Argonaute1敲除前后尖孢镰刀菌转录组分析比较
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摘要
Argonaute蛋白(AGO)介导的沉默复合体在RNA干扰(RNAi)中起着至关重要的作用。为了探究AGO1在尖孢镰刀菌RNAi中的作用机制,本文以粘团专化型尖孢镰刀菌生理1号小种FOX-A8野生型和其AGO1缺失突变体(FOX-A8-△Ago1)的菌丝和孢子为材料,分别进行了RNA提取、Illumina Hi Seq 2000高通量转录组测序、差异表达基因(DEGs)的显著富集分析;选择菌丝和孢子中的DEGs进行实时荧光定量PCR(Quantitative real-time PCR,qRT-PCR)验证。GO通路注释结果显示,相对于野生型菌株,AGO1缺失突变体菌丝中的醇脱氢酶(NADP~+)、孢子中的CAMK/CAMKL/CHK 1蛋白激酶均显著上调; KEGG通路注释结果显示,相对于野生型菌株,AGO1缺失突变体菌丝中与MAPK信号通路相关的基因、孢子中与PLD信号通路相关的基因均显著下调;另外,相对于野生型菌株,编码AGO2的基因下调,但是下调不显著。qRT-PCR检测DEGs的表达模式与RNA-Seq分析结果一致,证实了RNA-Seq结果的可靠性。
The Argonaute protein(AGO) mediated silencing complex plays a crucial role in RNA interference(RNAi). In order to explore the function of AGO1 in Fusarium oxysporum,F. oxysporum f. sp. Conglutinas race 1 FOX-A8 strain and the Ago1 deletion mutant(FOX-A8-△Ago1) were used as materials for RNA extraction from spores and mycelia. Transcriptome sequencing was conducted via Illumina Hi Seq 2000 platform and significant enrichment analysis of differentially expressed genes(DEGs) was performed by the RNA-seq analysis pipeline; The selected DEGs in hyphae and spores were validated by quantitative real-time PCR(qRT-PCR) approach. The results of GO annotation showed that the alcohol dehydrogenase(NADP~+) in mycelia of the AGO1 deletion mutant and the CAMK/CAMKL/CHK 1 protein kinase in the spore of the AGO1 deletion mutant were significantly up-regulated; The results of the KEGG pathway annotation showed that the genes associated with the MAPK in mycelia of the AGO1 mutant and the PLD signaling pathway in the spore of the AGO1 deletion mutant were significantly down-regulated compared to the wild-type strain; In addition,the gene encoding AGO2 was relatively down-regulated in compare with the wild-type strain,but not significant. The expression pattern of DEGs detected by qRT-PCR was consistent with the results of RNA-Seq analysis,confirming the reliability of the transcriptome data.
The Argonaute protein(AGO) mediated silencing complex plays a crucial role in RNA interference(RNAi). In order to explore the function of AGO1 in Fusarium oxysporum,F. oxysporum f. sp. Conglutinas race 1 FOX-A8 strain and the Ago1 deletion mutant(FOX-A8-△Ago1) were used as materials for RNA extraction from spores and mycelia. Transcriptome sequencing was conducted via Illumina Hi Seq 2000 platform and significant enrichment analysis of differentially expressed genes(DEGs) was performed by the RNA-seq analysis pipeline; The selected DEGs in hyphae and spores were validated by quantitative real-time PCR(qRT-PCR) approach. The results of GO annotation showed that the alcohol dehydrogenase(NADP~+) in mycelia of the AGO1 deletion mutant and the CAMK/CAMKL/CHK 1 protein kinase in the spore of the AGO1 deletion mutant were significantly up-regulated; The results of the KEGG pathway annotation showed that the genes associated with the MAPK in mycelia of the AGO1 mutant and the PLD signaling pathway in the spore of the AGO1 deletion mutant were significantly down-regulated compared to the wild-type strain; In addition,the gene encoding AGO2 was relatively down-regulated in compare with the wild-type strain,but not significant. The expression pattern of DEGs detected by qRT-PCR was consistent with the results of RNA-Seq analysis,confirming the reliability of the transcriptome data.
引文
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[2]Liang L Q,Du H Y,Duan J Y,et al.Research advance on the generation and mechanism of small RNAs in fungi(in Chinese)[J].M icrobiology China(微生物学通报),2018,45(7):1563-1573．
[3]Grishok A,Pasquinelli A E,Conte D,et al.Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C.elegans developmental timing[J].Cell,2001,106(1):23-34．
[4]Tabara H,Sarkissian M,Kelly W G,et al.The rde-1gene,RNA interference,and transposon silencing in C.elegans[J].Cell,1999,99(2):123-132．
[5]Hutvagner G,Simard M J.Argonaute proteins:key players in RNA silencing[J].Nat Rev M ol Cell Biol,2008,9(1):22-32．
[6]Parker J S,Roe S M,Barford D.Structural insights into mRNA recognition from a PIWI domain-siRNAguide complex[J].Nature,2005,434(7033):663．
[7]Zeng L S.Functional analysis of genes of small RNApathw ays in Coniothyrium minitans(in Chinese)[D].Wuhan:Huazhong Agricultural University(武汉:华中农业大学),2012．
[8]Zhang X,Segers G C,Sun Q,et al.Characterization of hypovirus-derived small RNAs generated in the Chestnut Blight Fungus by an inducible DCL-2-dependent Pathw ay[J].Journal of Virology,2008,82(6):2613．
[9]Liang L Q.Function analysis of Dicer,Argonaute and Rdrp involved in small RNAs biogenesis in Fusarium oxysporum(in Chinese)[D].Beijing:China Agricultural University(北京:中国农业大学),2014．
[10]Trapnell C,Pachter L,Salzberg S L.Bioinformatics Advance Access published M arch 16,2009 TopHat:discovering splice junctions w ith RNA-Seq[J].Bioinformatics 25:1105-1111．
[11]Zeng F.Identification of effectors in race of Fusarium oxysporum f.sp.conglutians(in Chinese)[D].Beijing:Chinese Academy of Agricultural Sciences(北京:中国农业科学院),2016．
[12]Mortazavi A,Williams B A,Mccue K,et al.Mapping and quantifying mammalian transcriptomes by RNA-Seq[J].Nature M ethods,2008,5(7):621-628．
[13]Faehnle,Christopher R,Elkayam,et al.Slicer and the Argonautes[J].Biophysical Journal,2014,106(2):455．
[14]Azlan A,Dzaki N,Azzam G.Argonaute:The executor of small RNA function[J].Journal of genetics and genomics,2016,43(8):481-494．
[15]Zhou J H.Prediction and validation of milRNA in Sclerotinia sclerotiorum and function analysis of gene encoding cinnamic acid dehydrogenase(in Chinese)[D].Wuhan:Huazhong Agricultural University(武汉:华中农业大学),2016．
[16]Wu D,Zhao Z T,Li Z Y,et al.Identification and expression pattern analysis of calcium/calmodulin-dependent protein kinases(CaM Ks)gene family in Setosphaeria turcica(in Chinese)[J].Journal of Agricultural Biotechnology(农业生物技术学报),2015,23(8):1020-1030．
[17]Solomon P S,Rybak K,Trengove R D,et al.Investigating the role of calcium/calmodulin-dependent protein kinases in Stagonospora nodorum[J].M olecular M icrobiology,2006,62(2):367-381．
[18]Liu X H,Lu J P,Dong B,et al.Disruption of MoC-M K1,encoding a putative calcium/calmodulin-dependent kinase,in Magnaporthe oryzae[J].M icrobiological Research,2010,165(5):402-410．
[19]Fan H Y,Tong C,Sun Q Y.Mitogen-activated protein kinase(M APK)signaling pathw ays:state-of-theart(in Chinese)[J].Chinese Journal of Zoology(动物学杂志),2002,37(5):98-102．
[20]Lin C H,Cai Z Y,Huang G Z.MAPK Cascades genes in Colletotrichum graminicola and C.higginsianum genome(in Chinese)[J].Chinese Journal of Tropical Crops(热带作物学报),2012,33(4):674-680．
[21]Chang L,Karin M.Mammalian MAP kinase signalling cascades[J].Nature,2001,410(6824):37-40．
[22]Brunet A,Pouysségur J.Identification of MAP kinase domains by redirecting stress signals into grow th factor responses[J].Science,1996,272(5268):1652-1655．
[23]Gustin M C,Albertyn J,Alexander M,et al.MAP kinase pathw ays in the yeast Saccharomyces cerevisiae[J].Microbiol Mol Biol Rev,1998,62(4):1264-1300．
[24]Di P A,Madrid M Z,Delgado-Jarana J,et al.Fusarium oxysporum:exploring the molecular arsenal of a vascular w ilt fungus[J].M olecular Plant Pathology,2003,4(5):315-325．
[25]Zhang Y H.Transcriptome study on the specialization of flax and Fusarium oxysporum(in Chinese)[D].Inner mongolia:M ongolian University(内蒙古:内蒙古大学),2017．
[26]Chen Z L,Xu Z H.Research progress of phospholipase D(in Chinese)[J].Industrial microorganism(工业微生物),1999(4):47-50．
[27]Dolan J W,Bell A C,Hube B,et al.Candida albicans PLD I activity is required for full virulence[J].M edical M ycology,2004,42(5):439-447．
[28]Li X P.Study on Aspergillus fumigatus Phospholipase D Gene Knockout and Its Function(in Chinese)[D].Qingdao:Qingdao University(青岛:青岛大学),2012．
[29]Rose K,Rudge S A,Frohman M A,et al.Phospholipase D signaling is essential for meiosis[J].Proceedings of the National Academy of Sciences of the United States of America,1995,92(26):12151-12155.
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