| The oriental river prawn,Macrobrachium nipponense,the most important prawns in freshwater aquaculture in China,is widely distributed in China,Japan,Korean Peninsula,northern Vietnam and other regions in East Asia.M.has the characteristic of rapid gonad development.From the July to August,young prawns can grow up to sexual maturity and ready to reproduce for many generations after 45 days.In high water temperature seasons,it only takes 15 days for female prawns to complete an ovarian maturation cycle(from ovarian emptying to next egg holding),which is an ideal model for the study of crustacean reproduction and development.However,"Sexual fast ripening" leads to over propagation in autumn,multiple generations of the same pond,thus causing problems such as high breeding density,increased risk of hypoxia and feed consumption,etc..Under such circumstance,these problems become the bottleneck limiting the discovery of the current prawns breeding industry.In this study,the ovarian development process was divided into five stages(I～V stage).Using transcriptome and metabolome technology,15 samples of transcriptome library and 50 samples of metabolome library were constructed respectively.Based on the gene expression and metabolite change,we study the screening and regulation mechanism of genes related to ovary development of prawn:1.Screening of genes related to ovary development based on transcriptome sequencingIn this study,363,957,240 high-quality clean reads were obtained by using Illumina hiseq high-throughput sequencing platform.255,966 transcripts and 105,082 unigenes were generated,with an average length of 1,173.15 bp and 988.46 bp,respectively.30,878 unigenes with clear functions were obtained by annotation with 7 databases including COG,GO and KEGG.In addition,752,353 SNP loci and 17,362 unigenes with SSR loci were obtained from the samples.Through the phase by phase comparison from the I and V periods,we found that the number of pathways enriched in the second to third periods was the largest,which was the period with the most differences.17 differentially express genes related to ovarian development,such as β-galactosidase,n-sulfoglucosamine sulfohydrolase and glutathione S-transferase,were obtained from Q-value sequencing of enrichment pathway.In addition to kappa opioid receptor and myosin heavy chain 67,the expression patterns of other 15 genes in the process of ovarian development showed the rule of first rising and then falling.Cathepsin B,cathepsin D,cathepsin L and so on are simultaneously enriched in the lysosome pathway,which supports the conclusion that the cathepsin family is related to the ovarian development of M nipponense,and so is the lysosome.2.Metabonomics based screening of metabolites related to ovary development of prawnIn this study,567 compounds were detected by GC/MS and 83-162 metabolic pathways were obtained.The results of principal component analysis of metabolites in different stages of development showed that there were significant quadrant differences between stages Ⅰ,Ⅱ,Ⅴand Ⅲ,Ⅳ.It was revealed that the metabolism patterns in stages Ⅰ,Ⅱ,Ⅴ were different with those in stages Ⅲ,Ⅳ.In the "biosynthesis of plant secondary metabolites" pathway,which was enriched many times in the phase Ⅰ-Ⅴ comparison,the changes of the contents of metabolites related to TCA cycle and fatty acid synthesis were detected frequently.After analyzing the expression patterns of the genes of the speed limiting enzymes and key enzymes in the upstream and downstream of these metabolites,it was found that the changes of the cDNA expression of the two subunits of succinate dehydrogenase complex and succinyl coenzyme A synthetase were consistent with the changes of the abundance of succinic acid and fumaric acid,which decreased in the fourth stage of ovarian development and then increased.The expression of fatty acid synthetase gene increased at first and then decreased during the development of ovary,which was as same as that of linoleic acid,palmitic acid and stearic acid.Therefore,this study speculated that the two pathways of TCA cycle and fatty acid synthesis might be the new perspectives of regulating ovarian development in M.nipponense.3.Screening of ovarian development related pathways in shrimp based on transcriptome and metabolome analysisIn this study,95 pathways such as "steroid biosynthesis" and "steroid biosynthesis"were identified to be closely related to the development of ovary in M nipponense by comparing the top 200 differential genes and metabolites from previous researches,according to the screening conditions of Pearson coefficient greater than 0.999 and pvalue less than 0.01.According to the conclusion obtained by the transcriptome,the differences were most concentrated during the period Ⅱ to Ⅲ of ovarian development.Focusing on the changes in genes during these periods,the progesterone mediated oocyte pathway with the largest number of genes was found as a key signal pathway responding to ovarian development,qRT-PCR showed that the mRNA expression of phosphatidylinositol-3 kinase and cyclin-dependent-2-kinase decreased first and then increased,the expression of cyclin-b2 reached the lowest level in stage III and then increased gradually,and there was no obvious rule which promoted the expression of complex subunit-5 in the later stage.The gene expression of these genes in gonad tissue was significantly higher than that in other tissues,whether in reproductive or non-reproductive season.We speculate that steroid non genomic signal transduction is involved in the regulation of ovarian development in M.nipponense.Our study is the first systematic and comprehensive report on the information of ovary stage transcriptome and metabolome of M.nipponense,which not only laid a foundation for the study of the regulate mechanism of rapid gonad development in M.nipponense,also provided important references for crustaceans. |
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