CN101921766A - 应答H2O2的miRNA-1425及其应用 - Google Patents
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Abstract
本发明公开了一种应答H2O2的miRNA-1425及其应用。本发明保护的miRNA-1425是序列表的序列1所示的RNA。本发明还保护序列1所示RNA在抑制PPR蛋白基因表达和/或促进PPR蛋白基因的mRNA降解中的应用;所述PPR蛋白如序列表的序列2所示。应用miRNA-1425有望获得对氧化胁迫、干旱、高盐等有较强耐受性的植株,造福于农业生产。
Description
技术领域
本发明涉及一种应答H2O2的miRNA-1425及其应用。
背景技术
过氧化氢(H2O2)是一种双功能分子,在植物体内起双重作用,既是一种毒性分子能导致细胞氧化损伤,又是一种信号分子在信号转导中发挥作用(Mittler R.Oxidative stress,antioxidants and stress tolerance.Trends Plant Sci.,2002,7:405-410.;Mittler R,Vanderauwera S,Gollery M,Breusegem F V.Reactiveoxygen gene network of plants.Trends Plant Sci.,2004,9:490-498.)。H2O2的双功能性决定了植物体内必然存在着H2O2相关的复杂网络,使它的浓度处于相对稳态。在H2O2引起氧化胁迫的条件下,网络中许多基因或蛋白都会对H2O2产生应答,自身的表达发生变化从而影响植物多种生理活动,最终使植物对H2O2胁迫产生适应。目前,转录组和蛋白质组的数据已经揭示出植物中相当多的应答H2O2的基因和蛋白,主要集中在细胞防御、氧化还原平衡等功能(Vanderauwera S,Van Der Kelen K,Dat J,Gadjev I,Boonefaes T,Morsa S,Rottiers P,Slooten L,Van Montagu M,ZabeauM.et al.A comprehensive analysis of hydrogen peroxide-regulated geneexpression in tobacco.Proc.Natl.Acad.Sci.USA,2003,100:16113-16118.;Wan X Y,Liu J Y.Comparative proteome analysis reveals an intimate proteinnetwork provoked by hydrogen peroxide stress in rice seedling leaves.Mol.CellProteomics,2008,7:1469-1488.)。Wan等通过比较蛋白质组学方法鉴定出144个在H2O2处理的水稻幼苗叶片中差异表达的蛋白,这些蛋白大多与细胞防御、氧化还原平衡、信号转导、蛋白合成和降解、光合成和光呼吸以及能量代谢相关,通过这些差异表达的蛋白以及它们的功能和参与的生理过程,构建了水稻幼苗应答H2O2的蛋白网络,该网络阐明了水稻幼苗叶片通过提高抗氧化相关蛋白的表达和抑制代谢相关蛋白的表达来适应氧化胁迫(Wan X Y,Liu J Y.Comparative proteome analysis revealsan intimate protein network provoked by hydrogen peroxide stress in riceseedling leaves.Mol.Cell Proteomics,2008,7:1469-1488.)。
miRNA(microRNA,微小RNA)是一类长度约为20-24nt的内源单链非编码小分子RNA,在生物体中广泛存在(Bartel D P.MicroRNAs:genomics,biogenesis,mechanisms,and function.Cell,2004,116:281-297.)。近年来大量研究表明,miRNA参与调控植物生长发育以及应答生物和非生物胁迫等许多重要生物学过程(Bushati N,Cohen S M.MicroRNA functions.Annu.Rev.Cell Dev.Biol.,2007,23:175-205.;金龙国,王川,刘进元.植物MicroRNA.中国生物化学与分子生物学报,2006,22:609-614.)。因此,有理由相信miRNA一定在植物应答H2O2的过程中起着关键作用。但是,目前的植物应答H2O2的相关研究主要集中在基因和蛋白,还很少涉及到植物小分子RNA、特别是miRNA领域。植物中是否存在着应答H2O2的miRNA,这些miRNA的作用是什么,目前还没有明确的答案。寻找和鉴定植物体内应答H2O2的miRNA,对于完善植物H2O2相关的调控网络,全面解析植物应答H2O2的分子机制具有重要意义。
水稻不仅是世界最重要的粮食作物之一,同时也是一种重要的模式生物,在植物研究特别是单子叶植物研究中占有重要地位。
发明内容
本发明的目的是提供一种应答H2O2的miRNA-1425及其应用。
本发明保护的miRNA-1425的序列如下(5’→3’):
UAGGAUUCAAUCCUUGCUGCU(序列表的序列1)。
本发明还保护序列1所示RNA在抑制PPR蛋白基因(Os10g0497300)表达中的应用;所述PPR蛋白如序列表的序列2所示。所述PPR蛋白基因可如序列表的序列3自5’末端第107至2491位核苷酸所示。所述PPR蛋白基因也可如序列表的序列3所示。
本发明还保护序列1所示RNA在促进PPR蛋白基因(Os10g0497300)的mRNA降解中的应用;所述PPR蛋白如序列表的序列2所示。所述PPR蛋白基因可如序列表的序列3自5’末端第107至2491位核苷酸所示。所述PPR蛋白基因也可如序列表的序列3所示。
序列表的序列1所示的RNA可用于水稻种质改良。
本发明采用国际上先进的Solexa高通量测序技术结合生物信息学分析、Northern杂交、实时定量PCR等多种生物学手段,首次从基因组水平鉴定到应答H2O2的miRNA-1425,并且证实该miRNA在植物体内调控的靶基因。H2O2胁迫下,miRNA-1425表达上调抑制了细胞器形成,实际上起到了延缓生长发育的效果,有利于植株适应H2O2胁迫。本发明将miRNA-1425的表达变化、靶基因的表达变化以及靶基因功能联系起来,发现H2O2胁迫下应答H2O2的miRNA-1425主要是通过延缓生长发育来帮助水稻幼苗适应氧化胁迫。植物应对外界胁迫条件的有效策略之一是延缓生长发育,这一措施不仅可以增加能量储备以更好地抵御胁迫,还能减少氧化损伤向新生细胞扩散的危险(May MJ,Vernoux T,Leaver C,Montagu M V,InzéD.Glutathione homeostasis in plants:implications for environmental sensing and plant development.J.Exp.Bot.,1998,49:649-667.)。
本发明揭示了应答H2O2的miRNA-1425在植物应对氧化胁迫所起的重要作用。将miRNA-1425基因转入水稻中过量表达或将miRNA-1425基因缺失,就可能获得在耐受氧化胁迫能力方面有明显变化的转基因植株,并有望通过改造获得对氧化胁迫、干旱、高盐等有较强耐受性的植株,造福于农业生产。
附图说明
图1为Northern杂交检测不同浓度H2O2处理的水稻幼苗中miRNA-1425的表达。
图2为miRNA-1425的的靶基因5’RACE验证;序列上方的箭头表示发生切割的位点,数值表示该切点处发生切割的克隆数与克隆总数比值。
图3为实时定量RT-PCR检测靶基因的表达;误差杆表示相对标准偏差。
具体实施方式
以下的实施例便于更好地理解本发明,但并不限定本发明。下述实施例中的实验方法,如无特殊说明,均为常规方法。下述实施例中所用的试验材料,如无特殊说明,均为自常规生化试剂商店购买得到的。下述实施例中的%,如无特殊说明,均为质量百分含量。
以下实施例中所用水稻品种均为籼稻品种93-11(Oryza sativaL.ssp indica cv.93-11),水稻种子购自国家农作物种质保存中心(中国农业科学院作物科学研究所,邮编:100081联系电话:010-68919715)。该水稻品种已经由中国北京基因组研究所完成基因组测序工作。
实施例1、miRNA-1425的发现
一、H2O2处理
水稻种子经表面消毒后,37℃浸泡24h,然后催芽萌发45h。萌发后将水稻转移到光照培养箱中,培养箱条件设定为:温度28℃/21℃(白天16h/夜晚8h),光照强度400μmol/m2·s,相对湿度70%,由Hogland营养液提供水稻生长所需的全部营养,营养液每2天更换一次。
12天龄的水稻幼苗采用H2O2处理:分别浸泡在0.6mM、3.0mM和15.0mM H2O2水溶液中,将浸泡在蒸馏水中的水稻幼苗作为对照;各种处理的幼苗同时置于25℃摇床中处理6h。处理完毕后,将各个处理的水稻样品按0.5g分装,液氮速冻后保存于-80℃备用。
二、miRNA-1425的发现
1、RNA提取
将水稻样品在液氮中研磨,用TRIZOL试剂盒(Invitrogen)提取总RNA,操作步骤按TRIZOL试剂盒自带的说明书进行。使用Ultrospec 3000型紫外分光光度计(Amersham Biosciences)测定提取的RNA在260nm(OD260)和280nm(OD280)波长的吸光度值以确定RNA的纯度和浓度。质量合格的RNA浓度应在1μg/μl以上,OD260/OD280的比值在1.8-2.0之间,且经电泳检测条带清晰,无明显降解和DNA污染。
2、小RNA文库的构建
将质量检验合格的水稻幼苗总RNA用于构建小RNA文库。3种浓度(0.6mM、3.0mM和15.0mM)H2O2处理的水稻样品提取的RNA各取10μg等量混合,总共30μg用于构建水稻幼苗H2O2处理的小RNA文库。对照样品提取的RNA取30μg,用于构建水稻幼苗对照小RNA文库。小RNA文库的构建按照Illumina Sample Preparation Protocol文库构建方法进行,构建好的文库采用Solexa高通量测序(北京华大基因研究中心),获得高质量的18-30nt的小RNA序列(两个小RNA文库均获得了五百多万条序列)。
3、两个小RNA文库中应答H2O2的miRNA的鉴定
参考之前国外文献对高通量测序数据分析的成功方法(Jones-Rhoades M W,Bartel D P.Computational identification of plant miRNAs and their targets,including a stress-induced miRNA.Mol.Cell,2004,14:787-799.),建立一套计算机分析方法用来发现和鉴定测序数据中的水稻miRNA。①将获得的两个小RNA文库中的原始序列通过计算机方法去掉3’接头,并过滤掉序列长度在18nt以下的序列;②通过SOAP程序(Li R,Li Y,Kristiansen K,Wang J.SOAP:short oligonucleotidealignment program.Bioinformatics,2008,24:713-714.)将序列与水稻93-11基因组(http://rice.genomics.org.cn/rice/)进行匹配,保留能与基因组完全匹配的序列,进行后续分析;③将与基因组匹配的序列与国际权威的miRNA数据库miRBase(http://microrna.sanger.ac.uk/sequences/)中公布的水稻miRNA成熟序列及前体序列进行BLAST,从而发现哪些序列来自于已知的水稻miRNA。鉴定出近300个水稻miRNA序列,分属于100余个miRNA家族。
由于高通量测序能给出每个miRNA的测序次数,通过比较水稻幼苗H2O2处理小RNA文库和对照小RNA文库中miRNA的测序数(测序数已经根据小RNA文库测序总数进行了标准化),可以从中发现两个库中测序数相差较大的miRNA,这部分miRNA可能就是应答H2O2的miRNA。基于此方法,比较了两个小RNA文库中miRNA的测序数,并采用以下两个限制条件来提高发现应答H2O2的miRNA的成功率:①miRNA在H2O2处理小RNA文库或对照小RNA文库中测序数要大于100,从而可以保证比较的可靠性;②miRNA在H2O2处理小RNA文库和对照小RNA文库中测序数相对差异要大于30%。发现有7个miRNA家族的测序数存在显著差异,可能是应答H2O2的miRNA家族,其中一个为osa-miR1425(miRNA-1425;miR1425)。
osa-miR1425的序列如下(5’→3’):UAGGAUUCAAUCCUUGCUGCU(序列1)。
H2O2处理小RNA文库的测序次数(QH2O2)为811,对照小RNA文库的测序次数(Q对照)为396,相对差异【(QH2O2/Q对照-1)×100%】为+104.8%。
三、miRNA Northern杂交
为了进一步验证osa-miR1425具有应答H2O2的表达模式,采用miRNA Northern杂交检测几种H2O2处理浓度下(0、0.6、3.0、15.0mM)miRNA的表达情况。
1、制备探针
检测osa-miR1425的探针(5’→3’)的序列:AGCAGCAAGGATTGAATCCTA。
采用T4多聚核苷酸激酶(New England Biolabs)对上述序列末端磷酸基团进行同位素(γ-32P ATP)标记,得到探针,用Microspin G-25柱(GE Healthcare)纯化探针,去除未标记的同位素,纯化后的探针用于Northern杂交。
2、miRNA Northern杂交
Northern杂交中,每个泳道上样量为20μg低分子量RNA,U6基因作为内参,与miRNA在同一张膜上检测。U6基因的探针序列为:TATGCGTGTCATCCTTGCGCAG。
(1)分别提取步骤一制备的各水稻样品的总RNA。
(2)采用Park等报道的PEG8000/NaCl沉淀法(Park W,Li J,Song R,MessingJ,Chen X.CARPEL FACTORY,a Dicer homolog,and HEN1,a novel protein,act inmicroRNA metabolism in Arabidopsis thaliana.Curr.Biol.,2002,12:1484-1495.)从总RNA中富集低分子量RNA(有利于提高miRNA的检测能力)。
(3)miRNA Northern杂交
①富集的低分子量RNA溶于DEPC水,再加入等体积的2×RNA上样缓冲液(95%甲酰胺,18mM EDTA,0.1%溴酚蓝和0.1%二甲苯青),混合后95℃变性5min,得到RNA样品。
②RNA样品用15%尿素变性聚丙烯酰胺凝胶电泳分离,随后用电转移装置(BIO-RAD)转移到Hybond N+尼龙膜(Amersham Biosciences)上。
③转移有RNA样品的尼龙膜经6×SSC溶液短暂漂洗,紫外交联(Stratagene)5min,再80℃烘烤2h,使RNA完全固定在尼龙膜上。
④将转移了RNA的尼龙膜放入杂交管中,加入5ml ULTRAhyb-Oligo杂交液(Ambion)在杂交炉中42℃预杂交2h,然后加入探针,混匀,42℃杂交过夜。
⑤杂交结束后,小心倒出杂交液,加入含0.5%SDS的2×SSC溶液,42℃洗涤三次,每次10min。
⑥洗膜结束后,将膜用保鲜膜包裹,平整压于X光片下,附加增感屏,-70℃曝光1周。
⑦曝光结束后,冲洗X光片,进行光密度扫描,以对照组的杂交信号为1,计算各个处理组杂交信号的相对强度。
结果见图1。Northern杂交结果和测序数据很吻合,osa-miR1425受H2O2诱导表达。
实施例2、应答H2O2的miRNA的靶基因预测与验证
由于植物miRNA与靶基因mRNA近乎完全互补,因此可以通过生物信息学方法预测osa-miR1425的靶基因。采用Schwab等描述的方法预测miRNA的靶基因(Schwab R,Palatnik J F,Riester M,Schommer C,Schmid M,Weigel D.Specific effects ofmicroRNAs on the plant transcriptome.Dev.Cell,2005,8:517-527.)。使用Patscan程序在水稻93-11全长cDNA和基因库(http://rice.genomics.org.cn/rice/)中寻找能与miRNA序列近乎完全互补的cDNA或基因,即为miRNA的靶标;参数设置为:Patscan程序参数为默认设置,允许最大错配数为3个,miRNA的第10和11位碱基不允许有错配,靶标的功能通过NCBI(http://www.ncbi.nlm.nih.gov/)同源性检索,以同源性最高的已知功能基因进行注释。
预测结果见表1。
表1osa-miR1425的靶基因及功能
miRNA家族 | NBb | 预测靶基因 | 靶基因功能 |
miR1425 | ↑ | Os10g0497300 | PPR蛋白(Pentatricopeptide repeat protein) |
注:(↑)表示在H2O2处理下表达量上调;bNB,Northern杂交分析得出的结果。
osa-miR1425的靶标是PPR蛋白(Pentatricopeptide repeat protein)基因Os10g0497300(GENBANK ACCESSION NO.Os10g0497300,见序列表的序列3;编码序列表的序列2所示的蛋白质)。PPR基因家族在高等植物中优势表达,是含有上百个成员的大基因家族,参与了许多转录后加工过程比如剪切、编辑和翻译等。大多数PPR基因定位在包括线粒体和叶绿体在内的各种细胞器,在细胞器基因表达的各个阶段都起至关重要的作用,在某种程度上可以控制细胞器的形成(O’Toole N,Hattori M,Andres C,Iida K,Lurin C,Schmitz-Linneweber C,Sugita M,Small I.On theexpansion of the pentatricopeptide repeat gene family in plants.Mol.Biol.Evol.,2008,25:1120-1128.)。
实施例3、miRNA对靶基因mRNA的切割
osa-miR1425对靶基因Os10g0497300(见序列表的序列3)mRNA的切割用5’RACE方法进行验证(Jones-Rhoades M W,Bartel D P.Computational identification ofplant miRNAs and their targets,including a stress-induced miRNA.Mol.Cell,2004,14:787-799.)。靶基因mRNA被miRNA切割后,其较为稳定的3’切割产物5’端核苷酸磷酸集团暴露,用T4RNA连接酶在该切割产物5’端连接上5’RACE专用接头;通过反转录反应合成cDNA;通过靶基因特异的巢式外引物和试剂盒所带的巢式外引物进行第一轮PCR,靶基因特异的巢式内引物和试剂盒所带的巢式内引物进行第二轮PCR;将5’RACE获得的PCR产物连接到pMD 19-T载体(TaKaRa)后测序,就能知道精确的靶基因mRNA切割位点。
1、分别提取实施例1的步骤一制备的各水稻样品的总RNA。
2、按Firstchoice RLM-RACE试剂盒(Ambion)操作说明进行5’RACE,靶基因特异的巢式外引物的序列为:CCTCCCTTCTTTGCAATGACTGTCA,靶基因特异的巢式内引物的序列为:CAGATGCCTCGATCCAACATTTCA。
3、PCR产物进行琼脂糖电泳,回收250bp左右的特异条带(图2中泳道1所示)。
4、将回收的PCR产物连接到pMD 19-T载体(TaKaRa),并转化大肠杆菌DH5α(TaKaRa)。
5、挑单克隆测序,根据测序结果确定靶基因mRNA的切割位点。
测序结果显示的切割位点见图2。osa-miR1425的靶基因在与其互补的区域发生了切割,这个结果强有力的证明了Os10g0497300确实是osa-miR1425体内真正调控的靶基因。
实施例4、靶基因的表达量分析
为了进一步考察osa-miR1425的功能,用实时定量RT-PCR检测靶基因Os10g0497300在几种H2O2处理浓度下(0、0.6、3.0、15.0mM)的水稻幼苗中的表达情况。
1、分别提取实施例1的步骤一制备的各水稻样品(2g)的总RNA。
2、总RNA加入DNase I(TaKaRa),室温放置30min以除去基因组DNA的污染。
3、加入终止缓冲液(50mM EDTA)后,70℃加热10min以变性DNase I和RNA。
4、采用TaKaRa RNA PCR Kit,用RNA合成cDNA模板,操作按试剂盒说明书进行。
5、采用Power SYBR Green PCR Master Mix(Appl ied Biosystems)在iCycler iQ5Multicolor实时定量PCR检测仪(Bio-Rad)上进行PCR扩增反应,通过比较CT值法(ΔΔCT值法)(Schmittgen T D.Real-Time Quantitative PCR.Methods,2001,25:383-385.)计算靶基因在不同样品中的相对表达量(对照组基因的表达量设定为1)。靶基因的检测设置3个重复,结果取平均值。以水稻β-tubulin基因作为内参(Zhao B,Ge L,LiangR,Li W,Ruan K,Lin H,Jin Y.Members of miR-169 family are induced by highsalinity and transiently inhibit the NF-YA transcription factor.BMC Mol.Biol.,2009,10:29.)。
扩增靶基因的引物如下(5’→3’):
上游引物:TACGAAACGGTATCCACCCTAATC;
下游引物:CAGATGCCTCGATCCAACATTTCA。
扩增β-tubulin基因引物如下(5’→3’):
上游引物:CCTCCAAGGATTTCAAGTCTGC;
下游引物:TTGTAAGGTTCCACCACGGTA。
结果见图3。靶基因Os10g0497300在H2O2处理下表达量发生了明显的变化,通过和osa-miR1425的表达(图1)进行比较,发现Os10g0497300的表达和osa-miR1425的表达呈现出明显的负相关性,即osa-miR1425表达上调的时候,Os10g0497300的表达就相应下调,这与miRNA对靶基因的负调控作用是完全一致的。osa-miR1425的表达随着H2O2处理浓度从0-3.0mM提高而增加,如果浓度进一步增大(3.0-15.0mM),osa-miR1425的表达会下降;相应地,Os 10g0497300的表达随着H2O2处理浓度增加而呈现出先下降后上升的表达模式,与osa-miR1425的表达完全负相关。实时定量PCR的结果进一步证明了Os10g0497300确实是osa-miR1425的靶基因。
Claims (8)
1.序列表的序列1所示的RNA。
2.序列1所示RNA在抑制PPR蛋白基因表达中的应用;所述PPR蛋白如序列表的序列2所示。
3.如权利要求2所述的应用,其特征在于:所述PPR蛋白基因如序列表的序列3自5’末端第107至2491位核苷酸所示。
4.如权利要求2所述的应用,其特征在于:所述PPR蛋白基因如序列表的序列3所示。
5.序列1所示RNA在促进PPR蛋白基因的mRNA降解中的应用;所述PPR蛋白如序列表的序列2所示。
6.如权利要求5所述的应用,其特征在于:所述PPR蛋白基因如序列表的序列3自5’末端第107至2491位核苷酸所示。
7.如权利要求5所述的应用,其特征在于:所述PPR蛋白基因如序列表的序列3所示。
8.序列表的序列1所示的RNA在水稻种质改良中的应用。
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