RNAi for insect-proof plants

RNA interference induced in insects after ingestion of plant-expressed hairpin RNA offers promise for managing devastating crop pests

The roles of plant dsRNA-binding proteins in RNAi-like pathways

Dicers are associated with double-stranded RNA-binding proteins (dsRBPs) in animals. In the plant, Arabidopsis, there are four dicer-like (DCL) proteins and five potential dsRBPs. These DCLs act redundantly and hierarchically. However, we show there is little or no redundancy or hierarchy amongst the DRBs in their DCL interactions. DCL1 operates exclusively with DRB1 to produce micro (mi)RNAs, DCL4 operates exclusively with DRB4 to produce trans-acting (ta) siRNAs and 21nt siRNAs from viral RNA. DCL2 and DCL3 produce viral siRNAs without requiring assistance from any dsRBP. DRB2, DRB3 and DRB5 appear unnecessary for mi-, tasi-, viral si-, or heterochromatinising siRNA production but act redundantly in a developmental pathway. © 2008 Federation of European Biochemical Societies.

Host-delivered RNAi: An effective strategy to silence genes in plant parasitic nematodes

Root-knot nematodes (Meloidogyne spp.) are obligate, sedentary endoparasites that infect many plant species causing large economic losses worldwide. Available nematicides are being banned due to their toxicity or ozone-depleting properties and alternative control strategies are urgently required. We have produced transgenic tobacco (Nicotiana tabacum) plants expressing different dsRNA hairpin structures targeting a root-knot nematode (Meloidogyne javanica) putative transcription factor, MjTis11. We provide evidence that MjTis11 was consistently silenced in nematodes feeding on the roots of transgenic plants. The observed silencing was specific for MjTis11, with other sequence-unrelated genes being unaffected in the nematodes. Those transgenic plants able to induce silencing of MjTis11, also showed the presence of small interfering RNAs. Even though down-regulation of MjTis11 did not result in a lethal phenotype, this study demonstrates the feasibility of silencing root-knot nematode genes by expressing dsRNA in the host plant. Host-delivered RNA interference-triggered (HD-RNAi) silencing of parasite genes provides a novel disease resistance strategy with wide biotechnological applications. The potential of HD-RNAi is not restricted to parasitic nematodes but could be adapted to control other plant-feeding pests.

Towards conditional RNAi : shape and sequence transduction with small conditional RNAs

RNA interference (RNAi) is a powerful biological pathway allowing for sequence-specific knockdown of any gene of interest. While RNAi is a proven tool for probing gene function in biological circuits, it is limited by being constitutively ON and executes the logical operation: silence gene Y. To provide greater control over post-transcriptional gene silencing, we propose engineering a biological logic gate to implement “conditional RNAi.” Such a logic gate would silence gene Y only upon the expression of gene X, a completely unrelated gene, executing the logic: if gene X is transcribed, silence independent gene Y. Silencing of gene Y could be confined to a specific time and/or tissue by appropriately selecting gene X.

To implement the logic of conditional RNAi, we present the design and experimental validation of three nucleic acid self-assembly mechanisms which detect a sub-sequence of mRNA X and produce a Dicer substrate specific to gene Y. We introduce small conditional RNAs (scRNAs) to execute the signal transduction under isothermal conditions. scRNAs are small RNAs which change conformation, leading to both shape and sequence signal transduction, in response to hybridization to an input nucleic acid target. While all three conditional RNAi mechanisms execute the same logical operation, they explore various design alternatives for nucleic acid self-assembly pathways, including the use of duplex and monomer scRNAs, stable versus metastable reactants, multiple methods of nucleation, and 3-way and 4-way branch migration.

We demonstrate the isothermal execution of the conditional RNAi mechanisms in a test tube with recombinant Dicer. These mechanisms execute the logic: if mRNA X is detected, produce a Dicer substrate targeting independent mRNA Y. Only the final Dicer substrate, not the scRNA reactants or intermediates, is efficiently processed by Dicer. Additional work in human whole-cell extracts and a model tissue-culture system delves into both the promise and challenge of implementing conditional RNAi in vivo.

Lipid Nanoparticles as Carriers for RNAi against Viral Infections: Current Status and Future Perspectives

The efforts made to develop RNAi-based therapies have led to productive research in the field of infections in humans, such as hepatitis C virus (HCV), hepatitis B virus (HBV), human immunodeficiency virus (HIV), human cytomegalovirus (HCMV), herpetic keratitis, human papillomavirus, or influenza virus. Naked RNAi molecules are rapidly digested by nucleases in the serum, and due to their negative surface charge, entry into the cell cytoplasm is also hampered, which makes necessary the use of delivery systems to exploit the full potential of RNAi therapeutics. Lipid nanoparticles (LNP) represent one of the most widely used delivery systems for in vivo application of RNAi due to their relative safety and simplicity of production, joint with the enhanced payload and protection of encapsulated RNAs. Moreover, LNP may be functionalized to reach target cells, and they may be used to combine RNAi molecules with conventional drug substances to reduce resistance or improve efficiency. This review features the current application of LNP in RNAi mediated therapy against viral infections and aims to explore possible future lines of action in this field.

植物油脂积累机理和品质改良—利用RNAi改良大豆油脂品质—四合木茎积累三脂酰甘油特征

利用RNAi改良大豆油脂品质 大豆[Glycine max (L.) Merr.]起源于中国，栽培历史悠久，是重要的粮食作物， 同时也是植物油和蛋白的重要来源。随着经济的发展和生活水平的提高，人们不但对大豆的需求量大大增加，同时对大豆的品质也提出了更高的要求。近年来，我国大豆进口量逐年攀升，已远远超过本国生产量。国外转抗除草剂转基因大豆大面积种植大大降低了生产成本，直接影响了我国大豆生产。因此，提高产量和改良品质是当前中国大豆生产所面临的重要课题。基因工程是大豆品种改良更为有效和快速的方法，但是由于历史原因我国的大豆转基因育种与发达国家尚存在一定差距，对我国的大豆生产贡献十分有限。因此，建立高效的大豆转化体系，加强大豆基因工程研究和育种是解决大豆面临困境的关键。 本研究的目的是以我国主要栽培大豆品种（黑农、合丰和东农等）为材料，利用GUS（β-glucuronidase）报告基因和RNAi技术，建立高效的大豆基因转化体系和基因功能研究体系。为大豆产量和品质基因工程改良提供技术手段和理论基础。结果如下： 以大豆下胚轴为外植体，对分生组织产生不定芽的频率进行了研究。培养基中添加高浓度BAP（6-benzylaminopurine）可以诱导外植体分生组织增殖产生不定芽的发生率;在培养基中添加银离子可以明显地促进大豆单个外植体多芽的产生，使得诱导不定芽总数目显著增加;不同基因型大豆再生不定芽能力有着较大区别，黑农44，黑农37，合丰35，合丰39等品种再生能力强;相对于大豆子叶节等再生系统，大豆下胚轴体系具有高效高频的再生特点（总的再生频率高于80%），且重复性好，容易操作。 以大豆下胚轴为外植体，用含有GUS报告基因的根癌农杆菌对其进行遗传转化，并重点对农杆菌菌液浓度、农杆菌侵染时间、乙酰丁香酮（AS）和抗氧化剂浓度等因素对农杆菌大豆转化效率的影响进行了研究。组织化学染色结果显示GUS基因在外植体顶端表达强烈，表达位置主要位于初生芽基部周围的分生组织。 农杆菌浸染时间以 4h 为最佳，此时的GUS瞬时表达频率可达73.0%;培养基中添加浓度为200μmol/L的乙酰丁香酮，可以显著增加GUS瞬时表达频率。抗氧化剂可以显著降低共培养阶段外植体的褐化和坏死率，进而显著提高农杆菌转化效率。用根癌农杆菌转化大豆下胚轴的方法得到了表达GUS基因转基因大豆株系。 利用大豆油酸去饱和酶基因(FAD2-1;Genbank, L43920)在第315-852碱基之间的基因片断构建了反向重复的RNAi表达载体，以农杆菌介导大豆下胚轴转化方法进行转化，并且获得转基因植株。经过PCR，Southern杂交和转基因后代的脂肪酸分析，表明沉默结构已经成功整合到大豆基因组中，并成功抑制了内源基因的表达。与栽培大豆品种相比较，转基因大豆种子的脂肪酸组成发生显著变化，油酸含量由栽培大豆的18.1％增加到71.5％¬-81.9％;亚油酸含量从栽培大豆的46.4％降到了约3.4％。 栽培大豆种子中油酸去饱和比率（ODP, oleic desaturation proportion）为0.76 到 0.84，转基因大豆种子的油酸去饱和比率降为0.06-0.26，表明Δ12-去饱和酶活性降低了74%-94%。上述结果表明，我们构建的RNAi反向重复序列沉默结构高效地抑制了大豆种子FAD2-1基因。 在本研究中，我们通过外源GUS基因的表达和内源FAD2基因的抑制，成功地建立了以大豆下胚轴为外植体的高效农杆菌介导大豆转化体系，并获得了相应的转基因株系。本研究对我国大豆品种基因工程改良以及进一步大豆功能基因组研究有重要参考价值。 四合木茎积累三脂酰甘油特征 四合木(Tetraena mongotica Maxim)是蒺藜科(Zygophyllaceac)四合木属唯一的种，是地球上最具代表性的古老残遗濒危珍稀植物。由于四合木极易燃烧，当地居民称其为“油柴”。 通过对四合木内可能存在的“油”成分进行了分析，我们发现其茎组织含有大量的三脂酰甘油（Triacylglycerols），含量达到46 mg/g DM。在韧皮部中更高，达到90 mg/g DM。我们通过半薄切片对四合木中三脂酰甘油在不同组织的分布和存在形式进行了研究，发现三脂酰甘油主要以油体形式存在于木质部和韧皮部的薄壁组织中。在韧皮部中，几乎所有的薄壁细胞都含有大量的油体。 三脂酰甘油在植物的生长发育中起着非常重要的作用。作为植物生长发育所需的碳源和能量，三脂酰甘油一般储存在植物的种子和果实中。虽然也有关于其在茎和叶中发现的报道，但是含量很少。四合木茎组织含有大量的三脂酰甘油，这种现象可能与四合木茎中存在茎特异油脂合成酶系统有关。因此，克隆相关基因并在作物中表达，将对能源植物的开发具有重要意义。

RNAi沉默光合膜磷脂合成酶基因的研究

磷脂是动物和植物非光合组织细胞膜系统的主要组成成分，在细胞生命过程中扮演着重要角色。尽管绿色植物光合膜的的甘油脂主要是糖脂，但是它仍然含有大约10％的磷脂，说明磷脂在光合膜的结构和功能中起重要作用。构成生物膜的磷脂有多种，但是，光合膜只含有磷脂酰甘油（PG）一种磷脂。光合膜中的PG有其特殊性，即：在PG的sn-2位上总连着一个棕榈酸（16:0）或者反式十六碳烯酸（16:1trans），说明了这种具有特殊结构的甘油脂在维持类囊体膜的结构和功能方面具有重要的作用。 叶绿体中有两个重要酶参与了PG的生物合成，它们分别是胞嘧啶二脂酰甘油合成酶（CDS）和磷脂酰甘油合成酶（PGS）。本实验以烟草和马铃薯为材料，利用RNAi技术，对CDS和PGS基因的表达进行抑制，通过PG缺失突变体，研究其功能。 对转含有PGS片段的沉默结构的转基因烟草叶片膜脂进行了分析，结果表明，与野生型烟草相比较，其PG含量下降了约20%，同时，SQDG和PC的含量增加。PG含量的降低没有引起MGDG和DGDG含量的变化。另外，我们还对转基因植株目的基因片段的RNA表达水平进行了RT-PCR分析，发现其表达量大幅度降低。这些结果表明，在转基因株系中，PGS基因的表达受到了抑制，说明我们获得了PG部分缺失的烟草PGS突变体。 对烟草PG缺失体的PG脂肪酸组成进行分析，表明其特征性脂肪酸反式十六碳烯酸含量明显下降，比野生型降低了44%，C18:0、C18:1和C18:2的相对含量增加，整个变化与总脂脂肪酸变化基本一致。 为了研究PG缺失对光合作用的影响，我们分析了多种光合指标。对叶绿素含量的分析表明，PG含量的降低影响了光合色素的组成。PG部分缺失的转基因烟草中的叶绿素总的含量下降，其中叶绿素b含量下降更为明显，结果，叶绿素a与叶绿素b的比值较野生型高。转基因植株净光合速率下降，二氧化碳利用率降低;PSII的最大光化学效率（Fv/Fm）和实际光化学效率（фPSII）降低，光化学猝灭下降，非光化学猝灭增加，尤其老叶的变化更为明显。这些结果说明了PG的部分缺失影响了植株的光合能力，捕光色素蛋白复合体的结构受到了影响，PSII功能遭受损伤。 同时，我们根据已经报道的马铃薯CDS基因，克隆了一个片段，构建沉默结构，并对沉默结构进行了转化。通过抗性基因的筛选以及RT-PCR检测，证明了沉默结构转化成功，目的基因的表达受到抑制，获得了马铃薯CDS转基因植株。 对马铃薯野生型和CDS转基因植株进行膜脂和脂肪酸分析表明，转基因植株叶片的PE、PG和PC等磷脂含量降低，SQDG和DGDG含量增加;C16:1（3t）、C16:2、C16:3、C18:1和C18:2含量下降，C16:0和C18:3含量增加，而C16:1和C18:0变化不明显。马铃薯CDS转基因植株的叶绿素荧光分析表明，PSII最大光化学效率降低，从野生型的0.82下降到0.77。

RNAi, DRD1, and histone methylation actively target developmentally important Non-CG DNA methylation in Arabidopsis

Cytosine DNA methylation protects eukaryotic genomes by silencing transposons and harmful DNAs, but also regulates gene expression during normal development. Loss of CG methylation in the Arabidopsis thaliana met1 and ddm1 mutants causes varied and stochastic developmental defects that are often inherited independently of the original met1 or ddm1 mutation. Loss of non-CG methylation in plants with combined mutations in the DRM and CMT3 genes also causes a suite of developmental defects. We show here that the pleiotropic developmental defects of drm1 drm2 cmt3 triple mutant plants are fully recessive, and unlike phenotypes caused by met1 and ddm1, are not inherited independently of the drm and cmt3 mutations. Developmental phenotypes are also reversed when drm1 drm2 cmt3 plants are transformed with DRM2 or CMT3, implying that non-CG DNA methylation is efficiently re-established by sequence-specific signals. We provide evidence that these signals include RNA silencing though the 24-nucleotide short interfering RNA (siRNA) pathway as well as histone H3K9 methylation, both of which converge on the putative chromatin-remodeling protein DRD1. These signals act in at least three partially intersecting pathways that control the locus-specific patterning of non-CG methylation by the DRM2 and CMT3 methyltransferases. Our results suggest that non-CG DNA methylation that is inherited via a network of persistent targeting signals has been co-opted to regulate developmentally important genes. © 2006 Chan et al.

小鼠Bicc1基因RNAi序列的筛选与鉴定

通过网上提供的生物信息学分析软件进行搜索和比对,初步筛选到3个较好的针对小鼠双尾-C(Bicc1)基因的RNA干扰(RNAi)序列。合成这3个干涉序列片段后克隆到pRS-HushshRNA载体中。构建Bicc1基因的真核表达载体pEGFP-C3-Bicc1,将绿色荧光蛋白(GFP)标签标记在Bicc1蛋白上。利用细胞转染技术将pEGFP-C3-Bicc1与3个干涉序列载体共转染至体外培养的HEK-293细胞中,最后通过细胞荧光强度、半定量PCR和Westernblotting鉴定出其中两个序列(pRS-Hush-RNAi-Bicc1-N/-C)能明显降低Bicc1蛋白在HEK-293细胞中的表达水平,为下一步建立起低表达Bicc1的稳定细胞株和研究小鼠Bicc1的功能提供了良好的材料。

Grass carp reovirus activates RNAi pathway in rare minnow, Gobiocypris rarus

Dicer catalyzes the initiation step of RNA interference (RNAi) which is known to play a significant role in innate immune response to viral infection in many organisms. To study the RNAi-related pathway after virus infection in fish, we identified a partial cDNA sequence of dicer from rare minnow, Gobiocypris rants. Real-time quantitative RT-PCR (qRT-PCR) demonstrated the Dicer transcript level was the highest at zygote stage, decreased at prim-5 stage, and was stable from the protruding mouth to adult stage. Regular RT-PCR analysis showed that the Dicer gene expressed widely in the tested tissues, including brain, gill, heart, intestine, kidney, liver, muscle, ovary, spleen and testis. The expression of Dicer mRNA was significantly increased in the early period of Grass carp reovirus (GCRV) infection, and declined from 24 It post-injection (h p.i.) (P<0.05). The mRNA expression returned to control levels at 48 h p.i. (P>0.05). Under transmission electron microscope, virions were difficulty to find out in 12 h p.i., and virus inclusion bodies and few scattered viral particles were easily visualized from 24 h p.i. to moribund. These results implied GCRV triggered the RNAi pathway in the early stages of infection and perhaps virus inclusion bodies suppressed the antiviral functions of RNAi mechanism. (C) 2009 Published by Elsevier B.V.

RNAi suppression of zebrafish peptidoglycan recognition protein 6 (zfPGRP6) mediated differentially expressed genes involved in Toll-like receptor signaling pathway and caused increased susceptibility to Flavobacterium columnare

In Drosophila, Toll signaling cascade, which resembles the mammalian Toll-like receptor (TLR)/IL-1R signaling pathways and regulates the expression of anti-microbial peptide genes, mainly relies on peptidoglycan recognition proteins (PGRPs) for the detection of bacterial pathogens. To explore the effect of zebrafish peptidoglycan recognition protein 6 (zfPGRP6) on Toll-like receptor signaling pathway, RNA interference (siRNA) and real time quantitative PCR (RQ-PCR) methods were used to identify differentially expressed genes regulated by zfPGRP6. The target genes included TLR2, TLR3, TLR5, TLR7, TLR8, IL1R, Sterile-alpha and Armadillo motif containing protein (SARM), myeloid differentiation factor 88 (MyD88) and nuclear factor (NF)-kappa B2 (p100/p52). The results of RQ-PCR showed that RNAi-mediated Suppression of zfPGRP6 significantly down-regulated the expression of TLR2, TLR5, IL1R, SARM, MyD88 and p100/p52. The expression of beta-defensin-1 was also down-regulated in those embryos silenced by zfPGRP6. In challenge experiments to determine the anti-bacterial response to Gram-negative bacteria, RNAi knock-down of zfPGRP6 markedly increased susceptibility to Flavobacterium columnare. (C) 2008 Elsevier B.V. All rights reserved.

RNAi沉默烟草GA 20-氧化酶基因研究

赤霉素是一种高效能的广谱植物生长调节剂，为五大植物激素之一，具有重要的生物学功能。目前利用赤霉素突变体研究生物合成途径和信号转导已经成为热点。 GA 20-氧化酶是GA生物合成中的一类关键酶，它位于GA合成途径的中心位置。本研究根据烟草（Nicotiana tabacum）GA 20-氧化酶基因序列，设计2对分别含有特定酶切位点的特异引物，以烟草基因组DNA为模板，扩增目的基因(约250 bp)片段。将正、反向目的片段分别插入中间载体的内含子两侧，再经BamH I和Sac I双酶切回收约700 bp的目的片段，插入到双元载体质粒p2355中，成功构建了含GA 20-氧化酶基因片段反向重复序列的植物表达载体p23700。分别将p2355质粒和p23700质粒导入根癌农杆菌（Agrobacterium tumefaciens）EHA105中并转化烟草叶片细胞，经卡那霉素选择培养，PCR及GUS组织染色鉴定，获得转基因烟草植株。以EHA105-p2355转化的烟草，获得41株转基因植株，均没有矮化表型；而以EHA105-p23700转化的烟草，获得转基因植株14株，其中具有矮化表型的烟草10株，表明反向重复序列转录产物能形成发夹RNA(hpRNA)，产生小分子干扰RNA（small interferring RNA，简称siRNA），干扰目的基因的表达。 赤霉素含量测定表明矮化植株中赤霉素合成途径的最终产物GA3总含量明显低于野生型烟草植株。荧光定量PCR结果表明，矮化转基因烟草的GA 20-氧化酶基因表达量受到明显抑制，表达量明显低于野生型对照。同时对上游内根-贝壳杉合成酶（Ent-kaurene synthase，KS）基因，下游的GA-3β羟化酶基因进行了RT-PCR分析，结果显示上游基因的表达没有规律性变化，而下游基因表达量亦降低。上述结果表明，GA 20-氧化酶基因的表达被有效地干扰了，表达受到抑制，从而影响植株体内GA3的合成，影响植株的生长发育，导致植株矮化。并推测，GA 20-氧化酶基因受到抑制，可能影响下游基因的表达。并且通过干旱胁迫测试，发现矮化植株相对于野生型植株及不含干扰片段的转基因植株，对干旱的耐受力有了很大的提高，具有更强的耐受力。 研究结果为进一步进行相关研究奠定基础。 Gibberellin(GA) is an efficient plant growth regulator. As one of five major plant hormones, it plays an important biological function. Using GA mutant for investigating biosynthetic pathways and signal transduction has become high lights. GA 20-oxidase is a crucial enzyme involved in gibberellin biosynthesis. According to tobacco (Nicotiana tabacum) GA 20-oxidase enzyme gene sequence and based on binary vector p2355, we constructed a plant expression vector p23700, which habors an inverted repeat DNA fragment of GA 20-oxidase gene drivered by Cauliflower mosaic virus promtor (CaMV 35Sp). Binary plasmid p2355 had no inverted repeat DNA fragment of GA 20-oxidase gene. The vector p2355 and p23700 were introduced into Agrobacterium tumefaciens EHA105 and tobacco leaf transformation was conducted. After selected by kanamycin and characterized by PCR and GUS hischemical reaction, transsgenic plants were obtained. Fourtheen transgenic plants, which were transformed by EHA105-p23700, were obtained. Among them, 10 were dwarf mutants. However, 41 transgenic plants with the same normal phenotype as wild type,which were transformed by EHA105-p2355, were obtained. Analysis of Gibberellin contents showed that it was lower in dwarf mutants than in normal phenotype plants. Moreover, comparing to normal phenotype plants including wild type and transgenic plants with no interference fragment, the drought tolerance of dwarf plants have greatly increased. And their proline content increased obviously after drought test. Fluorescence quantitative real time PCR (RT-PCR) showed that GA 20-oxidase gene expression was significantly inhibited in dwarf transgenic tobacco. Meanwhile, the expression of the upstream gene ent-kaurene synthase (KS) gene and downstream gene GA-3β hydroxylase gene was also detected by RT-PCR. The results presented that KS gene expression had no regular change while GA-3β hydroxylase gene expression reduced. It implied that inhibiting GA 20-oxidase gene probably reduce the expression of downstream genes. The results showed that the transcriptional products of the foreign inverted repeat fragment can form hairpin RNA (hpRNA) to induce RNAi. It presented that GA 20-oxidase gene expression was effectively interfered, resulting in reducing GA3 synthesis and inhibiting plant growth and development, then dwarf plants were produced. However, the dwarf plants had higher tolerance of drought.

干扰RNA(RNAi)作用机理及其在植物细胞工程中的应用进展

随着RNAi调控目的基因表达机理研究的深入,RNAi技术也发展为一种强有力的实验工具,用来控制目的基因的表达以获取预期的生物表型。目前在植物中至少发现存在三种不同的RNAi途径,这些途径中基因沉默信号可以放大、传递和自我调控。为了建立高效、经济的RNAi技术体系,必须解决以下几个问题:即RNAi的有效传递,稳定性的提高,非目标效应出现的减小以及目标RNA敏感位点的确定等。综述了RNAi作用机制及其在植物细胞工程中的最新应用进展,并详细探讨了其技术体系。