斑节对虾肌肉中虾青素积累的转录组测序分析

Transcriptome sequencing analysis on astaxanthin accumulation in muscle of Penaeus monodon

【目的】筛选出斑节对虾肌肉中与虾青素积累相关的基因及其信号通路,为揭示虾青素在对虾肌肉积累的分子机制提供基础数据。【方法】分别剪取注射虾青素斑节对虾(试验组)和未注射虾青素斑节对虾(对照组)的肌肉,提取RNA后等量混合构建cDNA文库,采用Illumina HiSeq X Ten测序仪进行转录组测序,序列拼接与注释后以DESeq筛选差异表达基因(DEGs),然后进行GO功能注释分析和KEGG信号通路富集分析,并通过实时荧光定量PCR验证转录组数据的准确性。【结果】在试验组和对照组中分别获得48415832和48532230条有效序列(Clean reads),拼接后得到20495条Unigenes;分别有8761(占42.75%)、7658(占37.37%)、6933(占33.83%)、5584(占27.25%)和7238(占35.32%)条Unigenes在Nr、Swiss-Prot、KOG、KEGG和GO数据库中注释成功,其中在GO数据库的生物学过程、细胞组分、分子功能三大类别55个功能条目上均注释到Unigenes。经DESeq筛选,试验组与对照组间存在1468个DEGs,其中1209个DEGs在对照组呈上调表达、259个DEGs在对照组呈下调表达;与虾青素相关的基因主要有细胞色素b5基因(CYB5)、细胞色素P450基因(CYP450)、葡糖基/葡萄糖醛酸基转移酶基因(UDPGT)、类kelch蛋白基因(KLHL)、ATP结合盒转运蛋白基因(ABCT)、脂质蛋白受体基因(LipR)、脂肪酶基因(lipase)及半胱天冬酶3基因(CASP3)等。DEGs聚集最多的GO功能条目是多聚糖分解过程,富集最多的KEGG信号通路是信号转导。【结论】CYP450、CYB5、ABCT、LipR、KLHL、UDPGT、CASP3和lipase等DEGs,以及信号转导、运输与分解代谢、内分泌系统及辅酶因子和维生素代谢等信号通路与斑节对虾体内虾青素的积累相关,后续宜通过原位杂交、原核表达等技术验证DEGs的功能,进一步揭示虾青素在对虾体内积累的分子机制。

【Objective】This study aimed to screen genes and signaling pathways that related to astaxanthin accumulated in muscle of Penaeus monodon,so as to provide basic data for elucidating the molecular mechanism of astaxanthin accu-mulation in muscle of shrimp.【Method】The muscle tissue were clipped from P.monodon individuals injected with asta-xanthin(experimental group)and P.monodon individuals untreated(control group).After RNA extraction from the muscle of the experimental group and the control group,equal amounts were mixed to build cDNA libraries.Transcriptome sequencing was performed using Illumina HiSeq X Ten sequencer.After sequence assembly and annotation,differentially expressed genes(DEGs)were screened by DESeq.Then GO functional annotation analysis and KEGG signal pathway en-richment analysis were performed.The transcriptome data were confirmed by real-time quantitative PCR.【Result】The clean reads from experimental group and control group were 48415832 and 48532230,respectively.All clean reads were assembled into 20495 unigenes.There were 8761(42.75%),7658(37.37%),6933(33.83%),5584(27.25%)and 7238(35.32%)Unigenes successfully annotated in Nr,Swiss-Prot,KOG,KEGG and GO databases respectively.Many Unigenes were mapped to 55 sub-categories of 3 terms of biological process,cellular component and molecular function in GO database.After DESeq screening,a total of 1468 DEGs were identified in the experimental group and the control group,of which 1209 DEGs were up-regulated and 259 DEGs were down-regulated in the control group.The genes rela-ted to astaxanthin accumulation in muscle were cytochrome b5(CYB5),cytochrome P450(CYP450),glucosyl/glucuro-nosyl transferase(UDPGT),kelch-like protein(KLHL),ATP-binding cassette transporter and lipoprotein receptor(ABCT),lipid protein receptor gene(LipR),lipaseand caspase 3(CASP3),et al.The GO function categories with the most DEGs was polysaccharide catabolic process.The most enriched KEGG signal pathway was signal transduction.【Conclusion】The DEGs(including CYP450,CYB5,ABCT,LipR,KLHL,UDPGT,CASP3 and lipase)and signal path-ways(such as signal transduction,transport and catabolism,endocrine system,coenzyme factor and vitamin metabo-lism)are found to be related to astaxanthin accumulation in P.monodon.In the future,the function of these DEGs using techniques such as in situ hybridization,prokaryotic expression will be analyzed to further reveal the molecular mecha-nism of astaxanthin accumulation in muscle of shrimp.