RNA interference reveals chloride channel 7 gene helps short-term hypersalinity stress resistance in Hong Kong oyster Crassostrea hongkongensis

The chloride channel 7 gene(CLC 7)of the Hong Kong oyster Crassostrea hongkongensis was cloned and named ChCLC 7.The cDNA was 2572 bp in length,with a 5′non-coding region containing 25 bp,a 3′non-coding region containing 327 bp,and an open reading frame of 2298 bp.ChCLC 7 has 96.8%and 92.1%homology with CLC 7 of Crassostrea gigas and Crassostrea virginica,respectively,and it was clustered with CLC 7 of C.gigas and C.virginica.QRT-PCR showed that ChCLC 7 was expressed in all eight tissues,with the highest in adductor muscle and second in gill.The ChCLC 7 expression pattern in gill was altered significantly under high salinity stress with an overall upward and then downward trend.After RNA interference,the expression of ChCLC 7 and survival rate of oyster under high salinity stress was reduced significantly,and so did the concentration of hemolymph chloride ion in 48-96 h after RNA interference.We believed that ChCLC 7 could play an important role in osmoregulation of C.hongkongensis by regulating Cl^(-)transport.This study provided data for the analysis of molecular mechanism against oyster salinity stress.

RNAi技术在马铃薯害虫防控中的应用和风险

害虫对农作物的危害严重威胁全球粮食安全,为满足日益增长的全球粮食需求,迫切需要安全有效的害虫绿色防控技术。RNAi技术(RNA interference)又叫RNA干扰技术,是一种转录后调控基因沉默的分子生物学技术,其原理是基于由19-25对核苷酸组成的小分子双链RNA与目标靶基因mRNA结合并引发mRNA降解,从而导致靶基因沉默。目前RNAi技术已被广泛应用于农作物害虫治理,在针对马铃薯靶标害虫方面,主要研究集中在防控鞘翅目、半翅目、鳞翅目害虫。2023年12月22日,世界上首款RNAi生物农药正式批准商业化,用于防控抗药性日益严重、国际公认的马铃薯重要毁灭性检疫害虫马铃薯甲虫,是世界上首款被允许在农作物上商业使用的可喷洒RNA生物农药,对马铃薯害虫的绿色防控具有划时代的里程碑意义。基于RNAi技术的产品被用于农业害虫防控的同时,仍需考虑其抗药性、脱靶效应和对环境安全的潜在风险。本文以RNAi技术在马铃薯害虫防控应用的可行性、RNAi技术在马铃薯害虫防控中的应用以及潜在的风险等方面进行了综述,以期阐述RNAi技术在马铃薯害虫防控中的应用现状与前景,为RNAi技术纳入防控马铃薯害虫综合治理体系提供理论参考。

RNAi技术在烟粉虱基因功能和防控研究中的应用

烟粉虱(Bemisia tabaci)是一种世界性蔬菜害虫,可传播多种植物病毒,影响植物生长发育,导致蔬菜产量下降。烟粉虱的防治以化学防治为主,但过度使用杀虫剂导致烟粉虱的抗药性不断增强,绿色防控的需求日益增加。RNA干扰(RNA interference,RNAi)是一项特异性沉默靶标基因技术,广泛应用于昆虫的基因功能研究和绿色防控。通过介绍RNAi作用原理和RNAi在烟粉虱的基因功能,总结了RNAi在烟粉虱的防控应用和RNA农药的应用现状,为烟粉虱的新靶点研究和开发RNA农药进行烟粉虱防控应用提供参考。

Cflac1基因的RNAi对白蚁肠道氧化还原电位响应病原菌感染的影响

台湾乳白蚁Coptotermes formosanus又称为地下白蚁,它以木质纤维素为主食,是广泛分布于全球的经济害虫。地下白蚁对病原微生物强大的免疫防御能力一直是其生物防治应用的瓶颈之一。植食性昆虫的解毒酶在其消化和免疫调节中起着至关重要的作用。针对具有木质素代谢活性的解毒酶在白蚁肠道免疫过程中氧化还原稳态的调控研究对揭示其肠道免疫机制有重要意义。本研究以金龟子绿僵菌Metarhizium anisopliae为免疫诱导物,基于肠道氧化还原态的微电极检测系统,首次对台湾乳白蚁感染真菌后前肠/唾液腺、中肠及后肠中氧化还原电位的变化进行了描述;并利用RNA干扰技术,抑制了具有木质素代谢活性的乳白蚁漆酶基因Cflac1在感染前后的表达,并确定了Cflac1在乳白蚁肠道免疫防御真菌过程中对氧化还原稳态维系的重要作用。本研究丰富了白蚁肠道免疫机制的研究内容,为拓宽白蚁防治新靶点及生物防治技术的研发提供了理论支撑。

RNAi——对抗细菌耐药的新途径

近年来细菌感染问题日益严重,给人类和畜禽带来极大的危害。当前治疗细菌感染的手段仍以使用抗生素为主,但抗生素的滥用会造成细菌耐药性的增强。RNAi的出现或许给治疗细菌感染带来希望,小分子RNA通过干扰耐药基因的合成阻止其耐药及增殖过程。目前该技术在哺乳动物的病毒感染研究中较为常见,且在治疗鱼类细菌感染中也有应用,但并未直接作用于特定抗生素耐药基因。阐明RNAi机制及其在防控耐药细菌感染方面的有关研究进展,进一步揭示该技术对抗耐药病原菌感染的可观前景。

Split-GFP双分子荧光互补技术在鸡MSTN基因RNAi检测中的应用

【目的】以Split-GFP双荧光互补技术检测鸡肌肉生长抑制素基因(MSTN)RNA干涉(RNAi)效果,并与其他常用检测方法比较,以验证Split-GFP双分子荧光互补技术在RNAi效果评估中的有效性和可行性。【方法】将合成的3个shRNA慢病毒载体(shRNA-a、shRNA-b和shRNA-c)分别转染稳定表达GFP11-MSTN融合蛋白的HEK 293TGFP11-MSTN细胞,经实时荧光定量PCR筛选出最佳shRNA慢病毒载体并包装为慢病毒,然后以慢病毒感染HEK 293TGFP11-MSTN细胞,采用潮霉素B筛选mCherry阳性(mCherry+)细胞,再以实时荧光定量PCR和Western blotting检测RNAi效果;得到的mCherry+细胞再转染pcDNA3.1(+)-GFP1-10质粒,通过荧光显微镜观察和流式细胞术评估RNAi效果。【结果】3个shRNA慢病毒载体对MSTN基因表达均有极显著的抑制作用(P<0.01,下同),其中又以Anti-MSTN shRNA-a慢病毒载体的干涉效果最佳。Anti-MSTN shRNA-a慢病毒感染HEK 293TGFP11-MSTN细胞的最适MOI=3,该条件下MSTN基因相对表达量及GFP11-MSTN融合蛋白表达量均受到抑制;得到的mCherry+细胞再转染pcDNA3.1(+)-GFP1-10质粒,荧光显微镜观察和流式细胞术检测结果表明,GFP+细胞数量明显减少,GFP+细胞百分率由31.1%降至11.5%。Split-GFP检测结果与实时荧光定量PCR及Western blotting检测结果相符,说明Anti-MSTN shRNA-a慢病毒能有效抑制GFP11-MSTN融合蛋白表达,发挥了RNAi作用。【结论】以Anti-MSTN shRNA-a慢病毒对MSTN基因的干涉效果最佳,其感染HEK 293TGFP11-MSTN细胞后MSTN基因表达极显著下调,且再转染pcDNA3.1(+)-GFP1-10质粒后细胞中的GFP+细胞百分率明显下降,与实时荧光定量PCR和Western blotting检测结果相符,证实Slipt-GFP双分子荧光互补技术是一种可靠的可视化RNAi检测方法。

Development of Studies on RNA Interference

RNA interference （RNAi）, caused by endogenous or exogenous double- stranded RNA （dsRNA） homologous with target genes, refers to gene silencing widely existing in animals and plants. It was first found in plants, and now it has developed into a kind of biotechnology as well as an important approach in post- genome era. This paper is to summarize the achievements of studies on RNAi tech- nology in basic biology, medicine, pharmacy, botany and other fields.

家蚕RNAi效率相关核酸酶基因对RNAi效率的影响

【目的】鳞翅目(Lepidoptera)昆虫消化系统存在的核酸酶是导致RNAi低效的主要原因之一,本研究旨在探索家蚕Bombyx mori RNAi效率相关核酸酶(RNAi efficiency-related nuclease, REase)BmREase对家蚕RNAi低效的影响。【方法】利用RT-PCR对家蚕BmREase cDNA全长序列进行同源克隆和生物信息学分析,并采用最大似然法进行系统发育分析;利用qRT-PCR检测BmREase在游走期家蚕不同组织(头、表皮、脂肪体、中肠、气管、马氏管和丝腺)中的特异性表达。通过向游走期家蚕注射BmREase,家蚕蜕皮激素受体(ecdysone receptor, EcR)基因BmEcR,超气门蛋白(ultraspiracle, USP)基因BmUSP和细胞因子sp?tzle1(BmSpz1)基因BmSpz1的dsRNA进行RNAi,分析干扰BmREase表达后是否可以提高靶基因dsRNAs的干扰效率。【结果】RT-PCR扩增得到家蚕BmREase(GenBank登录号:XM_021350017.2)全长cDNA序列,其ORF长2 241 bp,编码746个氨基酸残基;生物信息学分析发现,BmREase与人核酸外切酶I 3qe9.1具有极其相似的结构,其Thr7, His33, Ala37, Arg93, Lys97, Tyr159, Asp160, Ser161和Asn174组成的活性结构域能够与核苷酸序列结合,暗示BmREase具有核酸酶活性。qRT-PCR结果显示,BmREase在家蚕游走期的中肠和马氏管中高表达,说明BmREase主要在家蚕消化系统中表达。在家蚕游走期注射dsRNA,BmREase的表达量比空白对照组的高,RNAi干扰BmREase提高了dsBmEcR, dsBmUSP和dsBmSpz1的干扰效率。【结论】家蚕体内存在与人类核酸外切酶相似功能的酶BmREase,其存在可能影响dsRNA在家蚕体内的干扰效果。本研究为利用RNAi进行家蚕基因功能的研究以及进一步开发RNAi防治害虫具有指导性意义。

EGFR siRNA序列的筛选及其对HepG2细胞活性的影响

目的构建EGFR shRNA重组真核表达载体,并筛选抑制效果最好的EGFR shRNA序列。方法应用在线工具设计人EGFR siRNA序列,采用限制性内切酶BamHI和HindIII切割真核表达质粒pcDNA3.1(+),构建三种人EGFR的siRNA干扰序列载体(psilencer 4.1-CMV neo-EGFR siRNA)。将重组质粒载体转化大肠杆菌DH5α感受态并筛选阳性克隆,通过DNA测序鉴定重组质粒。应用脂质体lipo2000将三种人EGFR siRNA干扰序列载体转染到HepG2细胞,通过荧光显微镜观察转染效率,实时定量PCR检测EGFR mRNA表达水平,MTT法检测细胞活性。结果Psilencer 4.1-CMV neo-EGFR siRNA重组质粒被成功克隆。EGFR shRNA-1、EGFR shRNA-2和EGFR shRNA-3敲低EGFR mRNA的效率分别是80%、60%和70%以上。shRNA-2和shRNA-3使细胞活性分别下降50%(P<0.05),但shRNA-1对细胞活性无明显影响(P>0.05)。结论重组psilencer 4.1-CMV neo-EGFR siRNA质粒可下调肝癌细胞株EGFR表达水平和细胞活性。EGFR shRNA-3较EGFR shRNA-1和shRNA-2对HepG2细胞的抑制作用更显著。

RNAi技术在寄生蜂中的应用研究进展

RNA干扰技术问世至今20余年,已被广泛用于基因功能研究。膜翅目寄生蜂是农林害虫防治领域重要的天敌昆虫资源。RNAi技术对靶基因的昆虫沉默效果依赖于dsRNA的递送效率。寄生蜂幼期通过摄取寄主昆虫营养完成其生长发育,并与寄主昆虫发生相互作用,给RNAi技术的实施带来挑战。目前,RNAi技术已在丽蝇蛹集金小蜂(Nasonia vitripennis)、中红侧沟茧蜂(Microplitis mediator)、菜蛾盘绒茧蜂(Cotesia vestalis)、蝶蛹金小蜂(Pteromalus puparum)、斑痣悬茧蜂(Meteorus pulchricornis)等至少13种寄生蜂中开展。本文归纳了RNAi技术在寄生蜂基因调控方面的研究进展,并总结了在寄生蜂类群中影响RNAi效率的因素,以期为RNAi技术在寄生蜂类群中的应用提供参考。

Lentivirus vectors construction of SiRNA targeting interference GPC3 gene and its biological effects on liver cancer cell lines Huh-7

Objective:To build GPC3 gene short hairpin interference RNA(shRNA)slow virus veclor.observe expression of Huh-7 GPC3 gene in human liver cell line proliferation apoptosis and the effect of GPC3 gene influencing on liver cancer cell growth,and provide theoretical basis for genc therapy of liver cancer.Methods:Hepatocellular carcinoma cell line Huh-7 wsa transfected by a RNA interference technique.GPC3 gene expression in a variety of liver cancer cell lines was detected by fluorescence quantitative PCR.Targeted GPC3 gene seqnences of small interfering RNA(siRNA)PGC-shRNA-GPC3 were restructured.Stable expression cell linse of siRNA were screened and established with the heplp of liposomes(lipofectamine^(TM2000))as carrier transfcetion of human liver cell lines.In order to validate siRNA interference efficiency.GPC3 siRNA mRNA expression was detected after transfection by using RT-PCR and Western blot.The absorbance value of the cells of blank group,untransfection group and transfection group,the cell cycle and cell apoptosis were calculated,and effects of GPC3 gene nn Huh-7 cell proliferation and apoptosis were observed.Results:In the liver cancer cell lines Huh-7 GPC3 gene showed high expression.PGC-shRNA-GPC3 recombinant plasmid was constructde successfully via sequencing validation.Stable recombinant plasmid transfected into liver cancer cell linse Huh-7can obviously inhibit GPC3 mRNA expression level.Conclusions:The targeted GPC3 siRNA can effectively inhibit the expression of GPC3.

褐飞虱RNAi显微注射中麻醉方法的比较

显微注射dsRNA以实现系统性干扰,是一种有效且能准确评估基因功能的常用方法。试验靶标昆虫由于个体较小、活力强,注射前需要先适当麻醉。待显微注射dsRNA完成后,麻醉效应解除,方进一步开展表型测定试验。不同麻醉方法可对昆虫的麻醉效果、麻醉后生理和行为等产生不同程度的影响。因此,选用合适的麻醉方法对显著减少RNAi显微注射试验误差,提升干扰效率具有重要意义。本文以褐飞虱为评估对象,比较应用不同配比的乙醚与乙酸乙酯混合液麻醉,以及应用低温麻醉对褐飞虱的麻醉效应,并测定不同的麻醉方法对RNAi显微注射试验中褐飞虱存活率的影响。麻醉剂处理随乙酸乙酯的浓度升高,褐飞虱的苏醒时间延长。应用不同麻醉方法对RNAi显微注射试验中褐飞虱存活率的影响程度从小到大依次为乙醚:乙酸乙酯(1:2),乙醚:乙酸乙酯(1:1),乙醚,乙醚:乙酸乙酯(2:1),乙醚:乙酸乙酯(3:1),冰上20min,冰上40min。推荐乙醚:乙酸乙酯(1:2)混合液作为最优麻醉处理应用于褐飞虱RNAi显微注射试验。

Effects of RNA interference targeting transforming growth factor-beta 1 on immune hepatic fibrosis induced by Concanavalin A in mice

BACKGROUND: Previous studies have shown that transforming growth factor-beta 1 (TGF-beta 1) is the most potent means of stimulating liver fibrogenesis by myofibroblast-like cells derived from hepatic stellate cells. Thus, TGF-beta 1 could be a target for treating hepatic fibrosis. This study aimed to investigate the inhibitory effects of specific TGF-beta 1 small interference RNA (siRNA) on immune hepatic fibrosis induced by Concanavalin A (Con A) in mice. METHODS: Three short hairpin RNAs targeting different positions of TGF-beta 1 were designed and cloned to the plasmid pGenesil-1 to obtain three recombinant expression vectors (pGenesil-TGF-beta 1-ml, pGenesil-TGF-beta 1-m2 and pGenesil-TGF-beta 1-m3). Thirty male Kunming mice were randomly divided into 6 groups: normal, model, control, and three treatment groups. The immune hepatic fibrosis models were constructed by injecting Con A via the tail vein at 8 mg/kg per week for 6 weeks. At weeks 2, 4 and 6, pGenesil-TGF-beta 1-ml, pGenesil-TGF-beta 1-m2 or pGenesi1-TGF-beta 1-m3 was injected by a hydrodynamics-based transfection method via the tail vein at 0.8 ml/10 g within 24 hours after injection of Con A in each of the three treatment groups. The mice in the control group were injected with control plasmid pGenesil-HK at the same dose. All mice were sacrificed at week 7. The levels of hydroxyproline in liver tissue were determined by biochemistry. Liver histopathology was assessed by Van Gieson staining. The expression levels and localization of TGF-beta 1, Smad3, and Smad7 in liver tissue were detected by immunohistochemistry. The expression of TGF-beta 1, Smad3, Smad7 and alpha-smooth muscle actin (alpha-SMA) mRNAs in the liver were assessed by semi-quantitative RT-PCR. RESULTS: The levels of hydroxyproline in the liver tissue of the treatment groups were lower than those of the model group (P<0.01). Histopathologic assay showed that liver fibrogenesis was clearly improved in the treatment groups compared with the model group. The expression levels of TGF-beta 1 and Smad3 of liver tissue were also markedly lower in the treatment groups than in the model group (P<0.01), while the levels of Smad7 were higher in the treatment groups than in the model group (P<0.01). RT-PCR further showed that the expression of TGF-beta 1, Smad3 and alpha-SMA mRNA was significantly inhibited in the treatment groups compared with the model group, while the levels of Smad7 were increased. There was no difference in the above parameters among the three treatment groups or between the control and model groups (P>0.05), but the inhibitory effect of pGenesil-TGF-beta 1-ml was the highest among the treatment groups. CONCLUSIONS: Specific siRNA targeting of TGF-beta 1 markedly inhibited the fibrogenesis of immune hepatic fibrosis induced by Con A in mice. The anti-fibrosis mechanisms of siRNAs may be associated with the down-regulation of TGF-beta 1, Smad3 and alpha-SMA expression and up-regulation of Smad7 expression in liver tissue, which resulted in suppressing the activation of hepatic stellate cells. (Hepatobiliary Pancreat Dis Int 2009; 8: 300-308)

Preliminary experimental study of urethral reconstruction Nith tissue engineering and RNA interference techniques

This study investigated the feasibility of replacing urinary epithelial cells with oral keratinocytes and transforming growth factor-β1 （TGF-β1） small interfering RNA （siRNA）-transfected fibroblasts seeded on bladder acellular matrix graft （BAMG） in order to reconstruct tissue-engineered urethra. Constructed siRNAs, which expressed plasmids targeting TGF-β1, were transfected into rabbit fibroblasts. The effective siRNA was screened out by RT-PCR and was transfected into rabbit fibroblasts again. Synthesis of type I collagen in culture medium was measured by enzyme-linked immuno sorbent assay （ELISA）. Autologous oral keratinocytes and TGF-β1 siRNA-transfected fibroblasts were seeded onto BAMGs to obtain a tissue-engineered mucosa. The tissue-engineered mucosa was assessed morphologically and with the help of scanning electron microscopy. The TGF-β1 siRNA decreased the expression of fibroblasts synthesis type I collagen. Oral keratinocytes and TGF-β1 siRNA-transfected fibroblasts were seeded onto sterilized BAMG to obtain a tissue-engineered mucosa for urethral reconstruction. The compound graft was assessed using scanning electron microscope. Oral keratinocytes and TGF-β1 siRNA-transfected fibroblasts had a good compatibility with BAMG. The downregulation of fibroblasts synthesis type I collagen expression by constructed siRNA interfering TGF-β1 provided a potential basis for genetic therapy of urethral scar. Oral keratinocytes and TGF-β1 siRNA-transfected fibroblasts had good compatibility with BAMG and the compound graft could be a new choice for urethral reconstruction.

Suppression of starch synthase I(SSI) by RNA interference alters starch biosynthesis and amylopectin chain distribution in rice plants subjected to high temperature

Based on known cDNAs of rice starch synthase isoforms,we constructed dsRNA interference vectors for starch synthase I(SSI)to produce transgenic plants containing starch with a moderately high amylose content.We investigated the effect of SSI suppression on grain quality traits,starch biosynthesis,and amylopectin chain distribution in rice plants exposed to two different temperature regimes.The activities and transcripts of BEs,DBEs,and other SS isoforms were further investigated to clarify the effect of SSI suppression on these key enzymes and their specific isoforms under different temperature treatments.Suppression of SSI by RNAi altered grain starch component and amylopectin chain distribution,but it exerted only a slight effect on total starch content(%)and accumulation amount(mg kernel?1)and on starch granule morphology and particle size distribution.Under normal temperature(NT),insignificant differences in kernel weight,chalky kernel proportion,chalky degree,and starch granule morphology between SSI-RNAi line and its wild type(WT)were observed.However,amylose content(AC)level and granule-bound starch synthase(GBSS)activity in rice endosperms were markedly increased by SSI-RNAi suppression.The chalky kernel proportion and chalky degree of SSIRNAi lines were significantly higher than those of WT under high temperature(HT)exposure at filling stage.Inhibition of SSI by RNAi affected amylopectin chain distribution and raised starch gelatinization temperature(GT)in two ways:directly from the SSI deficiency itself and indirectly by reducing BEIIb amounts in an SSI-deficient background.The deficiency of SSI expression led to an alteration in the susceptibility of grain chalkiness occurrence and starch gelatinization temperature to HT exposure,owing to a pleiotropic effect of SSI deficiency on the expression of other genes associated with starch biosynthesis.

Down-Regulated Expression of RACK1 Gene by RNA Interference Enhances Drought Tolerance in Rice

The receptor for activated C-kinase 1 （RACK1） is a highly conserved scaffold protein with versatile functions, and plays important roles in the regulation of plant growth and development. Transgenic rice plants, in which the expression of RACK1 gene was inhibited by RNA interference （RNAi）, were studied to elucidate the possible functions of RACK1 in responses to drought stress in rice. Real-time PCR analysis showed that the expression of RACK1 in transgenic rice plants was inhibited by more than 50%. The tolerance to drought stress of the transgenic rice plants was higher as compared with the non-transgenic rice plants. The peroxidation of membrane and the production of malondialdehyde were significantly lower and the superoxide dismutase activity in transgenic rice plants was significantly higher than those in non-trangenic rice plants It is suggested that RACK1 negatively regulated the redox system-related tolerance to drought stress of rice plants.

植物RNA沉默抗病机制与应用研究进展

RNA沉默是真核生物基因表达调控的保守机制,在植物生长发育以及响应生物和非生物胁迫过程中发挥着非常重要的作用。跨界RNA沉默与种间RNA沉默为开发基于RNA沉默的作物病害防控体系提供了理论基础。本文概括了植物RNA沉默保守途径,归纳了RNA沉默在植物-病原互作研究中的代表性研究,阐述了基于RNA沉默开发的宿主诱导基因沉默、喷施诱导基因沉默和微生物诱导基因沉默技术的原理,以及应用研究现状,以期为开发基于RNA沉默的新型作物病害防控技术提供参考。

RNA interference and antiviral therapy

RNA interference (RNAi) is an evolutionally conserved gene silencing mechanism present in a variety of eukaryotic species. RNAi uses short double-stranded RNA (dsRNA) to trigger degradation or translation repression of homologous RNA targets in a sequence-specific manner. This system can be induced effectively in vitro and in vivo by direct application of small interfering RNAs (siRNAs), or by expression of short hairpin RNA (shRNA) with non-viral and viral vectors. To date, RNAi has been extensively used as a novel and effective tool for functional genomic studies, and has displayed great potential in treating human diseases, including human genetic and acquired disorders such as cancer and viral infections. In the present review, we focus on the recent development in the use of RNAi in the prevention and treatment of viral infections. The mechanisms, strategies, hurdles and prospects of employing RNAi in the pharmaceutical industry are also discussed.

Rice Repetitive DNA Sequence RRD3:a Plant Promoter and Its Application to RNA Interference

Previously, a moderately repetitive DNA sequence （RRD3） was cloned from rice （Oryza sativa L.） by DNA renaturation kinetics. Sequence analysis revealed several conserved promoter motifs, including four TATA-boxes and a CAAT-box, and promoter activity was shown in Escherichia coli and mammalian expression systems. Here, we inserted the RRD3 fragment into the plant promoter-capture vector, pCAMBIA1391Z, and examined whether the RRD3 fragment has promoter activity in plants. Transgenic tobacco and rice calli both showed β-glucuronidase （GUS） activity, indicating that RRD3 can act as a promoter in both monocot and dicot plants. Based on the promoter characteristic of RRD3, we designed a plant universal binary vector, pCRiRRD3, which is suitable for performing researches on plant RNA interference. This vector has two multiple cloning sites to facilitate sense and antisense cloning of the target sequence, separated by an intron fragment of 200 bp. The efficiency of the vector for gene silencing was assayed by histochemical and quantitative fluorometric GUS assays in transgenic tobacco. These research results suggested that this plant RNAi vector pCRiRRD3 can effectively perform gene silencing researches on both monocot and dicot plants.

RNA interference mediated silencing of α-synuclein in MN9D cells and its effects on cell viability

Objective To silence the expression of α-synuclein in MN9D dopaminergic cells using vector mediated RNA interference （RNAi） and examined its effects on cell proliferation and viability. Methods We identified two 19-nucleotide stretches within the coding region of the α-synuclein gene and designed three sets of oligonucleotides to generate doublestranded （ds） oligos. The ds oligos were inserted into the pENTR^TM/Hl/TO vector and transfected into MN9D dopaminergic cells, α-Synuclein expression was detected by RT-PCR, real-time PCR, immunocytochemistry staining and Western blot. In addition, we measured cell proliferation using growth curves and cell viability by 3-（4, 5）-dimethylthiahiazo （-z-y 1）-3, 5-diphenytetrazoliumromide （M＇FF）. Results The mRNA and protein levels of α-synuclein gene were significantly down-regulated in pSH2/α-SYN-transfected cells compared with control MN9D and pSH/CON-transfected MN9D cells, while pSHI/α-SYN- transfected cells showed no significant difference. Silencing α-synuclein expression does not affect cell proliferation but may decrease cell viability. Conclusion Our results demonstrated pSH2/α-SYN is an effective small interfering RNA （siRNA） sequence and potent silencing of mouse α-synuclein expression in MN9D cells by vector-based RNAi, which provides the tools for studying the normal function of α-synuclein and examining its role in Parkinson＇s disease （PD） pathogenesis. α-Synuclein may be important for the viability of MN9D cells, and loss of α-synuclein may induce cell injury directly or indirectl