凡纳滨对虾转录组测序分析及肌肉生长发育相关基因的筛选

Transcriptome sequencing and screening of genes related to muscle growth and development in Litopenaeus vannamei

  • 摘要: 【目的】筛选出凡纳滨对虾生长发育的功能基因及代谢调控网络,揭示其生长发育的分子机制,为后续开展凡纳滨对虾分子生物学研究提供宝贵的基因数据来源。【方法】以快速生长群体和慢速生长群体的凡纳滨对虾肌肉组织为研究材料,通过Illumina HiSeqTM2500平台对构建的cDNA文库进行高通量测序分析,以StringTie进行拼接组装后利用DESeq2筛选差异表达基因,并基于KOG、GO、nr、COG、Swiss-Pro、KEGG和Pfam等数据库进行差异表达基因功能注释分析。【结果】经拼接组装共获得53458844条Clean reads,各样品Clean reads的Q30均在93.00%以上;Illumina测序获得的Clean reads与参考基因组的比对效率在87.75%~87.80%,说明转录组测序数据真实可靠。采用SnpEff进行SNP/InDel变异注释分析,结果显示,SNP位点中以A>G、G>A、C>T和T>C等4种类型的数量较多(14950~21562个),InDel位点则以SYNONYMOUS_CODING的数量最多(33630个)。使用StringTie对Mapped reads(比对到参考基因组的Reads)进行拼接,共发掘到4607个新基因,分别输入COG、GO、KEGG、KOG、Pfam、Swiss-Prot、eggNOG和nr数据库中进行序列比对,最终发现共有1098个新基因被注释,以被nr数据库注释的新基因数量最多(1077个),而被COG数据库注释的新基因数量最少(416个)。基于Q-value<0.05且Fold Change>2的筛选条件,共获得1408个差异表达基因(661个为显著上调表达基因,747个为显著下调表达基因);1408个差异表达基因被注释到53个GO功能条目中,其中,22条被注释到生物学过程(Biological process),16条被注释到细胞组分(Cellular component),15条被注释到分子功能(Molecular function);KEGG信号通路富集分析发现3条重要的信号通路,分别是溶酶体通路(Lysosome)、氨基糖和核苷酸糖新陈代谢(Amino sugar and nucleotide sugar metabolism)及鞘脂类代谢(Sphingolipid metabolism)。在溶酶体通路中,CTSL基因、Nramp基因、MyoG基因和Myf5基因在凡纳滨对虾快速生长群体肌肉组织中呈上调表达,而Trypsin基因呈下调表达。【结论】通过转录组测序分析从快速生长群体和慢速生长群体的凡纳滨对虾肌肉组织中筛选出1408个差异表达基因(661个为显著上调表达基因,747个为显著下调表达基因),主要富集在溶酶体、氨基糖和核苷酸糖新陈代谢及鞘脂类代谢等通路上,在对虾肌肉生长发育过程中发挥重要作用。

     

    Abstract: 【Objective】 To screen out the functional genes and metabolic regulation network of growth and development in Litopenaeus vannamei,reveal the molecular mechanism of its growth and development,and provide a valuable source of genetic data for the subsequent molecular biology research of L. vannamei.【Method】 The muscle tissue of the fast-growing group and the slow-growing group of L. vannamei was used as the research material,high-throughput sequencing analysis was performed on the cDNA library through the Illumina HiSeqTM2500 platform,and the DESeq2 was used to screen differentially expressed genes after assembly using StringTie,and the functional annotation of differentially expressed genes was performed based on KOG,GO,nr,COG,Swiss-Pro,KEGG and Pfam databases.【Result】 A total of 53458844 Clean reads with Q30 more than 93.00% were obtained after splicing assembly. The comparison efficiency of Clean reads obtained by Illumina sequencing and the reference genome was 87.75% to 87.80%,indicating that the transcriptome sequencing data were true and reliable. SNPEff was used to annotate SNP and InDel variation,results showed that the number of 4 types of SNP loci,A>G,G>A,C>T,and T>C,was large(14950-21562),and the number of synonymous_coding points of InDel loci was the largest(33630). Using StringTie software to splice Mapped Read(Reads that were aligned to the reference genome),a total of 4607 new genes were discovered. The genes were input into COG,GO,KEGG,KOG,Pfam,Swiss-Prot,eggNOG and nr databases for sequence alignment. Finally,it was found that a total of 1098 new genes were annotated,with the largest number of new genes annotated by the nr database(1077),and the smallest number of new genes annotated by the COG database(416). Based on the screening conditions of Q-value<0.05 and Fold Change>2,a total of 1408 differential expression genes were detected,in which 661 genes were significantly upregulated and 747 genes were significantly down-regulated. A total of 1408 differentially expressed genes were annotated to 53 GO function items,in which 22 items were annotated to biological process,16 items were annotated to cellular component,15 items were annotated to molecular function. KEGG signal pathway enrichment analysis revealed three important signal pathways,namely lysosome pathway,amino sugar and nucleotide sugar metabolism pathway and sphingolipid metabolism pathway. In the lysosomal pathway,CTSL gene,Nramp gene,MyoG gene and Myf5 gene were up-regulated in the muscle tissue of fast-growing L. vannamei,while Trypsin gene was down-regulated.【Conclusion】 Through transcriptome sequencing analysis,1408 differentially expressed genes(661 are significantly up-regulated genes and 747 are significantly down-regulated genes)were screened from the muscle tissues of fast-growing and slow-growing L. vannamei,which are mainly enriched in lysosomes,sphingolipid metabolism,amino sugar and nucleotide sugar metabolism pathways. They play an important role in the growth and development of L. vannamei muscle.

     

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