文章摘要
红螯螯虾响应长期水体盐度胁迫的基因和代谢通路分析
Comparative transcriptome analysis of intestine of Cherax quadricarinatus in response to different salinities
投稿时间:2020-09-22  修订日期:2021-03-17
DOI:
中文关键词: 红螯螯虾  盐度胁迫  肠道  高通量测序  差异表达分析
英文关键词: Cherax quadricarinatus  Hypersaline stress  Intestine  High-throughput sequencing  Differential expression analysis
基金项目:国家重点研发计划(2018YFD0901305)、海南大学科研启动经费(KYQD(2R)1924)
作者单位邮编
刘树彬 海南大学海洋学院 570228
徐 畅 海南大学海洋学院 
贾永义 浙江省淡水水产研究所 
顾志敏 浙江省淡水水产研究所 
李二超 海南大学海洋学院 570228
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中文摘要:
      【目的】试验探究了长期盐度胁迫条件下红螯螯虾肠道基因表达和代谢通路的变化。【方法】采用Illumina Hiseq 2500平台对0‰、5‰、10‰和15‰四个盐度下养殖5周的红螯螯虾的肠道组织进行了高通量测序。【结果】研究共获76 534个unigenes,通过与NR、NT、KO、Swissprot、PFAM、GO和KOG数据库进行比对,成功注释37 378个unigenes。通过引入FPKM(Fragments Per Kilo bases per Million fragments)来估算基因表达水平,基于NR和Pfam两个数据库的蛋白注释结果,448 362个unigenes在GO数据库得到注释。根据KO功能注释与Pathway的联系,9 483个unigenes在KEGG数据库被注释分类到34条通路途径。通过DEGseq进行基因表达差异分析,以0‰为对照组,显著差异基因筛选条件设置为Q value<0.05且差异倍数|Fold Change|>2,5‰、10‰和15‰组分别获得差异基因2 733、91和236个,其中5‰组共有2 068个基因显著上调,665个基因显著下调。将所有差异表达基因进行KEGG通路富集分析,5‰、10‰和15‰组分别富集到265、80和120条通路,仅有10‰和15‰组分别有一条显著富集的通路,均为军团杆菌病通路。经过鉴定,军团杆菌病通路显著下调,这与团队已有研究肠道菌群研究结果一致。表明红螯螯虾在盐度胁迫下,可通过抑制潜在病原菌相关通路以及丰度,达到保护肠道避免外环境病原菌侵染的效果。此外,eEF1α、udp、UPP、ACTB_G1、TUBA,TUBB、PFN、CALM等差异基因的变化说明盐度升高可显著影响红螯螯虾肠道的细胞形态、细胞内物质运输和细胞内信号转导途径,但红螯螯虾启动嘧啶补救合成途径可以修复尿嘧啶核苷酸比例失衡导致的遗传损伤。【结论】研究结果可为红螯螯虾的半咸水养殖提供重要的理论支持,同时也可为深入研究红螯螯虾盐度胁迫下的肠道免疫调控机制提供参考。
英文摘要:
      In order to explore the effect of salinity stress on the intestinal gene expression level of Cherax quadricarinatus, Illumina Hiseq 2500 high-throughput sequencing platform was used to conduct bidirectional sequencing of intestinal tissues of C. quadricarinatus under different salinity conditions (0, 5, 10 and 15 psu). After quality control and assembly of the obtained raw data, 76,534 unigenes was obtained. Blast in NR, NT, KO, Swissprot, PFAM, GO and KOG databases, 37,378 unigenes could be annotated. The amount of gene expression was estimated according to FPKM (Fragments Per Kilo bases Per Million Fragments). Based on the protein annotation results of NR and Pfam databases, 448,362 unigenes was functional annotation. According to the relationship between KO functional annotation and Pathway, 9,483 unigenes was annotated and classified into 34 pathways. Gene expression differences were analyzed by DEGseq. 0 psu was used as the control group, and the screening conditions for significantly different genes were set as Q value<0.05 and multiple |Fold Change|>2. The 5, 10 and 15 psu groups obtained 2,733, 91 and 236 differentially expressed genes respectively, among which 2,068 genes were significantly up-regulated and 665 genes were significantly down-regulated in the 5 psu groups with the most differentially expressed genes. The KEGG pathway enrichment analysis of all differentially expressed genes showed that 5, 10 and 15 psu groups were enriched to 265, 80 and 120 pathways respectively, and only 10 psu and 15 psu groups had a significantly enriched pathway respectively, all of which were legionella pathways. After identification, legionella pathway was significantly down-regulated, which was consistent with the previous results of enterobacteriaceae. The results showed that red chelicerae could protect the intestinal tract from infection by exogenous pathogenic bacteria by inhibiting the relevant pathways and abundance of potential pathogenic bacteria under the condition of increased salinity. In addition, changing in differential genes (eEF1α、udp、UPP、ACTB_G1、TUBA,TUBB、PFN、CALM) suggest that increased salinity seriously affects the cellular morphology, intracellular material transport, and intracellular signal transduction in the gut of red chelicerae, and activates pyrimidine remediation pathways to repair genetic damage caused by uracil nucleotide imbalance. The results provide important reference materials for the saline-alkali or brackish aquaculture of C. quadricarinatus in the future, and provide reference for the further study of the intestinal immune regulation mechanism of C. quadricarinatus under salinity stress.
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