植物Argonaute基因研究进展

Argonaute (AGO)蛋白是一类在小RNA介导的基因沉默中起关键作用的RNA结合蛋白。AGO家族在不同物种中高度保守,由可变N端、PAZ、MID和PIWI等4个结构域组成。根据系统进化学以及AGO基因结合的小RNA类型可将该家族分为3个亚族,即AGO-like、PIWI-like和Group~3亚家族,目前发现的植物中的AGO基因均属于AGO-like亚家族。研究表明,不同类型的小RNA结合特定种类的AGO蛋白在植物不同生长发育和响应胁迫过程中扮演着重要角色。最近发掘的许多AGO蛋白新的生物学功能表明,AGO在调控配子生成和发育过程中也具有重要意义。现对植物AGO家族的分类、结构特点、结合的小RNA类别以及最新的功能研究进行详细综述。     

玉米AGO基因的克隆与表达分析

以玉米自交系‘昌7-2’三叶期前后2个时间点(种子萌发后5d和8d)幼苗不同组织部位的总RNA为研究对象,采用实时荧光定量PCR技术,对玉米中6个Argonaute(AGO)蛋白家族基因(AGO1、AGO2、AGO4、AGO5、AGO7和AGO10)在幼苗不同发育时期及不同组织部位的表达谱进行了研究。结果表明:(1)AGO1、AGO2、AGO4和AGO7在种子萌发后5d和8d幼苗不同组织中均有表达,种子萌发后5d幼苗中的平均表达量均高于萌发8d的幼苗,且在地上部分新生组织或细胞分裂比较旺盛的组织中表达较多,表明AGO1、AGO2、AGO4和AGO7可能在玉米幼苗发育早期的分生组织分裂生长中发挥调控作用。(2)AGO5和AGO10只在叶片和茎尖中表达,其他组织中不表达;其中AGO5主要集中在新生叶和种子萌发后8d的茎尖中,AGO10在玉米叶发育过程中可能存在着迁移的现象。     

解读AGO蛋白结构及其功能

RNA沉默是由小RNA特异向导和RNA诱导的沉默复合物（RISC）切割或者抑制靶标mRNA翻译的一种调控系统. 作为RISC的核心成分,AGO蛋白(argonaute proteins)由N末端、PAZ、MID和PIWI 4个结构域组成. PAZ区能非序列特异性识别结合双链小RNA 3′末端悬垂的2个核苷酸,MID与PIWI界面处的“保守口袋”识别结合小RNA 5′端第1位核苷酸,PIWI区具有切割mRNA的催化中心. 根据系统进化学分析,AGO蛋白家族分为3个组：AGO like、PIWI-like和GROUP3. 拟南芥共编码10种AGO蛋白.目前已经证实,具有切割活性的为AtAGO1、AtAGO4和AtAGO7,三者参与的小RNA通路也已得到确认. 在拟南芥10种AGO蛋白中,AtAGO1与AtAGO10、AtAGO1与AtAGO7、AtAGO4与AtAGO6存在功能上的部分冗余.     

玉米AGO基因在籽粒发育过程中的表达分析

以玉米自交系‘昌7-2’授粉后4个时间点(授粉后7、10、14和20d)的籽粒总RNA为研究对象,采用实时荧光定量PCR技术,对玉米中5个Argonaute(AGO)蛋白家族基因(AGO1、AGO2、AGO4、AGO10和AGO18)在籽粒不同发育时期的表达谱进行了研究。结果表明:AGO1和AGO2在籽粒发育过程中呈现一致的表达趋势,在授粉后7d的籽粒中表达量最高,从授粉后7d到20d呈持续下降的趋势。AGO4、AGO10和AGO18具有一致的表达趋势,均呈现先下降后上升的趋势,在授粉后10d的籽粒中表达量最低。结合实验室前期获得的miRNA在玉米籽粒不同发育阶段的表达谱,发现不同AGO家族基因可协助其靶标miRNA参与玉米籽粒发育调控。     

AGO在非编码RNA功能中的作用

AGO(argonaute)蛋白家族存在于几乎所有的物种中,是一种高度保守的碱性蛋白。AGO蛋白在细胞整个生命进程中发挥重要角色,参与m RNA降解、基因沉默、蛋白翻译等多种细胞进程。AGO蛋白也可与不同的非编码RNA结合发挥重要的作用。研究非编码RNA基因的作用机制,有助于发现新的与器官形成、胚胎发育和生长相关的调控因子,进一步探究人类疾病发病机制,为开发新的治疗各种疾病的手段提供理论基础。该文主要对AGO在非编码RNA中的生物学作用加以综述。     

c-flag-ago3质粒在人293细胞系中的表达及AGO3蛋白复合物的细胞定位

将c-flag-ago3质粒转入人293细胞系中,使c-flag-ago3基因稳定表达,为进一步研究AGO3蛋白复合物的结构、功能奠定了基础.利用亲和标签flag对目标蛋白进行检测和监测,将已合成的c-flag-ago3质粒和用于对照的质粒si-ago3(能使ago3基因沉默的质粒)导入人293细胞系中,在荧光镜下观察转染效果.RT-PCR法检测基因含量,蛋白印迹法(WB)检测人293细胞表达的flag-ago3,并对其蛋白复合物进行细胞定位.结果显示,c-flag-ago3质粒和si-ago3质粒成功地导入人293细胞系中,免疫细胞化学显示c-flag-ago3基因在细胞中高表达、并检测到AGO3复合物定位于细胞质中.AGO3复合物在细胞中可稳定表达,为进一步研究AGO3蛋白复合物的构成及其在人体中的功能奠定了基础.     

香蕉枯萎病菌粗毒素对地衣芽胞杆菌生长和培养液上清蛋白组成的影响

本文研究了香蕉枯萎病菌4号生理小种湛江菌株(Foc 4-zj)产生的粗毒素对地衣芽胞杆菌R21菌株生长及其培养液中蛋白组成变化的影响。实验结果表明, Foc 4-zj的粗毒素能够抑制R21菌株的生长, 缩短其生长周期; 减少培养液上清蛋白含量以及改变蛋白质的种类; 低剂量的粗毒素有利于拮抗蛋白的积累, 而高剂量的粗毒素则相反。     

一种可用于外源基因功能鉴定的真菌分泌表达载体74HSP-EGFP的构建及验证

传统的植物遗传转化体系能够鉴定植物基因的功能,但该过程烦琐、耗时长,且存在一定的局限性.本研究构建具有蛋白分泌信号肽和荧光蛋白EGFP的真菌分泌载体74HSP-EGFP,通过尖孢镰刀菌古巴专 化型(Fusarium oxysporum f.sp.cubense,Foc)携带侵染巴西蕉(Musa nana)植株对载体上的外源基因功能进行快速鉴定.利用蛋白质组学技术鉴定获得来源于香蕉枯萎病致病菌Foc的主要分泌蛋白Secreted in xylem 1(SIX1d)N端分泌信号肽.基于pCT74载体、SIX1d分泌信号肽序列以及供外源基因插入的多克隆位点序列构建真菌分泌表达载体74HSP,将EGFP基因通过多克隆位点插入到该载体中获得分泌载体74HSP-EGFP.转化Foc后发现,Foc分泌载体74HSP-EGFP转化菌株在PDA培养基和KK培养基中生长4 d后,菌体中无绿色荧光信号,培养基中检测到绿色荧光信号.转化菌株侵染巴西蕉后,在植株根部组织切片中检测到绿色荧光信号.研究结果表明分泌载体74HSP-EGFP能够利用病原菌将携带的外源蛋白表达并分泌到被侵染的植物体内,实现对外源基因功能的快速分析.     

伯克霍尔德菌HQB-1抑制香蕉枯萎病菌的活性化合物分离鉴定

【背景】伯克霍尔德菌HQB-1对香蕉枯萎病菌(Fusariumoxysporumf.sp.cubenseTropical Race 4,Foc TR4)具有良好的防治效果。【目的】从菌株HQB-1发酵液中分离活性化合物并通过超高效液相联用质谱进行检测,获得该菌株具有良好生防作用的单体化合物。【方法】以HQB-1为目标菌株,大批量发酵并进行发酵液分离、提纯,通过超高效液相联用质谱法与核磁共振波谱法鉴定活性化合物。【结果】HQB-1菌株发酵液中的蛋白对Foc TR4无抑制作用;HQB-1菌株产生儿茶酚型铁载体,而不产生异羟肟酸型铁载体;对HQB-1菌株发酵液离心、浓缩、干燥,获得乙酸乙酯浸膏(粗提物),经过大孔树脂柱的充分吸附,在30%、60%及无水甲醇的洗脱下获得组分1-3,对Foc TR4的抑菌率分别为10.06%、27.82%和51.40%;选择抑菌率最大的组分3过硅胶柱层析,获得黄绿色晶体。该化合物在365 nm波长下具有最大吸收峰,将核磁共振图谱与SciFinder和SDBS信息数据库进行图谱比对,将该抑菌活性化合物鉴定为吩嗪-1-羧酸(Phenazine-1-Carboxylic Acid,PCA)。PCA对Foc TR4的最小抑菌浓度最低,仅为1.563μg/mL,说明PCA对Foc TR4的抑制效果较强。【结论】从HQB-1菌株中分离得到活性化合物PCA,PCA的发现为香蕉枯萎病的生物防治奠定了良好的理论基础。     

马铃薯卷叶病毒P0蛋白抑制RNA沉默及其与AGO蛋白的相互作用

马铃薯卷叶病毒(Potato leafroll virus,PLRV)P0是由开放阅读框1(ORF1)所编码,利用农杆菌介导的瞬时表达技术渗透注射转绿色荧光蛋白(GFP)基因的16c烟草叶片发现PLRV-P0能够抑制由GFP mRNA引起的基因沉默,结果表明PLRV-P0是马铃薯卷叶病毒编码的一个基因沉默抑制因子。通过序列分析发现PLRV-P0基因序列中含有两个重复的WG基序,我们将PLRV-P0基因序列中第87位和第140位的色氨酸(W)点突变为丙氨酸(A)(命名为P0WA),构建植物表达载体pCAMBIA1300-CE-P0,pCAMBIA1300-CE-P0WA,农杆菌渗透注射本氏(Nicotiana benthamiana)烟草叶片,通过荧光显微镜观察发现PLRV-P0和AGO共同注射后有绿色荧光出现而PLRV-P0WA和AGO共同注射后则没有绿色荧光出现。研究结果初步表明,PLRV-P0能够和AGO蛋白发生相互作用,重复的WG基序是其与AGO蛋白相互作用的关键氨基酸。     

The Arabidopsis RNA-Directed DNA Methylation Argonautes Functionally Diverge Based on Their Expression and Interaction with Target Loci

Argonaute (AGO) effectors of RNA silencing bind small RNA (sRNA) molecules and mediate mRNA cleavage, translational repression, or epigenetic DNA modification. In many organisms, these targeting mechanisms are devolved to different products of AGO multigene families. To investigate the basis of AGO functional diversification, we characterized three closely related Arabidopsis thaliana AGOs (AGO4, AGO6, and AGO9) implicated in RNA-directed DNA methylation. All three AGOs bound 5′ adenosine 24-nucleotide sRNAs, but each exhibited different preferences for sRNAs from different heterochromatin-associated loci. This difference was reduced when AGO6 and AGO9 were expressed from the AGO4 promoter, indicating that the functional diversification was partially due to differential expression of the corresponding genes. However, the AGO4-directed pattern of sRNA accumulation and DNA methylation was not fully recapitulated with AGO6 or AGO9 expressed from the AGO4 promoter. Here, we show that sRNA length and 5′ nucleotide do not account for the observed functional diversification of these AGOs. Instead, the selectivity of sRNA binding is determined by the coincident expression of the AGO and sRNA-generating loci, and epigenetic modification is influenced by interactions between the AGO protein and the different target loci. These findings highlight the importance of tissue specificity and AGO-associated proteins in influencing epigenetic modifications.     

Analysis of the C. elegans Argonaute family reveals that distinct Argonautes act sequentially during RNAi

Argonaute (AGO) proteins interact with small RNAs to mediate gene silencing. C. elegans contains 27 AGO genes, raising the question of what roles these genes play in RNAi and related gene-silencing pathways. Here we describe 31 deletion alleles representing all of the previously uncharacterized AGO genes. Analysis of single- and multiple-AGO mutant strains reveals functions in several pathways, including (1) chromosome segregation, (2) fertility, and (3) at least two separate steps in the RNAi pathway. We show that RDE-1 interacts with trigger-derived sense and antisense RNAs to initiate RNAi, while several other AGO proteins interact with amplified siRNAs to mediate downstream silencing. Overexpression of downstream AGOs enhances silencing, suggesting that these proteins are limiting for RNAi. Interestingly, these AGO proteins lack key residues required for mRNA cleavage. Our findings support a two-step model for RNAi, in which functionally and structurally distinct AGOs act sequentially to direct gene silencing.     

大丽轮枝菌VdSCH9基因的功能研究

大丽轮枝菌是一种土传性植物病原真菌,可侵染多种植物并引发黄萎病。目前,人们关于大丽轮枝菌的侵染和致病机制的了解还很不深入。本文通过敲除大丽轮枝菌编码丝氨酸/苏氨酸的蛋白激酶基因VdSCH9,阐明了其在大丽轮枝菌生长发育及致病过程中的作用。SCH9基因在酵母中的表达与cAMP-PKA途径和TOR信号通路相关,对酵母的生长、压力响应和寿命等有重要作用。大丽轮枝菌VdSCH9敲除突变体的生长速率显著下降,菌落边缘菌丝更为稀疏,菌丝分枝减少,对棉花植株为害的平均病情指数为56.6,显著低于野生型和互补突变体的平均病情指数90.5和82.8,对茄子植株为害的平均病情指数为65.9,也显著低于野生型和互补突变体的平均病情指数91.1和89.8。另外,敲除突变体对于高渗透压、氧化还原压力、细胞膜和细胞壁完整性等压力条件的敏感性增强。因此,VdSCH9对于大丽轮枝菌的生长、压力响应及致病力均有重要作用。     

尖孢镰刀菌亚麻专化型Folprp4基因参与调控菌丝生长和分生孢子发生

目的: 通过对尖孢镰刀菌中Folprp4基因的鉴定,揭示其在尖孢镰刀菌中的功能及致病相关性。方法: 基于同源重组原理,根据测定出的Folprp4基因序列,应用Split-Marker重组技术构建含有潮霉素抗性基因(hph)的基因缺失盒。将基因缺失盒经PEG介导转化到野生型原生质体中,在含有潮霉素B的TCC培养基上筛选转化子,通过PCR正负筛查获得Folprp4基因缺失突变株(ΔFolprp4)。构建含有Folprp4基因的载体pZDH1,并将其转化到敲除突变体中进行互补测验。结果: 与野生型(hm)和异位插入突变体(ecFolprp4)相比,敲除突变体菌丝生长受到严重阻碍,当野生型和异位插入突变体长满整个平板时,敲除突变体菌落呈小点状。敲除突变体的另一个显著变化是ΔFolprp4的分生孢子产量显著下降。侵染实验表明,ΔFolprp4对亚麻幼苗的毒力显著降低。互补实验表明,该互补载体的回复子(Folprp4-C)在菌落形态、生长速率、分生孢子产量和毒力方面均恢复到了野生型菌株。结论: Folprp4基因与尖孢镰刀菌的菌丝生长、分生孢子发生和致病性有关。     

Drosophila R2D2 mediates follicle formation in somatic tissues through interactions with Dicer-1

The miRNA pathway has been shown to regulate developmentally important genes. Dicer-1 is required to cleave endogenously encoded microRNA (miRNA) precursors into mature miRNAs that regulate endogenous gene expression. RNA interference (RNAi) is a gene silencing mechanism triggered by double-stranded RNA (dsRNA) that protects organisms from parasitic nucleic acids. In Drosophila, Dicer-2 cleaves dsRNA into 21 base-pair small interfering RNA (siRNA) that are loaded into RISC (RNA induced silencing complex) that in turn cleaves mRNAs homologous to the siRNAs. Dicer-2 co-purifies with R2D2, a low-molecular weight protein that loads siRNA onto Ago-2 in RISC. Loss of R2D2 results in defective RNAi. However, unlike mutants in other RNAi components like Dicer-2 or Ago-2, we report here that r2d2¹ mutants have striking developmental defects. r2d2¹ mutants have reduced female fertility, producing less than 1/10 the normal number of progeny. These escapers have normal morphology. We show R2D2 functions in the ovary, specifically in the somatic tissues giving rise to the stalk and other follicle cells critical for establishing the cellular architecture of the oocyte. Most interestingly, the female fertility defects are dramatically enhanced when one copy of the dcr-1 gene is missing and Dicer-1 protein co-immunoprecipitates with R2D2 antisera. These data show that r2d2¹ mutants have reduced viability and defective female fertility that stems from abnormal follicle cell function, and Dicer-1 impacts this process. We conclude that R2D2 functions beyond its role in RNA interference to include ovarian development in Drosophila.     

Identification of an ARGONAUTE for antiviral RNA silencing in Nicotiana benthamiana

ARGONAUTE proteins (AGOs) are known to be key components of the RNA silencing mechanism in eukaryotes that, among other functions, serves to protect against viral invaders. Higher plants encode at least 10 individual AGOs yet the role played by many in RNA silencing-related antiviral defense is largely unknown, except for reports that AGO1, AGO2, and AGO7 play an antiviral role in Arabidopsis (Arabidopsis thaliana). In the plant virus model host Nicotiana benthamiana, Tomato bushy stunt virus (TBSV) P19 suppressor mutants are very susceptible to RNA silencing. Here, we report that a N. benthamiana AGO (NbAGO) with similarity to Arabidopsis AGO2, is involved in antiviral defense against TBSV. The activity of this NbAGO2 is shown to be directly associated with anti-TBSV RNA silencing, while its inactivation does not influence silencing of transiently expressed transgenes. Thus, the role of NbAGO2 might be primarily for antiviral defense.     

Somatic small RNA pathways promote the mitotic events of megagametogenesis during female reproductive development in Arabidopsis

Female gamete development in Arabidopsis ovules comprises two phases. During megasporogenesis, a somatic ovule cell differentiates into a megaspore mother cell and undergoes meiosis to produce four haploid megaspores, three of which degrade. The surviving functional megaspore participates in megagametogenesis, undergoing syncytial mitosis and cellular differentiation to produce a multicellular female gametophyte containing the egg and central cell, progenitors of the embryo and endosperm of the seed. The transition between megasporogenesis and megagametogenesis is poorly characterised, partly owing to the inaccessibility of reproductive cells within the ovule. Here, laser capture microdissection was used to identify genes expressed in and/or around developing megaspores during the transition to megagametogenesis. ARGONAUTE5 (AGO5), a putative effector of small RNA (sRNA) silencing pathways, was found to be expressed around reproductive cells during megasporogenesis, and a novel semi-dominant ago5-4 insertion allele showed defects in the initiation of megagametogenesis. Expression of a viral RNAi suppressor, P1/Hc-Pro, driven by the WUSCHEL and AGO5 promoters in somatic cells flanking the megaspores resulted in a similar phenotype. This indicates that sRNA-dependent pathways acting in somatic ovule tissues promote the initiation of megagametogenesis in the functional megaspore. Notably, these pathways are independent of AGO9, which functions in somatic epidermal ovule cells to inhibit the formation of multiple megaspore-like cells. Therefore, one somatic sRNA pathway involving AGO9 restricts reproductive development to the functional megaspore and a second pathway, inhibited by ago5-4 and P1/Hc-Pro, promotes megagametogenesis.     

Differential effects of viral silencing suppressors on siRNA and miRNA loading support the existence of two distinct cellular pools of ARGONAUTE1

Plant viruses encode RNA silencing suppressors (VSRs) to counteract the antiviral RNA silencing response. Based on in-vitro studies, several VSRs were proposed to suppress silencing through direct binding of short-interfering RNAs (siRNAs). Because their expression also frequently hinders endogenous miRNA-mediated regulation and stabilizes labile miRNA* strands, VSRs have been assumed to prevent both siRNA and miRNA loading into their common effector protein, AGO1, through sequestration of small RNA (sRNA) duplexes in vivo. These assumptions, however, have not been formally tested experimentally. Here, we present a systematic in planta analysis comparing the effects of four distinct VSRs in Arabidopsis. While all of the VSRs tested compromised loading of siRNAs into AGO1, only P19 was found to concurrently prevent miRNA loading, consistent with a VSR strategy primarily based on sRNA sequestration. By contrast, we provide multiple lines of evidence that the action of the other VSRs tested is unlikely to entail siRNA sequestration, indicating that in-vitro binding assays and in-vivo miRNA* stabilization are not reliable indicator of VSR action. The contrasted effects of VSRs on siRNA versus miRNA loading into AGO1 also imply the existence of two distinct pools of cellular AGO1 that are specifically loaded by each class of sRNAs. These findings have important implications for our current understanding of RNA silencing and of its suppression in plants.