【摘要】 为了探究甲壳类幼体应对饥饿胁迫的分子机理，以拟穴青蟹(Scylla paramamosain)为研究对象，设置正常投喂组和饥饿组，处理3 d后对不同组别的拟穴青蟹大眼幼体进行转录组测序并开展生物信息学分析。De novo组装后共获得79 581个unigene,平均长度1 065.44 bp。Nr数据库共注释20 061个unigene。DESeq分析筛选出5 621个差异表达基因，饥饿组相比对照组2 641个上调、2 980个下调。基因本体论(GO)富集分析表明，共254条GO Term显著富集(矫正后的P值小于0.05)。京都基因与基因组百科全书(KEGG)富集分析表明，共60条KEGG pathway显著富集(矫正后的P值小于0.05)。基于KEGG富集分析结果筛选了一系列与饥饿胁迫相关的代谢通路，如DNA复制、细胞凋亡、缬氨酸，亮氨酸和异亮氨酸代谢、亚油酸代谢、淀粉和蔗糖代谢。研究表明，饥饿胁迫干扰了拟穴青蟹大眼幼体的细胞分裂和凋亡，影响了体内的氨基酸代谢、脂质代谢和碳水化合物代谢。研究结果可为拟穴青蟹苗种的健康培育提供理论参考，并丰富拟穴青蟹的分子生物学基础数据。更多还原

【Abstract】 In nature, food scarcity is one of the environmental stresses that crustaceans often face. Crustaceans often adjust metabolism, behavior and utilize internal storage to maintain basic physiological functions and achieve limited growth and reproduction in short-term or long-term nutritional limitations. Scylla paramamosain is an important marine breeding crab in the southeast coastal area of China. In this study, comparative transcriptome analysis was conducted on normally fed and starved megalopae of Scylla paramamosain to explore the effect of starvation stress on the metabolic process of megalopae, and to provide some theoretical references for the healthy cultivation of megalopae, and to enrich the molecular biological research data of Scylla paramamosain. The experiment was divided into two groups: the normal feeding group for 3 days（F3S0） and the starvation group for 3 days（S3F0）. Each group had 3 replicates, and each replicate had 8 megalopaes. Transcriptome sequencing was carried out on the collected samples of megalopae.RNA was extracted separately from each sample. RNA degradation and contamination were assessed on agarose gels. RNA concentration, OD_260/280, and OD_260/230 ratios were measured. The RNA integrity number（RIN） was determined. Oligo（dT） magnetic beads were used to enrich mRNA with polyA tails, and the RNA was cut into fragments of about 300 bp in length by ion interruption. The first strand of cDNA was synthesized using RNA as template with 6-base random primers and reverse transcriptase. The second strand of cDNA was then synthesized using the first strand of cDNA as template. Library preparation and sequencing were then performed. Gene function was annotated based on NCBI non-redundant protein sequences（Nr）, Gene Ontology（GO）, Kyoto Encyclopedia of Genes and Genomes（KEGG）, evolutionary genealogy of genes: Non-supervised Orthologous Groups（eggNOG）, Swiss-Prot, and Pfam databases. Differential expression was assessed using transcript abundances as inputs to the DESeq. The charting of volcano map and cluster analysis about differentially expresed genes（DEGs） were performed. Principal component analysis（PCA） of each sample was implemented on the genes significantly expressed. The enrichment analysis was performed using topGO. P-value was calculated by hypergeometric distribution method to find the GO significantly enriched term, to identify the major biological functions of differential gene exercise.The number of differentially expressed genes at different KEGG pathways was counted to determine the linked signaling pathways.The raw reads of transcriptome sequencing were filtered to obtain 150 103 944 and 128 924 890 high-quality sequences, respectively. Trinity software was used to do De novo assembly of high quality sequences and 158 201 transcripts were obtained. After removing the redundancy of transcripts, 79 581 unigenes were obtained, with a maximum length of 20 651 bp and an average length of 1 065.44 bp. Unigenes were annotated with major databases, including Nr, GO, KEGG, Pfam, EggNOG and Swiss-Prot. In total, 25.21%, 9.17%, 14.73%, 18.82%, 23.67% and 22.94% of unigenes were successfully annotated. GO analysis revealed that unigenes related to cellular process, metabolic process and single organism process were highly represented in the biological process（BP） category. Unigenes related to cell, cell part and organelle were highly represented in the cellular component（CC） category. Unigenes related to catalytic activity and binding were highly represented in the molecular function（MF） category. KEGG classification revealed that unigenes were involved in the following five pathways: metabolism, genetic information processing, environmental information processing, cellular processes, and organismal systems. In the transcriptome, 5 621 differentially expressed genes were screened, including 2 641 up-regulated and 2 980 down-regulated genes. GO enrichment analysis showed that a total of 254 GO terms were significantly enriched（corrected P<0.05）. Among them, the top five GO terms were catalytic activity, carbohydrate metabolic process, oxidation-reduction process, oxidoreductase activity and DNA replication initiation. The KEGG enrichment analysis showed that a total of 60 KEGG pathways were significantly enriched（corrected P<0.05）. Among them, the top five KEGG pathways were DNA replication, valine, leucine and isoleucine degradation, cell cycle, starch and sucrose metabolism and base exception repair. Based on the results of KEGG enrichment analysis, a series of metabolic pathways related to starvation stress were screened, such as DNA replication, apoptosis, valine, leucine and isoleucine metabolism, linoleic acid metabolism, starch and sucrose metabolism. In addition, some genes related to digestive system, smell and circadian rhythm were screened. RT-PCR verified five genes, and their expression trends were consistent with the results of transcriptome sequencing.In this study, the gene annotation of megalopae of Scylla paramamosain were obtained through transcriptome analysis. The metabolic pathways of amino acid metabolism, lipid metabolism, carbohydrate metabolism and cell growth and death related to starvation stress were screened, and some genes related to digestive system, olfactory and circadian rhythm were analyzed. The results showed that starvation stress interfered with cell division and death, and affected amino acid metabolism, lipid metabolism and carbohydrate metabolism in megalopae of Scylla paramamosain.更多还原