1 研究miRNA参与植物性状调控的主要途径
1.1 影响作物性状关键miRNA的发现和鉴定
自植物中首次发现miRNA以来,不断有新的miRNA被鉴定和验证. 植物miRNA的发现和鉴定最早是在模式植物拟南芥中,通过克隆DCL蛋白剪切产物以及与动物中发现的已知miRNA进行结构比对完
成[9] . 根据序列分析,后续在重要农作物中也预测和鉴定出一些植物中保守的miRNA[12,31] . 然而,无论是传统的克隆还是利用保守序列的预测都具有很大的局限性,不仅耗时耗力而且许多物种特异性或非序列保守的miRNA不能被有效鉴定. 随着高通量测序技术的发展和进步,许多研究者开始利用小RNA文库和高通量测序的方法,很大程度提高了miRNA鉴定的速度和通量[32,33] . 例如为了鉴定玉米雌蕊发育相关的miRNA,研究者通过构建玉米不同发育时期雌蕊的小RNA文库,进行高通量测序分析鉴定到98个关键的miRNA[34] . 为了研究与干旱胁迫相关的miRNA,对植物进行干旱胁迫处理,通过建立小RNA文库及高通量测序分析,得到可能参与植物干旱胁迫耐受响应的miRNA[35] . 目前,在九大作物中大约有2 560个成熟miRNA和2 063个miRNA前体已经被鉴定[36] . miRNA主要是通过抑制其靶基因的表达来发挥功能,因此miRNA靶基因的鉴定也非常重要. miRNA靶基因的鉴定主要是利用降解组联合高通量测序或者5’ RACE(5’ rapid amplification of cDNA ends)的方法验证靶基因被剪切后的转录本5’端的序列来进行[34,37] ,但这种方法只能鉴定到被miRNA通过转录剪切模式靶向的靶基因,不能获得被miRNA通过翻译抑制模式靶向的靶基因.另外,有关作物中调控农艺性状的miRNA的鉴定也可以通过借鉴模式植物中已有的研究结果. 由于植物中存在着许多在进化中高度保守的miRNA,它们在不同植物中的功能可能是相似的. 如miRNA396在拟南芥中的靶基因是生长调控因子GRF,GRF能与GIF互作共同组成miR396-GRF-GIF调控网络,调控拟南芥的生长发育过
程[38] . 参考拟南芥中的研究结果,在水稻中发现转录因子GRF4的编码基因GS2(GRAIN SIZE ON CHROMOSOME 2)也能够被miRNA396剪切. 在一种籼稻变种中,GS2转录本在miRNA396靶向的位点发生了2个碱基的自然突变,从而不能被识别剪切,最终导致该品种的籽粒的长度和重量都显著增加[39] . 但同一个保守的miRNA在不同的植物中也可能具有不同的功能,或者多种功能. 例如miR166在水稻中与营养离子的吸收与积累相关[40] ,而在玉米中却调控了叶极性的发育[41] . miR156除了参与植物分蘖调控外,还参与了花期、籽粒等方面性状的调控[42,43] . 通过以上方法鉴定到的参与调控作物性状的miRNA及其靶基因还需要通过基因沉默或者敲除等遗传学手段进行进一步的功能验证和研究.1.2 miRNA功能研究的主要手段
1.2.1 amiRNA(artificial miRNA)技术
利用amiRNA技术,可以实现对特定miRNA在植物体内进行组成型过量表达或组织特异性表达,从而为研究其功能提供材料. amiRNA技术通过修饰天然的pre-miRNA产生,将miRNA/miRNA*序列替换成对应的感兴趣的靶基因序列,但是保留pre-miRNA的茎环结构. amiRNA中的miRNA链序列与靶基因的mRNA序列互补,而miRNA*链的作用是使其miRNA/miRNA*二聚体的结构和天然的相同,所以amiRNA和miRNA相同,通过内源的miRNA途径对靶基因进行沉
默[44] . 通过改造amiRNA载体骨架,将其中原有的miRNA/miRNA*序列替换为所研究的miRNA/miRNA*序列,采用组成型表达启动子或组织特异性表达启动子驱动pre-miRNA的表达,可以获得miRNA过量表达或组织特异性表达的材料,为研究该miRNA的生物学功能奠定基础. 对miRNA进行过量表达也可以通过采用高表达的启动子直接驱动miRNA的前体(pri-miRNA)进行,但由于很多miRNA尤其是一些新发现的miRNA的前体序列未知,并且miRNA前体的表达量往往很低难以被克隆和测序,所以amiRNA是一种更为快速和精确的过量表达miRNA的方式. 但是amiRNA的应用具有一定的局限性,如果所研究miRNA的本底表达水平很高,在植物中已经达到饱和水平,过表达后可能对植物没有明显影响. 另外,amiRNA技术在育种中的应用也有一定的限制,过表达某种miRNA可能会给植物带来剧烈的性状改变,从而不适合育种;同时,由于一种miRNA可能有多种功能,组成型过表达特定miRNA可能会对植物产生多重影响,改变除目的性状之外的其他性状. 采用组织特异性启动子或者诱导表达型启动子在目的组织或特异条件下对miRNA进行特异性表达可能会减小异位表达对植物整体的影响,从而减少副效应的产生[45] .1.2.2 串联
短片段靶标模拟(short tandem target mimic,STTM)技术
STTM技术的作用原理是通过模拟靶基因序列结合miRNA而造成miRNA丰度的降低和功能的抑制,从而降低miRNA对其内源靶基因的沉默作用. STTM骨架由长约100 nt的片段组成,包括两个miRNA结合位点以及连接它们的一个不完全互补的linker结构,其中linker结构起到维持STTM结构稳定性的作
用[46] . STTM设计中,miRNA结合区域的序列是与成熟miRNA互补的,但在该序列相对于miRNA的5’至3’第10~11个核苷酸处(即miRNA切割靶基因的位点),设计一个不能与miRNA形成互补的凸起(bulge),使得该序列既可以结合miRNA但又不被miRNA切割,使miRNA不能正常行使功能[46] . 该设计使STTM既能结合miRNA,形成miRNA无功能复合体,减少了细胞中游离miRNA的数量,同时又能引起结合到STTM结构上的miRNA降解,该降解过程可能是SDN1[46] 蛋白介导的,从而使miRNA靶基因的表达量升高. 利用STTM技术可以同时靶向一个miRNA家族的多个成员,克服由于基因冗余带来的沉默单个miRNA成员性状改变不明显的问题[46] . 对于一些表达丰度很高的miRNA也能达到显著效果,如miR165/166[47] 可以用STTM技术有效进行沉默. 此外,STTM转基因的植株中,靶向的miRNA被抑制,造成miRNA靶基因表达水平升高,引起植物产生相应表型,能有效反映出miRNA的具体功能. STTM的表达载体一般使用组成型表达启动子,在植物整体及植物全生长周期都能起到沉默miRNA的作用;STTM的表达载体也可以使用组织特异表达启动子或诱导型启动子,实现对miRNA表达的精确调控,获得理想的农艺性状[46,48] . 但该方法也存在一些潜在的问题,如由于同一个miRNA家族的成员之间成熟序列非常保守,使用该方法将沉默整个家族,无法精确研究每一个miRNA家族成员的功能,并可能导致植物生长受到严重影响,不利于获得理想农艺性状. 因此,在研究miRNA的时候需要结合利用多种遗传学手段确定其具体的功能. 目前,STTM技术已成熟利用在miRNA的沉默中,并改变了模式植物和多种农作物的性状. 例如:利用STTM技术沉默番茄中的miR396,使番茄的果实重量增加[49] ;利用STTM技术沉默拟南芥中miR159的表达,改变其响应胁迫的能力[50] ;利用STTM技术沉默水稻中的miR156、miR159、miR166、miR398等,改变了水稻的株型及产量等[51,52] .1.2.3 CRISPR/Cas9基因编辑技术
CRISPR全称为成簇的、规律间隔的短回文重复序列(clustered regularly interspaced short palindromic repeats),与其关联蛋白(CRISPR-associated proteins, Cas)共同组成了CRISPR/Cas系统. 早在1987年,日本微生物学家石野良纯在大肠杆菌中就发现了这种串联间隔重复序列,但一直不清楚其功
能[53] . 后来的研究发现,这种重复序列广泛存在于细菌和古细菌中,直到2002年才正式命名为CRISPR. 而CRISPR序列附近存在的多个编码序列,则被命名为CRISPR相关基因,即Cas基因[54] . 后来的研究发现CRISPR系统实际是细菌的一种获得性免疫系统. 细菌被噬菌体侵染之后,可以获得噬菌体的DNA片段并将之整合进基因组中形成记忆,当细菌再次遭到噬菌体入侵时,就能形成免疫[55] . CRISPR/Cas系统有三种类型,CRISPR/Cas9属于Ⅱ型,Ⅰ型和Ⅲ型由于需要多种Cas蛋白参与,比较复杂故应用不广,而CRISPR/Cas9系统只需要一种Cas蛋白即Cas9的参与,操控简单,因此应用广泛. CRISPR/Cas9系统由单链的向导RNA(single guide RNA,即sgRNA)和有核酸内切酶活性的Cas9蛋白构成,sgRNA需有能与靶基因匹配的序列,Cas9蛋白则需带有核定位信号. 通过设计序列特异性的向导RNA,将Cas9蛋白招募到基因组特定位置,Cas9蛋白行使核酸内切酶功能使DNA双链断裂,而细胞在启动修复的过程中会造成碱基的突变或缺失,从而使靶基因的功能丧失,达到基因定向编辑的目的[56] . 该系统目前已经广泛运用到拟南芥、水稻、玉米、番茄等多种植物的基因编辑中,并取得较好的效果,是一种有效的定向基因编辑手段[57,58] . 用于基因编辑的外源DNA序列很容易在随后几代从被编辑的植物中去除,将它们转化为不含转基因成分的株系,有利于消除公众对转基因隐患的担忧. 类似于编码基因,利用CRISPR/Cas9基因编辑技术可以对特定的非编码基因miRNA进行敲除或碱基替换,敲除和替换的序列可以是miRNA的种子序列、茎环结构序列或启动子序列,替换种子序列可以影响其对于靶标的识别,替换茎环结构可以影响其生物合成,替换启动子可以影响其表达模式. 同一个miRNA不同的家族成员可能具有不同的表达模式,在植物的生长发育过程中具有不同的调控功能,利用CRISPR/Cas9系统可以特异性地针对miRNA基因家族的每个成员单独进行研究,这是STTM技术无法实现的. 但由于miRNA的序列短,某些miRNA在其序列上可能难以设计出满足条件的sgRNA,所以一般在成熟miRNA序列附近的上下游区域选取合适位置设计两个sgRNA位点,当两个位点同时被编辑时,它们之间的成熟miRNA序列将被删去,但这对CRISPR/Cas9系统的编辑效率提出了更高的要求. 目前CRISPR-Cas9技术已经被证明是一种有效的用来编辑植物miRNA的重要手段[59] ,该技术将会极大地拓展miRNA在作物分子育种领域内的应用.2 参与调控农作物性状的主要miRNA
miRNA是植物生长发育过程的重要调控因子,越来越多的证据表明,miRNA在调控农作物性状的过程中起着关键的作用. miRNA及其靶基因对农作物的性状有多方面的影响,在调控农作物的株型、花期、育性、产量、抗逆性状形成、品质等方面都起着关键的作用(图2). 表1中列举了近年来经过实验验证的参与调控农作物性状的主要miRNA及其靶基因.
图2 miRNA及其靶基因对农作物性状有多方面的影响
Fig. 2 Effects of miRNAs and their target genes on multiple traits of crops
表1 经实验验证的参与调控农作物性状的主要miRNA及其靶基因
Table 1 Major miRNAs and their target genes involved in regulation of crop traits verified by experiments
miRNA miRNA靶基因 物种 参考文献 调控株型性状 miR156 SPLs 水稻、小麦 [42,60,61] miR164 NAC2 水稻 [62] miR166 HB4 水稻 [63] miR171 GRAS24 番茄 [64] miR172 AP2 水稻 [65] miR393 TB1,AUX1 水稻 [66,67] miR394 LC4 水稻 [68] miR396 GRF6 水稻 [69] miR444 MADS57 水稻 [70] miR529 SPLs 水稻 [65] 调控花期 miR156 SPLs 水稻、玉米、大麦、大白菜、柳枝稷 [43,71,72,73] miR168 AGO1 番茄 [74] miR172 AP2 水稻、玉米、大麦、大豆、土豆 [75,76,77,78,79] miR393 TB1, AUX1 水稻 [66] miR408 TOC1 小麦 [80] 调控雄性育性 miR159 GAMYB 水稻 [81,82] miR167 ARF6/8 番茄 [83] miR1227 SMARCA3L3 小麦 [84] miR2118 PMS1T 水稻 [85] miR2275 CAF1 小麦 [84] 调控种子/果实发育 miR156 SPLs 水稻、番茄 [42,86,87,88] miR157 SPLs 番茄 [88] miR168 AGO1 番茄 [74] miR396 GRF4 水稻 [39,89] miR397 LAC 水稻 [90] miR1432 ACOT 水稻 [91] miR4376 ACA10 番茄 [92] 调控温度胁迫 miR159 GAMYB 小麦 [93] miR167 ARF 小麦 [94] miR319 PCFs 水稻 [95] miR396 GRFs 烟草 [96] 调控干旱胁迫 miR159 MYB55 玉米 [97] miR162 TRE1 水稻 [98] miR164 OMTNs 水稻 [99] miR168 AGO1 玉米 [71] miR169 NF-YA 玉米、番茄 [100,101] miR9654 DR733425 小麦 [102] 调控盐胁迫 miR171 ARFs 小麦 [103] miR393 TIR1, AFB2 水稻、小麦 [66,103,104] miR408 CLP1 小麦 [105] miR1848 CYP51G3 水稻 [106] 调控养分吸收 miR166 RDD1 水稻 [107] miR399 LTN1, PHO2 水稻、玉米 [107,108,109] miR827 SPX-MFSs 水稻 [110] 调控免疫应答 miR159 HiC-15 棉花 [111] miR164 NAC21/22 小麦 [112] miR166 CLP-1 棉花 [111] miR168 AGO1 水稻 [113] miR319 PCFs, TCP21 水稻 [114] miR398 SODs 水稻、大麦 [115,116] miR408 CLP1 小麦 [80] miR444 MADSs 水稻 [117] miR482 NB–LRRs 番茄 [118] miR528 AO 水稻 [119] miR5300 NB–LRRs 番茄 [118] miR6024 NB–LRRs 番茄 [120] miR7695 NRAMP6 水稻 [121] miR9863 MLA1 大麦 [122] 调控其他方面 miR159(拔节) GAMYB 水稻 [81] miR160(侧根生长) ARFs 大豆 [123] miR164(侧根生长) NAC1 玉米 [124] miR166(叶极性) RLD1 玉米 [41] miR319(结球特性) TCPs 大白菜 [125] miR395(硫酸盐稳态) SULTR2 烟草 [126] miR528(抗倒伏) LAC3,LAC5 玉米 [127] miR828(纤维生长) MYB2D 棉花 [128] miR858(花青素积累) MYB 番茄 [129] miR1848(蜡质合成) CYP51G3 水稻 [130] miR2111(根部结瘤) TML 百脉根 [131] 2.1 调控作物株型的miRNA
植物的株型与产量密切相关,植物的株型包括植物的分支模式、株高、叶片形状和排列方式(叶夹角)以及穗型等方面. 为了筛选出高产的水稻品种,人们定义了理想株型的概念:在理想的株型中,分蘖数少但几乎都是有效分蘖;穗大且籽粒多;茎粗
壮[132] . 目前已经鉴定出了一些与植物株型相关的基因,这些基因当中,有相当一部分已被证明受到miRNA的调控.禾本科植物的株型大部分由其分支模式决定,其中分蘖发生在营养生长期,穗分支则在生殖生长期,它们的模式与产量密切相关. 在水稻中,miR156、miR529及miR172协同调控了其分支模
式[65] . 其中miR156和miR529通过靶向SPL(SQUAMOSA PROMOTER BINDING PROTEIN LIKE)家族的基因,调控水稻的分蘖及穗分支,而miR172则通过靶向AP2(APETALA2)家族的基因调控水稻的分蘖及穗分支. miR156和miR529能使水稻的分蘖数增多,但是也会使它的穗变小,小穗的数目减少,它们负调控花序分生组织的活性及穗分支起始. miR172与分蘖数无关,但它也会使小穗的数目变少,它负调控花向小穗的转变. 小麦的miR156与水稻miR156有着相似的作用,也起到促进分蘖及抑制小穗形成的作用[33] ,其miR529和miR172也可能起着与水稻中相似的作用. 分蘖是受多基因控制的性状,多个miRNA参与调控这些基因,如miR393和miR444. 其中miR393通过靶向两个生长素受体基因,TIR1(TRANSPORT INHIBITOR RESPONSE 1)和AFB2(AUXIN SIGNALING F-BOX 2),正调控水稻的分蘖数[66] . miR444则抑制分蘖[70] ,其机制是:miR444抑制MADS57的表达,而MADS57是D14(Dwarf14)的表达抑制子,在miR444过量的情况下,D14的表达量增多,从而抑制水稻的分蘖. 另外,TB1(TEOSINTE BRANCHED1)与MADS57相互作用会降低MADS57对D14表达的抑制,使D14的表达量增多. 因此,TB1基因也是水稻分蘖的负调控因子.miR393除了调控分蘖外,还与miR394、miR396等一起调控叶夹角. 其中miR393通过靶向TIR1、AFB2基因的转录本,正向调控旗叶的叶夹
角[67] . 而miR394通过靶向一个F-box基因LC4(LEAF INCLINATION 4)负调控叶夹角[68] . miR396与miR393相似,也正调控叶夹角,此外miR396还负调控株高,它在水稻中的靶基因是GRF6(GROWTH REGULATING FACTOR 6),通过赤霉素和类固醇信号途径起到调控株型的作用[69] . 与分蘖和叶夹角相似,穗型和株高也受到多个miRNA的调控. 如miR164通过靶向NAC2(NAC-REGULATED SEED MORPHOLOGY 2)负调控穗的大小,过表达miR164会减少穗的长度及籽粒产量,而过表达抗miR164的靶基因模拟序列(target mimic),会增加穗长及籽粒产量[62] ;miR398与miR172则是正调控穗的大小,沉默它们后,穗都会变短. 其中miR398沉默株系穗变得短小,籽粒变少变轻,还会产生晚花的现象,而过表达则结果相反,而miR172沉默株系则只会使穗变短,但籽粒却变得更密集[133] . miR156与miR396都有调控多个性状的作用,且它们都负调控株高[42] . 除了它们两个外,miR171也是株高的负调控因子,在过表达miR171的番茄株系中,植株矮化且果实产量变少[64] . 另外,miR166也参与植物株型的调控. miR166通过靶向HB4(HOMEODOMAIN CONTAINING PROTEIN4)来调控水稻叶片的形状和木质部的大小,在miR166表达沉默的植株中,叶子卷曲且茎秆的木质部直径变小[63] . 植物的株型是一个复合性状,包含株高、分支模式、叶夹角等多个方面,由多个基因共同调控决定,这些基因的表达同时也在转录后水平受多个miRNA共同调控. 不同的miRNA可能通过协同作用或拮抗作用调控同一个性状,同一个miRNA也可能具有多种功能,参与调控多个不同的性状. 如miR156和miR529通过SPL基因协同促进作物的分蘖[65] ,而miR444则通过MADS57基因抑制分蘖[70] ,与它们形成拮抗关系. 同一个性状由多个miRNA共同调控,可能使植物通过多个途径去改变这个性状,更快地适应环境变化. 同一个miRNA也可能通过不同靶基因调控多个性状,如miR393参与分蘖、叶夹角等性状的调控,其中分蘖性状是与miR156等共同调控的,而叶夹角性状则是与miR394等共同调控的. 这些miRNA与它们的靶基因一起构成复杂的调控网络,在不同的环境中精确地控制植物的株型,使其更好地适应外界环境.2.2 调控作物花期的miRNA
在高等植物的生命周期中,开花是一个非常关键的事件. 植物从营养生长期到开花的转变受到严格的控制,这关系到它们的繁殖是否成功. 为保证在最有利的条件下开花结果,植物进化出复杂的调控网络,它整合了内源性的信号和环境信号,miRNA是其中的一个重要调控因
子[134] . 在调控作物花期的miRNA中,miR172的研究比较透彻,在拟南芥中其靶基因是AP2转录因子[26] . miR172在营养生长时期表达量很低,随着开花的进程表达量逐渐升高. 过表达miR172能够抑制AP2的翻译,从而导致植物早花[135] . 在玉米[76] 、大豆[78] 、土豆[79] 和水稻[75] 等作物中也有类似研究. 以水稻的miR172为例,Hd3a(Heading date 3a)和RFT1(Rice Flowering Locus T 1)编码水稻的成花素合成基因,它们能够促进水稻开花. Ehd1(Early heading date 1)是Hd3a和RFT1上游的正调控子,miR172是通过抑制AP2家族的两个成员IDS1 (INDETERMINATE SPIKELET 1)和SNB(SUPERNUMERARY BRACT)的表达,使Ehd1表达增多,从而导致成花素合成基因表达升高,使水稻的花期提前. miR156是个多功能的miRNA,它在植物中高度保守[136] ,除了调控作物的分蘖外,在调控作物的花期方面也起着重要的作用. 在水稻和玉米过表达miR156的株系中,植株花期延迟[43,71] . 此外,在柳枝稷中过表达玉米的miR156也会使其花期延迟[137] . 在拟南芥和大白菜中miR156也有延长花期的作用[73,138] ,说明它的功能在植物中与miR172一样也是保守的,而且两者可能存在着拮抗作用. 在拟南芥中,miR156在幼年期的表达量较成年期高,而miR172则刚好相反. 通过SPL基因的衔接(有些SPL基因,如AtSPL9、AtSPL10能直接激活miR172从而促进植物的生长发育和开花),它们在调控生长发育的时间上有着连续的作用[139] . 除了miR156和miR172外,作物中还有其他的miRNA在调控花期上起作用. 过表达miR393的水稻株系出现了早花的现象,说明它能使花期提前[66] . 在番茄中,沉默miR168能够使它的开花时间延后[74] ;在小麦中,过表达miR408的植株抽穗时间提前等[80] . 开花是植物生命周期的关键事件,由多个途径共同参与调控,miRNA与其靶基因构成复杂的调控网络参与这些途径,在同一时期或不同时期发挥调控作用. 如miR172和miR156通过SPL基因的衔接,分别作为正调控因子和负调控因子在时间上连续调控花期. 随着植物的生长,miR156的表达水平逐渐下调,使其靶基因SPL表达水平升高,进一步激活miR172从而抑制AP2的表达,最终使植物顺利完成开花. 不同miRNA通过靶基因精确地调控着植物的花期,使其能够正常生长发育繁衍后代. 研究miRNA调控花期的机制对确保作物正常繁殖及缩短作物的生长周期具有重要意义.2.3 调控作物育性的miRNA
植物的育性关系到其能否产生后代,是植物繁殖过程中的关键性状之一. 但在作物育种研究中,通常需要通过雄性不育的株系,培育杂交品种的作物,利用杂交优势提高作物的产
量[140] . 雄性不育分两种:细胞质雄性不育(CMS)和遗传性雄性不育(GMS). CMS产生的原因:线粒体和细胞核的相互作用,产生了无法存活的花粉[141] ,导致CMS的基因位于线粒体基因组中,但它们的表达受到Rf(restorer of fertility)基因的调控[142] . GMS的机制目前还不是很清楚,但近年来的研究发现,非编码RNA在GMS复杂的调控过程中起着重要作用,它可能充当了细胞核与线粒体间信使的角色[143] . 目前,有关调控作物雄性育性miRNA的报导较少,其中一个重要的是miR2118[85] . 长日照条件下,水稻的光周期敏感的雄性不育(PSMS)株系中,miR2118通过靶向PMS1T,一个长链的非编码RNA,产生一系列21 nt的phasiRNA,使这种phasiRNA特异地积累在PSMS系的水稻中,这些phasiRNA可能作用于Rf 基因,进而调控PSMS. 水稻中另一个影响雄性育性的miRNA是miR159[81] . 据研究miR159影响了水稻花药的发育,在水稻中过表达miR159会导致它的花发育畸形,雄蕊里面没有花粉. 另外,在水稻中过表达小麦的miR159,即tae-miR159也会导致水稻雄性不育[82] . 最近研究发现,miR1227和miR2275可能与小麦的雄性不育相关. miR1227和miR2275分别靶向CAF1(CCR4-associated factor 1)和SMARCA3L3(SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A, member 3-like 3),这2个基因都与减数分裂有关. CAF1和SMARCA3L3参与了DNA修复及转录,以维持染色体和基因组在减数分裂过程中的完整性,使减数分裂正常进行[84] . 此外,番茄中过表达miR167会引起花发育的缺陷及雄性不育[83] ,其靶基因是生长素响应因子ARF6(Auxin Response Factor 6)和ARF8(Auxin Response Factor 8),在拟南芥中ARF6和ARF8的作用是促进花序茎伸长及后期雄蕊雌蕊发育[144] . 除此之外,通过建库、高通量测序等方法也发现了一些在雄性不育系和它们保持系间差异表达的miRNA,它们也可能参与了作物雄性育性的调控,如玉米的miR397、miR601、miR604[145] ,水稻的miR528、miR1432[146] ,大豆的miR169、miR171、miR397、miR408[147] 等. 上述调控育性的miRNA基本都与植物的花发育相关,而在花发育过程中,无论是21 nt还是24 nt的phasiRNA均在花序中大量表达[148,149] . 如在玉米和非洲水稻中,21 nt的phasiRNA在花药减数分裂前期会在花粉囊中积累,而在减数分裂期,24 nt的phasiRNA则聚集在绒毡层和性目细胞中,这种phasiRNA积累的现象会持续到花粉性细胞成熟和单倍体配体分化成花粉. 其中21 nt的phasiRNA由miR2118触发产生,而24 nt的phasiRNA由miR2275触发产生[150,151] ,这两种miRNA都参与调控了作物的育性[84,85] . 其中由miR2118触发产生21 nt的phasiRNA已被证明与水稻的光周期敏感的雄性不育相关[85] ,其他的miRNA调控育性的机制目前虽然还不是很清楚,可能也与phasiRNA有关. 植物育性是其繁殖后代的关键,其背后必然存在一个复杂的调控网络,虽然现在有关miRNA调控育性的报道不多,但miRNA在其中所起的重要作用不可忽视,有关miRNA调控作物育性的机制亟待研究.2.4 调控作物种子/果实发育的miRNA
种子是植物生长的基础,也是农业生产中重要的生产资料,miRNA在种子发育过程中也起着重要的作用. miRNA可以通过多种途径调控种子的发育,如信号转导(ABA、生长素、油菜素甾醇等)、淀粉合成、抗氧化作用、糖转化、细胞生长
等[152] . 研究发现在萌发的种子中miR159能够负调控ABA信号的正向调控因子MYB33和MYB101,说明miR159可能通过ABA信号途径调控种子的发育[153] . 此外,miR159在水稻劣质籽粒中的表达量高于优势籽粒[154] ,而且在灌浆期对水稻进行ABA处理可以加快细胞分裂、增加细胞数量、提高灌浆率,从而增加劣势籽粒的重量[155] . 这表明miR159可能通过调控种子对ABA信号的转导,而影响了种子的灌浆. miR164在发育的小麦种子中的表达量呈上升趋势[156] ,miR167在玉米种子中的表达量很高[157] ,且它们的靶基因均是生长素信号途径相关的基因[12] ,说明它们可能通过生长素途径调控种子的发育. 在水稻中过表达miR397会使籽粒增大,且促进穗分枝,从而使水稻的产量升高. miR397的靶基因为LAC(LACCASE)编码类漆酶蛋白,参与油菜素甾醇信号转导,说明miRNA397很可能通过油菜素甾醇信号转导途径调控种子的发育[90] .还有一些miRNA是通过调控细胞生长的相关途径影响种子发育. 比如miR156,它是个多功能的miRNA,除了上文提到的调控水稻的分蘖外,它在调控水稻籽粒的大小、质量、形状方面也有重要的作用. miR156通过它的靶基因SPL家族基因来行使它的功能,如通过OsSPL14来调控籽粒的产
量[42] ,通过OsSPL13、OsSPL16来控制籽粒的大小、质量及形状等. OsSPL13能在谷壳中正调控细胞的大小,从而使水稻的籽粒长度增加且提高了籽粒的产量[87] . 而OsSPL16则是细胞增殖的正调控子,它能促进细胞分化和籽粒的灌浆,从而增加籽粒的宽度和产量[86] . 与miR156相似,miR396也是籽粒产量的负调控因子,它通过miR396-OsGRF4(GROWTH‐REGULATING FACTOR 4)-OsGIF1 (GRF‐interacting factors 1)模式调控籽粒的大小[39,89] . 在过表达miR396的水稻中,籽粒的大小和重量都下降了,说明miR396是籽粒长度和宽度的负调控子,且它是通过抑制细胞扩张来调控籽粒大小的. 另外,OsGRF4能与激活因子OsGIF1互作,且增加OsGIF1的表达量也会增加籽粒的大小. 此外,miR1432也是籽粒的负调控因子,在抑制miR1432的植株中,籽粒的总产量增加了[91] .在番茄中,miR156、miR157、miR168和miR4376被报道参与果实发育的调控. miR156和miR157均靶向SPL家族的基因,LeSPL-CNR,它们参与调控番茄果实的成熟. 其中miR157与果实成熟的起始相关,而miR156则与果实成熟后的软化相
关[88] ;而miR168通过靶向AGO1来调控果实的起始和发育[74] ;miR4376则通过靶向ACA10(autoinhibited Ca2+-ATPase 10),一种Ca2+ -ATPase来调控果实的发育[92] . 在草莓中过表达miR399能使其中葡萄糖、果糖及可溶性物质增多, 从而改善果实的品质[158] . 另外,miR408、miR1867等可能通过抗氧化、糖转化、淀粉合成等途径参与种子发育的调控[152,159] . 协同调控种子果实发育的miRNA应该存在着功能冗余的情况,以确保种子果实正常发育.种子果实的发育好坏关系着植物繁殖的成功与否,其背后必然有着一个复杂精细的调控网络,参与调控该过程的miRNA与其靶基因是其中重要的一环. 这些miRNA与靶基因中,有的可能是上下游关系,有的可能是协同或者相互拮抗的关系,具体的调控网络还有待进一步揭示. miR156、miR159、miR396、miR1432等均对籽粒大小有负调控的作用,它们可能协同负调控籽粒的大小,而miR164、miR397等则与它们的作用相反.
2.5 调控作物抗逆性状形成的miRNA
农作物每年减少的产量中有70%与生物和非生物胁迫相关,其中非生物胁迫占50%以
上[160] . 研究作物抗胁迫的机理,对提高作物产量有很大的帮助. miRNA是作物应对逆境胁迫的主要调控因子,它已经成为一种具有巨大潜力的遗传工具,可以利用它来理解作物在分子水平胁迫适应机理,并用于基因工程来提高作物的抗逆能力.在生长和发育期间,作物可能会经历洪水、干旱、极端温度、营养不平衡或盐分等逆境,越来越多的研究发现,miRNA在作物应对这些逆境的过程中发挥着重大的作用. 在水
稻[95] 、小麦[93] 、大麦[161] 、棉花[162] 、烟草[96] 等作物中均已有报道对温度胁迫响应的miRNA. 其中响应冷胁迫的有miR167、miR319、miR396、miR444等,它们中miR319和miR396都是通过活性氧(ROS)的水平来响应冷胁迫,且miR319和miR396的过表达植株的冷胁迫耐受力增强[95,96] . 响应热胁迫的miRNA则有miR159、miR160、miR166、miR167等,其中在小麦中过表达miR159会使它的对热胁迫更敏感[93] .在水稻、小麦、番茄、玉米等作物中也已有响应干旱胁迫miRNA的报道,但具体作用机制仍不清楚. 在干旱胁迫的小麦中,miR10和miR9654的表达量上
调[102] . 在干旱胁迫条件下,miR159、miR398、miR408、miR528等在耐旱品种的水稻中上调,而在敏感的品种中下调. 这些miRNA的靶基因有些是编码铜蛋白的,它们可能通过下调铜蛋白的含量使体内活性氧(ROS)增多,进而使气孔关闭程度增加以提高抗旱能力[35] . 此外,miR162能通过它的靶基因TRE1(TREHALASE 1)增强水稻的抗旱能力[98] ,而miR164则可能通过它的靶基因,几个OMTN(Oryza miR164-targeted NAC)基因来增强水稻的抗旱能力[99] . 玉米中miR159、miR168、miR169在响应干旱胁迫过程中起着重要作用[97,101] . 在番茄中过表达miR169会使它的气孔开放程度变小,从而增加它的抗旱能力[100] . 在大麦中过表达miR827也可以提高它的抗旱能力[163] .对于调控盐胁迫的miRNA,人们也已在多种作物中有过研究,但是具体机制仍不清楚. 如水稻的miR393、miR184
8[66,106] ,小麦的miR171、miR393和miR408等[103,104,105] ,玉米的miR164、miR167等[164] ,大麦的miR168、miR171、miR444等[165] . miRNA在作物遭受营养不足时也起着重要的调控作用,如miR166、miR399和miR827等[40,107,108,110] . 值得一提的是,miR166与营养离子的吸收和积累有关,它的靶基因RDD1(Dof daily fluctuations 1)过表达时会提高水稻籽粒的产量. 最近研究发现,玉米中PILNCR1会抑制miR399对PHO2(PHOSPHATE2)mRNA的切割. miR399/PILNCR1模块对玉米耐受低磷胁迫有着重要的调控作用[109] .除了上述几种逆境胁迫外,miRNA在调节植物免疫应答方面也有重要的作用,使得植物在病毒、细菌、真菌及虫害等生物胁迫的条件下生存下来. 如水稻的miR168、miR319、miR398
等[113,114,117] ,小麦的miR164、miR408[80,112] ,大麦的miR398、miR9863[116,122] ,番茄的miR482、miR5300、miR6024[118,120] ,棉花的miR159、miR166、miR482等[111] 在作物应对病原菌侵害的过程中起着重要的调控作用.综上所述,miRNA参与多种生物胁迫和非生物胁迫过程,在作物的逆境胁迫适应和抗逆性状形成中起着非常重要的作用. 不同的miRNA可能共同参与应答调控同一种胁迫,同一个miRNA也可能在多个胁迫应答中都发挥作用. 研究miRNA及其靶基因应对这些逆境胁迫的机理,对改良作物性状、提高作物的抗逆能力以及最终提高作物的产量和品质有着很大的帮助.
2.6 调控作物其他性状的miRNA
除了上述性状外,miRNA在调控作物的其他性状方面也有着重要的作用. 如水稻中miR159参与调控拔
节[81] ,miR1848参与调控蜡质的生物合成过程[130] ,玉米miR164参与调控侧根的生长[124] ,玉米miR166参与叶极性的决定[41] ,玉米miR528通过调控木质素合成影响其抗倒伏性等[127] ,棉花miR828和miR858参与调控纤维的生长[128] ,大豆miR160参与调控侧根的生长[124] ,大白菜中miR319通过调控叶片中的细胞分化影响其结球特性[125] ,烟草miR395参与调控体内硫酸盐的稳态[126] ,番茄中miR858参与调控花青素的积累[129] ,miR2111参与调控百脉根的结瘤[131] 等等. miRNA的功能多样化,几乎参与调控了植物各个方面性状的调控,对植物的生长发育和抗逆性状形成具有重要意义. 研究miRNA的作用机制,揭示其关键靶基因和下游基因调控网络,结合基因工程的手段,通过遗传转化和分子设计育种能有效改变作物的性状,为改良作物重要性状提供基础研究材料及理论支持.3 miRNA在改良作物性状中的应用前景和展望
目前,miRNA已成为分子生物学领域的研究热点,在植物中已经有大量的miRNA被发现和鉴定,随着研究手段的不断进步,越来越多植物miRNA的生物学功能及分子作用机制也将逐渐得以阐明,这些miRNA参与植物的生长发育、新陈代谢以及胁迫响应等多个过程,表明miRNA及其靶基因在作物性状改良方面具有不可低估的应用潜能. 一些影响作物重要农艺性状(如产量、株型、育性等)的miRNA陆续被发现,如miR156调控分蘖数、株高、花期和籽粒大
小[65] ,miR396调控叶夹角、株高和籽粒大小[89] ,miR2118调控育性等[85] . 基于miRNA的分子设计育种可通过增加农作物的胁迫耐受性、增加作物产量、提高品质等方面来发展优良的作物品种,从而提高边际土地的利用率,减少农药的使用量,促进农业生产力可持续发展. 另外,miRNA的相关理论也可应用于作物杂交育种. 研究发现,水稻、小麦等的杂交品种与它们的亲本相比,miRNA的整体表达量下调[166,167] . 研究与杂种优势相关的miRNA将有助于作物育种者选择最佳的杂交后代组合,以产生商业上可用的F1代杂交品种[36] . 还有一些与育性相关的miRNA,如Osa-miR2118[85] ,预示miRNA也可应用在培育雄性不育系作物上. 此外,CRISPR/Cas9基因编辑技术在作物miRNA及其靶基因上的应用[59,168] ,使获得性状发生改良且不含转基因成分的遗传材料成为可能,miRNA在作物性状的分子育种改良中具有广阔的应用前景.Advances in miRNAs Regulating Agricultural Traits for Crop
s* JIANG Zeng-Min
g1)** , HE Juan1,3)** , Mo Bei-Xin1,2) , LIU Lin1,2)*** , XU Xiao-Feng1)*** (1) Guangdong Provincial Key Laboratory for Plant Epigenetics, College of Life Sciences and Oceanography, Shenzhen University, Shenzhen 518060, China;2)Longhua Bioindustry and Innovation Research Institure, Shenzhen University, Shenzhen 518060, China;
3)Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province,
College of Optoelectronic Engineering, Shenzhen University, Shenzhen 518060, China)
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摘要
植物microRNA (miRNA) 是一类长度约为20~24 nt的内源非编码小RNA,它们通过在转录后水平调控靶基因的表达,在植物的生长发育、逆境响应和环境适应等过程中起着关键作用. miRNA对水稻、玉米、大豆等重要经济作物的农艺性状也起着重要的调控作用,在改良农作物性状上具有重大的应用潜能. 本文重点介绍了参与作物农艺性状(如株型、花期、种子发育及抗逆等)调控的关键miRNA及其调控途径,同时总结了miRNA参与作物性状改良的主要研究方法和手段,并讨论了miRNA在作物性状改良应用中的前景.
Abstract
Plant microRNAs (miRNA) are 21-24 nt endogenous small non-coding RNAs. They play key roles in plant development, adaptation to stresses and flexible environments through regulating the expression of their target genes post-transcriptionally. Besides, miRNAs are critical for regulating agronomic traits of important economic crops such as rice, maize and soybean, and have great potential in improving crop traits. This review focuses on research progresses of miRNAs involved in regulating important crop agronomic traits (including plant architecture, flowering, seeds development, stresses resistance, etc.) and their regulatory mechanisms. We also summarized the major research methods and strategies for taking advantage of miRNAs to improve crop traits and discussed the future prospects and problems for the application of miRNAs in crop traits improvement.
Keywords
microRNA; crop; regulatory mechanism; agronomic traits improvement
刘琳. Tel:13510529306, Email: linliu@szu.edu.cn
关键词 microRNA,农作物,调控机制,性状改良
microRNA(miRNA)是一类在动植物中广泛分布的长度在20~24 nt左右的非编码小RNA. 最早发现的miRNA编码基因是线虫(Caenorhabditis elegans)的lin-4,它的产物是24 nt的小RNA,能够与其靶基因lin-14 mRNA的3’非编码区序列以非完全互补的形式结合并抑制蛋白质的翻译过程,从而调控线虫早期发育时期的转
在植物中miRNA能够通过转录剪切或翻译抑制来负调控靶基因,从而精确控制其表达水平,这些靶基因主要包括转录因子、胁迫响应蛋白以及其他一些能够影响植物生长发育及生理机能的蛋白质编码基
miRNA主要通过与靶基因mRNA进行碱基互补配对,引导效应蛋白AGO1发挥切割靶基因mRNA或者抑制靶基因翻译的作用(图1
miRNA几乎参与植物的生长发育和新陈代谢各方面的调控,如叶片的发育和形成(miR15