriptome analysis of the optic gland and screening of neuropeptide genes in female Sepia esculentaThe Illumina transcriptome was sequenced in the optic gland of golden cuttlefish in spawning period(OS)and post-reproductive death period(OA).A total of 45.21 Gb clean data were obtained,with Q20>96%.Then,the transcriptome data of optic gland in the OS-vs-OA group of S.esculenta were compared and analyzed.The number of DEGs in OS-vs-OA group was 94,of which 53 were up-regulated genes and 41 were down-regulated genes.A further GO-significant enrichment analysis was performed on DEGs in OS-vs-OA group,and 35 GO entries were enriched to a significant level(corrected-P value-0.05).GO enrichment analysis of DEGs revealed that the regulation of cyclin-dependent protein serine/threonine kinase activity,oxidative phosphorylation,mitochondrial inner membrane and respiratory chain were significantly enriched.The KEGG metabolic pathway database was compared and annotated for all DEGs.The results showed that the OS-vs-OA group had 95 metabolic pathways,in which 53 DEGs were upregulated and 61 downregulated.Further,the relevant pathways with the highest degree of apparent enrichment of optic gland of golden cuttlefish in spawning and post-reproductive death stage were identified,including oxidative phosphorylation,amino acid metabolism(tryptophan metabolism),lipid metabolism(ketone body synthesis and degradation),estrogen signaling pathway,ubiquitin-mediated proteolytic pathway,MAPK signaling pathway,AMPK signal pathway.The optic glands and their secretions were related to their reproduction and death,but most importantly,were the optic gland secretions of golden cuttlefish a neuropeptide-like substance that had a neurohormonal-like effect on the body?To address these issues,this study used the BLAST tblastn program combined with the Openoffice software search procedure to share homology and conservation based on other crustacean and invertebrate neuropeptides.A comprehensive genetic screening was performed in the transcriptome data of optic gland.As a result,a total of 17 neuropeptide gene families with 51 neuropeptide genes were obtained in the optic gland transcriptome data of the spawning period;and 19 neuropeptide gene families with 56 neuropeptide genes were identified in the transcriptome data of post-reproductive death period.It can be seen that the optic glands transcriptome of S.esculenta had abundant neuropeptide genes,which provided a foundation for studying the endocrine function of optic glands.4.Transcriptome analysis of the ovary tissue and screening of key signaling pathways and key genes in Sepia esculentaThe Illumina transcriptome analysis was performed on the ovary tissue of female S.esculenta in the three key periods:growth period(IG),spawning period(IS),and post-reproductive death period(IA).Finally,66.65 Gb clean data were obtained,with Q20>96%.The transcriptome of ovary tissue in the three periods of IG,IS,and IA were compared and analyzed.In the IG-vs-IS group,10,290 DEGs were found,including 4,997 upregulated and 5,293 downregulated genes.In the IG-vs-IA group,17,107 genes were differentially expressed;the number of upregulated and downregulated genes was 5,490 and 11,617,respectively.In the IS-vs-IA group,of the 380 DEGs found,303 were upregulated genes and 77 were downregulated genes.In addition,the Venn diagrams of DEGs among the 3 comparison groups showed that 20,931 DEGs were identified in the 3 periods of ovary tissue comparison.GO-significant enrichment analysis of DEGs revealed that oxidative phosphorylation,NADH dehydrogenase(ubiquinone)activity and respiratory chain and other GO entries were significantly enriched.KEGG metabolic pathway database alignment and annotation revealed that IG-vs-IS involved 317 metabolic pathways;IG-vs-IA involved 317 metabolic pathways;IS-vs-IA involved 161 metabolic pathways.Pathway significant enrichment analysis was performed in conjunction with the KEGG functional annotation results of DEGs.A total of significantly enriched Pathway includes transport and catabolism,signal transduction,folding,classification and degradation,transcription,amino acid metabolism,endocrine system,immune system,disease and other metabolic pathways.The results showed that in addition to the oxidative phosphorylation pathway,signal pathways such as cell cycle,mTOR signaling pathway,AMPK signaling pathway,and ubiquitin-mediated proteolytic pathway were associated and interacted,and the post-reproductive death of S.esculenta was regulated by a complex signaling network function system.5.Transcriptome analysis of the nidamental gland tissue and screening of key signaling pathways and key genes in Sepia esculentaThe Illumina transcriptome was sequenced on the nidamental gland tissue in the three key periods:growth period(NG),spawning period(NS),and post-reproductive death period(NA),and a total of 65.59 Gb clean data was obtained.By comparing and analyzing the transcrip tome data of nidamental gland tissue in different periods(NG,NS and NA)of golden cuttlefish,we obtained a large number of DEGs.In the NG-vs-NS group,there were 1,889 DEGs;in the NG-vs-NA group,there were 6,574 DEGs,and in the NS-vs-NA group,95 DEGs were found,and the three-period ovarian tissue comparison group was compared.In addition,the Venn diagrams of DEGs among the 3 comparison groups showed that 7,021 DEGs were identified.The most extensive functional classification of the GO classification of DEGs in the nidamental gland tissue at the three periods are "binding" and"catalytic activity",respectively.KEGG pathway analysis revealed that NG-vs-NS involved 295 metabolic pathways,NG-vs-NA involved 318 metabolic pathways,and NS-vs-NA involved 90 metabolic pathways.It can be seen that there is a big difference between the various periods of the golden cuttlefish,and more metabolic pathways are involved.The pathway significant enrichment analysis was performed in conjunction with the KEGG functional annotation results of DEGs.The significantly enriched pathway included cell cycle,protein digestion and absorption,p53 signaling pathway,mTOR signaling pathway,PI3K-Akt signaling pathway,metabolic pathway,lysosome.The above results showed that the nidamental gland was a reproductive accessory gland,and the signal pathways related to energy metabolism(protein digestion and absorption and other metabolic pathways)were highly enriched in the three key periods of golden cuttlefish.This may be inseparable from providing rich nutrition for the reproductive activities of S.esculenta.In summary,the transcriptome sequencing data(245.843 Gb clean data)of the subpeduncle lobe and its adjacent area,optic gland,nidamental gland and ovary were obtained from three key periods of golden cuttlefish.Through comprehensive analysis of differentially expressed genes data and significant enrichment results of S.esculenta,the pattern diagram of signal pathways related to post-reproductive death of golden cuttlefish were drawn,and the role of mTOR in the regulation of energy homeostasis of golden cuttlefish was put forward.The importance of oxidative phosphorylation in the regulation of cell energy metabolism in reproductive process was clarified.It was revealed that the rapid post-reproductive death of female S.esculenta was controlled by a complex network system of energy homeostasis regulation. |
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