基于RNA-Seq技术的真菌病毒AfPV1感染对寄主黄曲霉基因表达的影响

Effect of mycovirus AfPV1 infection on gene expression in Aspergillus flavus based on RNA-Seq technique

目的基于转录组测序(RNA-Seq)技术探讨真菌病毒黄曲霉(A.flavus)partitivirus 1(AfPV1)感染对寄主A.flavus基因表达的影响。方法分别提取无病毒感染的A.flavus菌株LD-F1和真菌病毒AfPV1感染的A.flavus菌株LD-F1-b的RNA进行文库构建和RNA-Seq;以A.flavus菌株NRRL 3357(GCA_009017415.1)的基因组为参考,分析RNA-Seq测序结果中有差异表达的基因(DEGs),并对DEGs进行基因本体(GO)、京都基因与基因组百科全书(KEGG)功能富集分析;随机选取12个DEGs,使用实时荧光定量PCR(qRT-PCR)对转录组进行验证。结果样品RNA完整无污染,测序结果可靠,错误率0.02%,测序的结果能够成功比对到A.flavus的基因组序列(>56%);获得真菌病毒AfPV1感染的A.flavus的DEGs有4127个;GO富集分析显示,DEGs涉及A.flavus的氧化还原过程、单生物过程、单生物代谢过程、跨膜转运、谷氨酰胺家族氨基酸代谢过程、谷氨酰胺家族氨基酸生物合成过程、二羟酸代谢过程及血红素结合等;KEGG富集分析显示,DEGs参与A.flavus代谢通路中的戊糖与葡萄糖醛酸互相转换、非同源重组末端连接、氮代谢、错配修复、同源重组、DNA复制、次级代谢产物的生物合成及碱基切除修复等;qRT-PCR的结果与转录组测序结果趋势一致。结论真菌病毒AfPV1感染A.flavus能够引起基因表达差异,DEGs涉及A.flavus生命过程中多个基因和多个信号通路。

Objective To investigate the differential expressed genes of Aspergillus flavus(A.flavus)induced by mycovirus AfPV1 infection based on RNA-Seq technique.Methods Total RNA was extracted from virus-free A.flavus LD-F1 and AfPV1-infected A.flavus LD-F1-b,respectively,then used for constructing RNA library and sequencing RNA.The genome of A.flavus NRRL 3357(GCA_00901741.5)was used as a reference to analyze the differentially expressed genes(DEGs)determined by RNA-seq.Gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)functional enrichment analyses were run on DEGs.Twelve DEGs were randomly selected and verified by real-time fluorescence quantitative PCR(qRT-PCR)to confirm the transcriptome analysis.Results Extracted RNA was intact and contamination-free.The sequencing results were reliable with an error rate of 0.02%.The sequencing results successfully matched more than 56%of the genome sequences of Aspergillus flavus.A total of 4127 DEGs were obtained from Aspergillus flavus infected by fungal virus AfPV1.GO enrichment analysis showed that DEGs were involved in a variety of biological processes of Aspergillus flavus such as redox process,single organism process,single organism metabolism process,transmembrane transport,amino acid metabolism and amino acid biosynthesis of glutamine family,dihydroxy acid metabolism process and heme binding,etc..KEGG pathway enrichment analysis revealed that DEGs were involved in pentose and glucuronate interconversions,non-homologous end-joining,nitrogen metabolism,mismatch repair,homologous recombination,DNA replication,biosynthesis of secondary metabolites and base excision repair,etc..qRT-PCR results were consistent with RNA-seq.Conclusion Mycovirus AfPV1 infection can cause DEGs of A.flavus.DEGs involve multiple genes and signal pathways in the life courses of A.flavus.