植物小RNA研究进展—张荣志

植物小RNA研究进展张荣志2014/09/02小RNA的分类MicroRNA概述MicroRNA生源MicroRNA进化OutlineMicroRNASiRNA小RNA的分类OutlineUGACAGAAGAGAGAGAGCACA小RNA：小RNA（smallRNA）是一类长21-24nt的单链RNA主要在转录后水平、翻译水平和染色质修饰等基因的表达调节过程中发挥重要作用研究表明，小RNA对生物发育阶段的转换、细胞的分生与分化、细胞程序化死亡、器官的分化与极性的建成、分生组织的决定性建成、逆境的应答及维持染色体的稳定性等方面都起着重要作用根据其起源、结构特征及功能可将小分子RNA分为两类：microRNA(miRNA)和smallinterferingRNA(siRNA)Definition(Khraiweshetal.2011)microRNA基因起源FunctionofmiRNATarget(Khraiweshetal.2011)Development:幼年到成年的转换miR156-172花器官的形成：miR160-444侧根的发育miR160叶子的极性发育miR166Stress:抗旱：miR169N饥饿:miR169S吸收：395抗病过程：miR398-408MicroRNASiRNA小RNA的分类OutlinesiRNA是一类由长双链RNA（doublestrandRNA,dsRNA）在DCL4的切割下产生的小分子RNA。在植物中，内源siRNA一般来源于mRNA、转座子、病毒和异染色质DNA等序列。根据其产生机制和功能的不同(BrodersenandVoinnet,2006;Vaucheret,2006)，植物内源siRNAs被分为四类：heterochromaticsiRNA(hc-siRNA)、trans-actingsiRNA(ta-siRNA)、naturalantisensetranscript-derivedsiRNA(nat-siRNA)和phased-siRNASiRNA植物siRNA介导的DNA甲基化过程ModelforRNA-directedDNAmethylationinplants(LawandJacobsen,2010)heterochromaticsiRNASense-antisensetranscriptpairNon-antisensebidirectionaltranscriptpairnat-siRNAnat-siRNAtrans-actingsiRNAmiR173：TAS1和TAS2均由识别切割，产生的ta-siRNA调节PPR基因(Howelletal.,2007)。miR390：TAS3由miR390识别切割，产生ta-siRNA调节ARF基因(Allenetal.,2005;Hunteretal.,2006;Williamsetal.,2005)。miR828：TAS4的产生是由miR828起始(Rajagopalanetal.,2006)。trans-actingsiRNAPhased-siRNASongetal2012Songetal2012Phased-siRNA(Monocots)Phased-siRNA(Dicots)Zhaietal2011小RNA的分类MicroRNA概述MicroRNA生源MicroRNA进化Outline•MicroRNAprediction•Targetprediction•MicroRNAdatabaseMicroRNA(Khraiweshetal.2011)microRNA基因起源1020304050|AGC-AAAUAAACAGUCUUUAGAUGAGAAGUAGAUCAUAAUAGUGAGCGAAAUG\AUUUGUCAGAAGUCUACUCUUCGUCUAGUAUUGUUACUCGUUUUAUG^A-AGAGU10090807060microRNA判定标准ArmregionmiRNAregionLoopregionmiRNA和miRNA*来自同一个pre-miRNA颈环结构的两条不同臂，且miRNA/miRNA*duplex3'末端要有2个碱基的突出miRNA与茎环结构另一条臂的配对部分（包括miRNA*）错配碱基要少于四个miRNA和miRNA*配对部分的凸起一般不超过一个，且每个凸起不得超过两个碱基Meyersetal.,2008microRNA判定标准microRNA预测软件•MIREAP:•miRDEEP:•MIReNA:˜carbone/data8/Zhouetal.,2010aFriedlanderetal.,2008MathelierandCarbone,2010•MicroRNAprediction•Targetprediction•MicroRNAdatabaseMicroRNA•Allen等人在植物中定义了预测miRNA靶基因的罚分标准(Allenetal.,2005)：在miRNA与靶基因的配对二聚体中，•miRNA5‘端的第2-13位为核心区域，•在非核心区域，碱基错配罚1分，G:U配对罚0.5分，•若错配发生在核心区域，则罚分加倍。•最终，如果总罚分小于等于4，那么这个基因被认为是该miRNA的候选靶基因。•若在翻译水平沉默靶基因，这一标准可以适当放宽。Target预测Target预测软件•Targetfinder•miRU()•miRandaRACE:Degradome:Target预测验证Target验证Target验证Software：CleaveLandmicroRNA分析流程LengthDistributionofSmallRNAsBdRBdVSmallRNAcount02468101219202122232425Length(nt)051015202519202122232425uniqueredundantx104LengthDistributionofSmallRNAsDistributionof21-and24-ntsmallRNAsontheBrachypodiumgenome12345Ring1:chromosomesRing2:21-ntsmallRNAsRing3:24-ntsmallRNAsRing4:codingsequencesRing5:repetitivesequencesMicroRNApredictionMicroRNAdifferentialanalysisMICRORNA标准化：TPM=readsx(1000000/totalreads)TP2M=readsx(2000000/totalreads)TP10M=readsx(10000000/totalreads)Software:DEGSeq和DESeqGOEnrichmentanalysisCandidatemicroRNAWuetal2013Wuetal2013Model•Nat-siRNA？•Phased-siRNA？•Target？•Hc-siRNA？Noinsight?Jiaoetal2010InteractionbetweenMicroRNAandtargetgeneLuetal2012Lietal2014Hc-sRNAMethylation•MicroRNAprediction•Targetprediction•MicroRNAdatabaseMicroRNAMicroRNADatabaseMicroRNADatabaseMicroRNADatabase小RNA的分类MicroRNA概述MicroRNA生源MicroRNA进化OutlineMicroRNA生源•Invertedduplicationoftargetgene•Derivationfromtransposonelement•Spontaneousevolution(Allenetal.2004)Invertedduplicationoftargetgene(Jinetal.2011;Voinnet2009)MIRNAderivationfromtransposonelementSpontaneousevolution小RNA的分类MicroRNA概述MicroRNA生源MicroRNA进化OutlineMicroRNA扩增方式•Wholegenomeduplication•Segmentalduplication•TandemduplicationPaleo-polyploidevent(Woodhouseetal.2010)MoreWGDseventindicotsthaninmonocotsPaleo-tetraploidmaize(Schnableetal.2011)TwochrinmaizevsonechrinsorghumWGD:WholeGenomeDuplication(Zhangetal.2009)Sb:sorghumZm:maizeMIRNAfamilieswithmoremembersMIRNAfamilieswithlessmembersSegmentalduplication(Li&Mao2006)Tandemduplication(Guddetietal.2005)osa-miR395sbi-miR395bdi-miR395zma-miR395Nonsyntenicregion?TandemduplicationABC1kb1kb1kbS212S233S128S126CACTA_12333CACTA_10954PolyproteinmiR529miR156Ty1copiaTransposonproteinPolyproteinPolyproteinmiR529miR160miR172miR169Ty3gypsyTerminalrepeatS167S196LTRAbrouketal2012PlantCellTransposition:catchedbyTERoleofmicroRNAinpolyploidsOutlineConservationofMIRNAsinfourgrassspeciesMIRNAfamilieswithmorememberMIRNAfamilieswithlessmemberGenome-wideMIRNAconservationinfourgrassspecies50%miRNAslocatedinsyntenicregion31%conservedmiRNAslocatedintelomericorsubtelomericregionA1A1A5WGDWGD后非共保留WGD后共保留祖先miRNAMIRNAlocatedinthemicro-syntenicregion20%48%32%Genome-widepreferentialdistributionofconservedmiRNAsAncestralduplicationP-value=0.026RecentduplicationinmaizeP-value=0.038EnrichedtargetGOtermsofdiploidizationresistantmiRNAsEnrichedtargetsGOtermsofdiploidizationsensitivemiRNAs生物调节代谢过程应答刺激提高了植物的适应性(Abrou