王永江;李中安;张振臣;杨方云;周彦;曹孟籍;申晚霞;刘金香;周常勇;

• 1:国家柑桔工程技术研究中心
• 2:河南省农业科学院植物保护研究所

摘要(Abstract)：

【目的】预测鉴定甜橙miRNA及其作用机制和功能,为深入研究挖掘甜橙miRNA及其抗病相关miRNA的作用等提供翔实的基因信息。【方法】利用高通量测序技术、相关的生物信息学软件和数据库,构建甜橙小RNA文库,然后以甜橙基因组为参考基因组,进行甜橙miRNA鉴定和靶基因预测;利用GO和KEGG数据库对靶基因进行功能分析。【结果】成功构建了甜橙小RNA文库:获得12 617 944条小RNA序列分属于3 065 625种小RNA序列,获得序列中24 nt长的序列最多,占到序列的49.3%。鉴定出2 199 561条miRNA,其中已知miRNA638 722条,分属52种miRNA家族,候选miRNA1 560 839条,分属204种miRNA。已知的52种保守miRNA基因预测出927个靶基因位点,候选miRNA中有154种预测出1 777个靶基因位点。GO数据库对靶基因分类显示,甜橙miRNA调控的靶基因主要参与多细胞互作、转录本调控和细胞组成等生物学途经和分子功能方面。KEGG数据库中病原物与寄主互作亚库分析结果显示,靶基因主要与抗性蛋白、转录因子和抗病酶等关联。【结论】预测鉴定出一批甜橙miRNA及其调控的靶基因,为进一步研究提供了参考信息。

关键词(KeyWords)： 甜橙;miRNA;靶基因;高通量测序

Abstract:

Keywords:

基金项目(Foundation): 农业部行业公益项目(200903004-06,201203076-01);; 科技支撑计划(2012BAD19B00);; 教育部创新团队(IRT0976)

作者(Author): 王永江;李中安;张振臣;杨方云;周彦;曹孟籍;申晚霞;刘金香;周常勇;

Email:

参考文献(References)：

• [1]HUNTZINGER E,IZAURRALDE E.Gene silencing by microRNAs:contributions of translational repression and mRNA decay[J].NatureReviews Genetics,2011,12(2):99-110.
• [2]WU L,ZHOU H Y,ZHANG Q Q,ZHANG J G,NI F R,LIU C,QIY J.DNA methylation mediated by a microRNA pathway[J].Molecu-lar Cell,2010,38(3):465-475.
• [3]KIDNER C A,MARTIESSEN R A.The developmental role of mi-croRNA in plants[J].Current Opinion in Plant Biology,2005,8:38-44.
• [4]MALLORY A C,VAUCHERET H.Functions of microRNAs and re-lated small RNAs in plants[J].Nature Genetics,2006,38:31-36.
• [5]JOVER-GIL S,CANDELA H,PONE M R.Plant microRNAs anddevelopment[J].International Journal of Developmental Biology,2005,49:733-744.
• [6]MOXON S,JING R C,SZITTYA G,SCHWACH F,PILCHER R LR,MOULTON V,DALMAY T.Deep sequencing of tomato shortRNAs identifies microRNAs targeting genes involved in fruit ripen-ing[J].Genome Research,2008,18:1602-1609.
• [7]FAHLGREN N,HOWELL M D,KASSCHAU K D,CHAPMAN EJ,SULLIVAN C M,CUMBIE J S,GIVAN S A,LAW T F,GRANTS R,DANGL J L,CARRINGTON J C.High-throughput sequencingof Arabidopsis microRNAs:evidence for frequent birth and death ofmiRNA genes[J].PloS One,2007,2(2):e219.
• [8]FRIEDLANDER M R,CHEN W,ADAMIDI C,MAASKOLA J,EINSPANIER R,KNESPEL S,RAJEWSKY N.Discovering microR-NAs from deep sequencing data using miRDeep[J].Nature Biotechnol-ogy,2008,26(4):407-415.
• [9]LI X Y,WANG X,ZHANG S P,LIU D W,DUAN Y X,DONG W.Identification of soybean microRNAs involved in soybean Cyst nema-tode infection by deep sequencing[J].PloS One,2012,7(6):e39650.
• [10]XU Q,LIU Y L,ZHU A D,WU X M,YE J L,YU K Q,GUO WW,DENG X X.Discovery and comparative profiling of microRNAsin a sweet orange red-flesh mutant and its wild type[J].BMC Ge-nomics,2010,11(1):246.
• [11]SONG C N,WANG C,ZHANG C Q,KORIR N K,YU H P,MA ZQ,FANG J G.Deep sequencing discovery of novel and conservedmicroRNAs in trifoliate orange(Citrus trifoliata)[J].BMC Genomics,2010,11:431.
• [12]ZHANG J Z,AI X Y,GUO W W,PENG S A,DENG X X,HU CG.Identification of miRNAs and their target genes using deep se-quencing and degradome analysis in trifoliate orange[Poncirus trifo-liata L.Raf[J].Molecular Biotechnology,2012,51:44-57.
• [13]SUN L M,AI X Y,LI W Y,GUO W W,DENG X X,HU C G,ZHANG J Z.Identification and comparative profiling of miRNAs inan early flowering mutant of trifoliate orange and its wild type bygenome-wide deep sequencing[J].PloS One,2012,7(8):e43760.
• [14]WU X M,LIU M Y,GE X X,XU Q,GUO W W.Stage and tissue-specific modulation of ten conserved miRNAs and their targets dur-ing somatic embryogenesis of Valencia sweet orange[J].Planta,2011,233(3):495-505.
• [15]CHEN Xiao-dong.Germplasm conservation and microRNA identifi-cation of in vitro plantlets in citrus trees[D].Fuzhou:Fujian Agri-culture and Forestry University,2011.陈晓东.柑橘试管苗种质保存及其microRNA鉴定[D].福州:福建农林大学,2011.
• [16]LI Y,ZHANG Z,LIU F,VONGSANGNAK W,JING Q,SHEN BR.Performance comparison and evaluation of software tools for mi-croRNA deep-sequencing data analysis[J].Nucleic Acids Research,2012,40(10):4298-4305.
• [17]SONG Chang-nian,JIA Qi-dong,WANG Chen,LI Fei,ZHANGZhen,FANG Jing-gui.Computational identification and analysis ofthe putative microRNAs in 32 fruit crops[J].Acta Horticulturae Sini-ca,2010,37(6):869-879.宋长年,贾启东,王晨,李飞,章镇,房经贵.32种果树microRNA的生物信息学预测与分析[J].园艺学报,2010,37(6):869-879.
• [18]ALLEN E XIE Z X,GUSTAFSON A M,CARRINGTON J C.Mi-croRNA-directed phasing during trans-acting siRNA biogenesis inplants[J].Cell,2005,121:207-221.
• [19]SCHWAB R,PALATNIK J F,RIESTER M,SCHOMMER C,SCHMID M,WEIGEL D.Specific effects of microRNAs on the planttranscriptome[J].Develompmental Cell,2005,8(4):517-527.
• [20]WANG X S,TONG Y A,WANG S H.Rapid and accurate detectionof plant mirnas by liquid northern hybridization[J].International Jour-nal of Molecular Sciences,2010,11(9):3138-3148.
• [21]SUN Hai-xi,WANG Xiu-jie.The development and future perspec-tives of DNA sequencing technology[J].e-Science,2009,6:19-29.孙海汐,王秀杰.DNA测序技术发展及其展望[J].e-Science,2009,6:19-29.
• [22]CHINCHILLA D,BAUER Z,REGENASS M,BOLLER T,FELIXG.The Arabidopsis receptor kinase FLS2 binds flg22 and determinesthe specificity of flagellin perception[J].The Plant Cell Online,2006,18(2):465-476.
• [23]ZIPFEL C.Pattern-recognition receptors in plant innate immunity[J].Current Opinion in Immunology,2008,20(1):10-16.
• [24]ZIPFEL C,KUNZE G,CHINCHILLA D,CANIARD A,JONES J DG,BOLLER T,FELIX G.Perception of the bacterial PAMP EF-Tuby the receptor EFR restricts Agrobacterium-mediated transforma-tion[J].Cell,2006,125(4):749-760.
• [25]SWIDERSKI M R,INNES R W.The Arabidopsis PBS1 resistancegene encodes a member of a novel protein kinase subfamily[J].ThePlant Journal,2001,26(1):101-112.
• [26]BENT A F,KUNKEL B N,DAHLBECK D,BROWN K L,SCHMIDT R,GIRAUDAT J,LEUNG J,STASKAWICZ B J.RPS2of Arabidopsis thaliana:A leucine-rich repeat class of plant diseaseresistance genes[J].Science,1994,265(5180):1856-1860.
• [27]WARREN R F,HENK A,MOWERY P,HOLUB E,INNES R W.A mutation within the leucine-rich repeat domain of the Arabidopsisdisease resistance gene RPS5 partially suppresses multiple bacterialand downy mildew resistance genes[J].The Plant Cell Online,1998,10(9):1439-1452.
• [28]GRANT M R,GODIARD L,STRAUBE E,ASHFIELD T,LEWALD J,SATTLER A,INNES R W,DANGL J L.Structure ofthe Arabidopsis RPM1 gene enabling dual specificity disease resis-tance[J].Science,1995,269:843-846.
• [29]SHIRASU K,LAHAYE T,TAN M W,ZHOU F,AZEVEDO C,SCHULZE-LEFERT P.A novel class of eukaryotic zinc-bindingproteins is required for disease resistance signaling in barley and de-velopment in C.elegans[J].Cell,1999,99(4):355-366.
• [30]WANG W,VINOCUR B,SHOSEYOV O,ALTMAN A.Role ofplant heat-shock proteins and molecular chaperones in the abioticstress response[J].Trends in Plant Science,2004,9(5):244-252.
• [31]WANG G F,WEI X N,FAN R C,ZHOU H B,WANG X P,YU CM,DONG L L,DONG Z Y,WANG X J,KANG Z S,LING H Q,SHEN Q H,WANG D W,ZHANG X Q.Molecular analysis of com-mon wheat genes encoding three types of cytosolic heat shock protein90(Hsp90):Functional involvement of cytosolic Hsp90s in the con-trol of wheat seedling growth and disease resistance[J].New Phytolo-gist,2011,191:418-431.
• [32]CHICO J M,CHINI A,FONSECA S,SOLANO R.JAZ repressorsset the rhythm in jasmonate signaling[J].Current Opinion in PlantBiology,2008,11:486-494.
• [33]ABE H,URAO T,ITO T,SEKI M,SHINOZAKI K,YAM-AGUCHI-SHINOZAKI K.Arabidopsis AtMYC2(bHLH)and At-MYB2(MYB)function as transcriptional activators in abscisic acidsignaling[J].The Plant Cell Online,2003,15(1):63-78.
• [34]DOMBRECHT B,XUE G P,SPRAGUE S J,KIRKEGAARD J A,ROSS J J,REID J B,FITT G P,SEWELAM N,SCHENK P M,MANNERS J M.MYC2 differentially modulates diverse jasmonate-dependent functions in Arabidopsis[J].The Plant Cell Online,2007,19(7):2225-2245.
• [35]BIRKENBIHL R P,DIEZEL C,SOMSSICH I E.ArabidopsisWRKY33 is a key transcriptional regulator of hormonal and metabol-ic responses toward Botrytis cinerea infection[J].Plant Physiology,2012,159(1):266-285.
• [36]JIANG Y Q,DEYHOLOS M K.Functional characterization of Ara-bidopsis NaCl-inducible WRKY25 and WRKY33 transcription fac-tors in abiotic stresses[J].Plant Molecular Biology,2009,69(1):91-105.
• [37]YOSHIOKA H,NUMATA N,NAKAJIMA K,KATOU S,KAWAKI-TA K,ROWLAND O,JONES J D G,DOKE N.Nicotiana ben-thamiana gp91phox homologs NbrbohA and NbrbohB participate inH2O2 accumulation and resistance to Phytophthora infestans[J].ThePlant Cell Online,2003,15(3):706-718.
• [38]KAPLAN B,SHERMAN T,FROMM H.Cyclic nucleotide-gatedchannels in plants[J].FEBS letters,2007,581(12):2237-2246.
• [39]LIU Sheng-cai,LIN Yu-ling,CHEN Xiao-dong,LAI Zhong-xiong.Novel and conserved miRNAs i dentification during in vitroflowering of Amaranthus by solexa sequencing[J].Chinese Journal ofTropical Crops,2011,32(7):1296-1303.刘生财,林玉玲,陈晓东,赖钟雄.利用Solexa测序技术鉴定苋菜试管开花过程中的miRNAs[J].热带作物学报,2011,32(7):1296-1303.
• [40]CHEN Dong-ju.Analysis of small RNA deep sequencing data duringseed development in Arabidopsis thaliana[D].Fuzhou:Fujian Agri-culture and Forestry University,2011.陈冬菊.拟南芥种子发育过程小RNA深度测序数据的分析[D].福州:福建农林大学,2011.
• [41]ABDELALI B,PHILLIP K W,SCOTT D,CLAUDE W D,JOHN EC.Conservation and divergence of microRNAs in Populus[J].BMCGenomics,2007,8:481.
• [42]WEI B,CAI T,ZHANG R Z,LI A L,HUO N X,LI S,GU Y Q,VO-GEL J,JIA J Z,QI Y J,MAO L.Novel microRNAs uncovered bydeep sequencing of small RNA transcriptomes in bread wheat(Triticum aestivum L.)and Brachypodium distachyon(L.)Beauv[J].Functional Integrative Genomics,2009,9:499-511.