正向和反向重复RNA介导的抗马铃薯Y病毒基因工程比较研究

 RNA介导的病毒抗性与RNA沉默现象密切相关。反向重复cDNA序列(IR)的转录产物往往形成双链RNA结构,而双链RNA是诱发RNA沉默的有效因子。据此,本研究通过体外合成马铃薯Y病毒坏死株系衣壳蛋白基因(PVY^N-CP)5'端反向重复cDNA序列和正向重复cDNA序列(DR),分别构建植物表达载体pROK-IR和pROK-DR,利用农杆菌介导方法转化烟草NC89,比较这2种转基因烟草在RNA介导抗病性方面的差异。抗病性检测表明,转化IR和DR的转基因烟草均可获得抗病程度达到免疫的植株,但转化IR序列可大大提高抗病植株在转基因植株中的比例。分析结果表明所获得的抗病性为RNA介导的抗病性,是RNA沉默的结果。这一研究结果为利用IR策略进行抗病毒遗传育种提供了理论依据,并为讲一步开展RNA介导抗病性的机制研究奠宗了基础。     

心叶烟NPR1基因全长cDNA序列的克隆及表达分析

 NPR1(non-expressor of pathogenesis-related gene 1)基因在拟南芥系统获得抗性中起着关键作用,可调控拟南芥植株广谱抗性的发生。本文报道了从心叶烟中克隆NPR1同源基因(NgNPR1)及其表达特性的研究结果。NgNPR1 cDNA全长2253 bp,编码588个氨基酸。将NgNPR1基因组全长与cDNA序列进行比对发现,NgNPR1基因组DNA含有4个外显子和3个内含子。Southern杂交分析表明,在心叶烟基因组中NgNPR1为单拷贝基因。采用绿色荧光蛋白在洋葱表皮瞬时表达的试验,证明了NgNPR1蛋白在水杨酸诱导时会从细胞质转运到细胞核中。Northern杂交分析发现,NgNPR1基因可以被与植物抗病相关的信号分子如水杨酸、茉莉酸甲酯、过氧化氢和乙烯所诱导。进一步研究发现,植物病原物如赤星病菌、青枯病菌和烟草花叶病毒对心叶烟植株的侵染也会使NgNPR1表达量增加。这些结果表明,NgNPR1基因在心叶烟植株抵御病原物侵染过程中可能起着重要作用。     

HC Pro 3个参与抑制RNA沉默氨基酸位点的鉴定

 马铃薯Y病毒属病毒编码的辅助成分-蛋白酶（helper component-proteinase,HC-Pro）是第一个被鉴定的RNA沉默抑制因子。本研究通过定点突变的方法获得了马铃薯A病毒（Potato virus A,PVA）HC-Pro的3个突变体, 利用农杆菌共浸润的方法分析了这些突变对HC-Pro抑制RNA沉默活性的影响。与野生型HC-Pro处理相比,Phe⁶、Asn¹¹ 缺失突变体的处理中绿色荧光减弱,而Ile²⁵⁰-Gly²⁵¹-Asn²⁵²（IGN）基序中的Ile和Gly分别突变为Asp和Glu的处理中观察不到绿色荧光。该结果表明Phe⁶、Asn¹¹ 和IGN²⁵⁰  3个位点均参与调控HC-Pro的抑制RNA沉默活性。     

转基因烟草中PVYN CP基因沉默及其介导的抗病性受温度的调控

 本研究以转不可翻译的马铃薯Y病毒坏死株系外壳蛋白基因(PVY^N CP)烟草的T₃代植株为材料,在获得高度抗病植株并证明转基因植株的抗病性是由RNA沉默介导的基础上,采用Northern杂交及ELISA检测病毒的方法,分析了温度对转基因烟草中RNA沉默以及转基因植株抗病水平的影响。结果表明,低温可以改变转基因植株中已发生的RNA沉默和转基因植株的抗病状态。在15℃低温下生长的转基因植株,转基因产生的RNA沉默被抑制,转基因植株失去了对PVY^N高度抗病的特性,表现感病症状;而在25℃或以上高温(30℃、35℃)下生长的转基因植株,转基因产生的RNA沉默没有发生被抑制的现象,转基因植株对PVY^N病毒的侵染仍保持高度抗病性。     

龙山县烟田烟草潜叶蛾的发生为害和综防措施

烟草潜叶蛾[Phthorimaea operculella (Zeller)]又叫马铃薯块茎蛾、马铃薯麦蛾.它除为害烤烟、马铃薯之外,还为害茄子、辣椒、番茄、曼陀罗、龙葵、酸浆、枸杞等10种以上植物.本县由于经常从外省调进马铃薯种薯,不慎将烟草潜叶蛾随种薯带进来,现已在茨岩镇、农车乡等超过10个乡(镇)的烤烟上发生为害, 为害面积已近667 hm2.由于防治难度大,造成的损失逐年加重,一般减产5%～10%,严重的可达50%.已成为烤烟上主要虫害.     

黄瓜花叶病毒M株系引致烟草症状恢复的初步研究

 黄瓜花叶病毒M株系在白肋烟上具有典型的症状恢复现象,本研究用提纯病毒和DAS-ELISA建立了CMV-M病毒定量检测方法。研究发现,症状严重程度与叶片中的病毒浓度呈正相关:最早发病的黄化叶每克病组织中病毒可高达790 μg,而恢复叶片上部再发病的花叶症状病叶中每克病组织中病毒也可高达508 μg,恢复叶片中病毒含量很低,每克叶片中最高也没有超过6 μg,仅为发病叶片病毒浓度的1/85~1/135,远低于根和茎中的病毒浓度。RT-PCR和生物学检测结果表明恢复叶片中确实存在具侵染活性的病毒,而且病毒在长达半个月以上一直保持很低浓度。结果表明恢复叶中可能存在有效的病毒防御机制,其具体机理有待进一步研究。     

Depolymerization of the actin cytoskeleton induces defense responses in tobacco plants

Tobacco leaf sections were treated with actin inhibitors, i.e., cytochalasins, to determine the effects of actin depolymerization on tobacco defense responses. Inoculation of the leaf sections with the pathogen Erysiphe cichoracearum, depolymerized the actin cytoskeleton, priming the cells for a hypersensitive response-like cell death. Further, expression of the acidic PR1 and PR2 genes were induced in cytochalasin-treated leaf sections. The intensity of the cytochalasin effects on the defense responses was closely correlated with the extent of actin depolymerization. This suggests that plant cells may perceive perturbation of the actin cytoskeleton, and this stimulus may trigger plant defense responses.     

Shoot meristem tissue of tobacco inoculated with Cucumber mosaic virus is infected with the virus and subsequently recovers from infection by RNA silencing

Distribution of Cucumber mosaic virus (CMV) in shoot meristem tissue of CMV-inoculated tobacco was successively analyzed with immunohistochemical microscopy and in situ hybridization. CMV signals were detected in the tissue at 7 days postinoculation (dpi), but then they decreased and disappeared after 14dpi. Detailed observation confirmed CMV invasion of shoot apical meristem at 6–8dpi. Short interfering RNA corresponding to CMV RNAs was first detected at 7dpi and was detected up to 24dpi. These results suggest that the shoot meristem tissue is infected with CMV but subsequently recovers from the infection by RNA silencing.     

RNA interference in Nilaparvata lugens (Homoptera: Delphacidae) based on dsRNA ingestion

BACKGROUND: An efficient and convenient RNA interference (RNAi) technique involving double‐stranded RNA (dsRNA) ingestion is useful for gene function studies of non‐model insects. RESULTS: Three dsRNAs targeting different sites within a gene encoding vacuolar ATP synthase subunit E (V‐ATPase‐E, 21E01) were synthesised for RNAi in Nilaparvata lugens. dsRNA was found to be stable in 0.1 g mL^?1 sucrose solution, but unstable in artificial fodder. Therefore, dsRNAs were orally delivered into N. lugens in 0.1 g mL^?1 sucrose solution. RNAi was induced by all three of the dsRNAs at 0.05 µg µL^?1 in N. lugens. Time dynamics analysis of gene silencing indicated that significant suppression of the target gene began as early as 2 days after ingestion of ds2‐21E01 and ds3‐21E01. However, significant repressive effects were recorded up to 10 days after exposure to ds1‐21E01. The maximum reduction in target gene mRNA was observed after 10 days of treatment, with suppression ratios induced by ds1‐21E01, ds2‐21E01 and ds3‐21E01 of 41, 55 and 48% respectively. CONCLUSION: An efficient and convenient RNAi technique involving dsRNA ingestion has been successfully developed for N. lugens. This will be a useful tool for further functional genomic investigation in this organism. Copyright © 2011 Society of Chemical Industry     

利用siRNA高通量测序技术检测烟草病毒

<正>烟草是我国的主要经济作物之一,病毒病是烟草生产上的重要病害。常见的烟草病毒主要有马铃薯Y病毒(Potato virus Y,PVY)、黄瓜花叶病毒(Cucumber mosaic virus,CMV)、烟草花叶病毒(Tobacco mosaic virus,TMV)、烟草脉带花叶病毒(Tobacco vein banding mosaic virus,TVMBV)等[1]。此外,近年来在烟草上还发生一些新的病毒病害,如南美红辣椒脉斑驳病毒(Chilli veinal mottle virus,ChiVMV)[2]、番茄环纹斑点病毒(Tomato zonate spot virus,ZTSV)[3]和番茄斑萎病毒(Tomato spotted wilt virus,TSWV)[4]。这些病毒     

Validation of RNA interference in western corn rootworm Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae) adults

BACKGROUND: RNA interference (RNAi) is commonly used in insect functional genomics studies and usually involves direct injection of double‐stranded RNA (dsRNA). Only a few studies have involved exposure to dsRNAs through feeding. For western corn rootworm (Diabrotica virgifera virgifera) larvae, ingestion of dsRNA designed from the housekeeping gene, vacuolar ATPase (vATPase) triggers RNAi causing growth inhibition and mortality; however, the effect of dsRNA feeding on adults has not been examined. In this research, WCR adults were fed with vATPase‐dsRNA‐treated artificial diet containing a cucurbitacin bait, which is a proven feeding stimulant for chrysomelid beetles of the subtribe Diabroticina to which rootworms belong. RESULTS: Real‐time PCR confirmed suppression of vATPase expression and western blot analysis indicated reduced signal of a protein that cross‐reacted with a vATPase polyclonal antiserum in WCR adults exposed to artificial diet treated with dsRNA and cucurbitacin bait. Continuous feeding on cucurbitacin and dsRNA‐treated artificial diet resulted in more than 95% adult mortality within 2 weeks while mortality in control treatments never exceeded 20%. CONCLUSIONS: This research clearly demonstrates the effect of RNAi on WCR adults that have been exposed to dsRNA by feeding and establishes a tool to screen dsRNAs of potential target genes in adults. This technique may serve as an alternative to target screening of larvae which are difficult to maintain on artificial diets. Copyright © 2011 Society of Chemical Industry     

基于纳米递送系统的RNA农药及转基因植物研究进展

近年来,转基因植物和RNA农药作为绿色可持续发展农业的重要组成部分受到越来越多的关注。传统的基因工程元件和RNA农药递送策略往往会受到多种外界因素的干扰,造成效率偏低甚至失效,这也是限制植物基因工程和RNA农药发展的主要技术瓶颈。基于纳米载体的新型递送系统与植物基因工程和RNA干扰技术的结合展现出了广阔的应用前景。本文结合最新研究进展,重点综述了以纳米科技为核心的植物保护新理念、基于纳米递送系统的RNA农药以及纳米载体在植物基因工程中的应用,展望了纳米科技在未来植物保护中的应用前景。