Robust RNAi-based resistance to mixed infection of three viruses in soybean plants expressing separate short hairpins from a single transgene

Transgenic plants expressing double-stranded RNA (dsRNA) of virus origin have been previously shown to confer resistance to virus infections through the highly conserved RNA-targeting process termed RNA silencing or RNA interference (RNAi). In this study we applied this strategy to soybean plants and achieved robust resistance to multiple viruses with a single dsRNA-expressing transgene. Unlike previous reports that relied on the expression of one long inverted repeat (IR) combining sequences of several viruses, our improved strategy utilized a transgene designed to express several shorter IRs. Each of these short IRs contains highly conserved sequences of one virus, forming dsRNA of less than 150 bp. These short dsRNA stems were interspersed with single-stranded sequences to prevent homologous recombination during the transgene assembly process. Three such short IRs with sequences of unrelated soybean-infecting viruses (Alfalfa mosaic virus, Bean pod mottle virus, and Soybean mosaic virus) were assembled into a single transgene under control of the 35S promoter and terminator of Cauliflower mosaic virus. Three independent transgenic lines were obtained and all of them exhibited strong systemic resistance to the simultaneous infection of the three viruses. These results demonstrate the effectiveness of this very straight forward strategy for engineering RNAi-based virus resistance in a major crop plant. More importantly, our strategy of construct assembly makes it easy to incorporate additional short IRs in the transgene, thus expanding the spectrum of virus resistance. Finally, this strategy could be easily adapted to control virus problems of other crop plants.     

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。     

植物抗病毒的可能机制--基因沉默

根据目前RNA介导的植物病毒抗性(RMVR)、转录后基因沉默(PTGS)的研究成果,综述了基因沉默可能是植物抵抗病毒的一种机制,深入研究基因沉默不仅在抗病机制上有重要的理论意义,而且对彻底解决植物病毒问题有较大的潜在实用价值.     

RNAi‐based enhanced resistance to Cowpea severe mosaic virus and Cowpea aphid‐borne mosaic virus in transgenic cowpea

Cowpea (Vigna unguiculata) is one of the most important legumes cultivated in many parts of the world. The diseases caused by Cowpea severe mosaic virus (CPSMV) and Cowpea aphid‐borne mosaic virus (CABMV) are considered among the most important constraints on yield and quality, especially in Latin America and Africa. Here, the concept of using an RNA interference construct to silence the CPSMV proteinase cofactor gene and the CABMV coat protein gene is explored, in order to generate resistant transgenic cowpea plants. Ten cowpea transgenic lines were produced, presenting a normal phenotype and transferring the transgene to the next generation. Plants were tested for resistance to both CABMV and CPSMV by mechanical co‐inoculation. Seven lines presented milder symptoms when compared to the control and three lines presented enhanced resistance to both viruses. Northern analyses were carried out to detect the transgene‐derived small interfering RNA (siRNA) in leaves and revealed no correlation between siRNA levels and virus resistance. Additionally, in the symptomless resistant lines the resistance was homozygosis‐dependent. Only homozygous plants remained uninfected while hemizygous plants presented milder symptoms.     

RNA沉默转基因技术在植物抗病虫害研究中的作用

RNA沉默技术由于其简便、高效及高特异性在各种模式和非模式生物系统中得到了广泛的应用。植物转基因技术是将植物、动物或微生物中分离到的目的基因,通过各种手段整合到植物基因组上,使其稳定遗传并赋予植物新的优良性状的生物技术。RNA沉默在植物转基因工程领域的研究十分广泛,目前已经成功研究培育出许多抗病毒、抗虫转基因植物,为农业病虫害的防控提供了新的思路和方法。本文重点综述了近年来植物转基因介导RNA沉默在抗虫与抗病毒研究方面的进展,加深人们对转基因植物抗虫与抗病毒研究的认识。     

Hairpin-based virus resistance depends on the sequence similarity between challenge virus and discrete,highly accumulating siRNA species

Virus resistance can be effectively generated in transgenic plants by using the plant’s silencing machinery. To study the specificity of gene-silencing-based resistance, homozygous tobacco (Nicotiana tabacum L.) plants containing a 597-nt hairpin RNA construct of the Potato Virus Y (PVY) replicase sequence were challenged with a variety of PVY strains. The transgene-carrying tobacco line was immune to five potato PVY strains with high sequence similarity (88.3–99.5%) to the transgene. Infection with more distant tomato and pepper PVY field strains (86–86.8% sequence similarity) caused delayed symptom appearance in the transgenic tobacco. Transgene production of small interfering (si) RNA was detected by northern blot and measured using a custom-designed microarray for the detection of small RNAs. siRNA accumulation peaks were observed throughout the inverted-repeat transgene. In the resistance-breaking tomato and pepper strains there were nucleotide differences in the sequences correlated to siRNA transgene accumulation, indicating the role of siRNA specificity in resistance breaking. The log of transgene siRNA signal intensity increased with probe GC content, indicating that the accumulating siRNA molecules were GC-rich. Sequence similarity of highly accumulating siRNAs with the target virus strain appears to be important for both resistance and resistance-breaking characteristics.     

Production of anti-virus,viroid plants by genetic manipulations

Many pathogenic plant viruses are RNA viruses, which initiate production of double-stranded RNA intermediates when they replicate in host plant cells. Introduction of double-stranded RNA-specific ribonucleases such as the Schizosaccharomyces pombe derived pac I protein and animal cell derived interferon-induced 2',5'-oligoadenylate synthetase (2-5 Aase)/ribonuclease L (RNase L) system into various plants may make plants resistant to various pathogenic viruses and viroids. We have demonstrated that pac I and 2-5 Aase/RNase L transgenic tobacco plants are resistant to various viruses including tobacco mosaic virus, cucumber mosaic virus and potato virus Y. In addition, pac I transgenic potato plants are resistant to potato spindle tuber viroid. Using Agrobacterium-mediated transformation, we have established a transformation system for chrysanthemum plants and have recently developed pac I transgenic chrysanthemum (Dendranthema grandiflora cv Reagan) resistant to chrysanthemum stunt viroid and have grown them in isolated fields for an evaluation of their effects.     

Near immunity to rice tungro spherical virus achieved in rice by a replicase-mediated resistance strategy

ABSTRACT Rice tungro disease is caused by rice tungro bacilliform virus (RTBV), which is responsible for the symptoms, and rice tungro spherical virus (RTSV), which assists transmission of both viruses by leafhoppers. Transgenic japonica rice plants (Oryza sativa) were produced containing the RTSV replicase (Rep) gene in the sense or antisense orientation. Over 70% of the plants contained one to five copies of the Rep gene, with integration occurring at a single locus in most cases. Plants producing antisense sequences exhibited significant but moderate resistance to RTSV (60%); accumulation of antisense RNA was substantial, indicating that the protection was not of the homology-dependent type. Plants expressing the full-length Rep gene, as well as a truncated Rep gene, in the (+)-sense orientation were 100% resistant to RTSV even when challenged with a high level of inoculum. Accumulation of viral RNA was low, leading us to conclude that RTSV Rep-mediated resistance is not protein-mediated but is of the cosuppression type. Resistance was effective against geographically distinct RTSV isolates. In addition, RTSV-resistant transgenic rice plants were unable to assist transmission of RTBV. Such transgenic plants could be used in an epidemiological approach to combat the spread of the tungro disease.     

hpRNA的茎环比例对RNA介导的病毒抗性产生的影响

 以马铃薯Y病毒坏死株系(PVY^N)的外壳蛋白(coat protein,CP)基因3'端50bp片段为hpRNA的茎,以pUC19不同长度的序列为hpRNA的环,构建了茎环比例分别为4:1、2:1、1:1、1:2、1:4和1:8的植物表达载体。利用农杆菌介导法转化烟草品种NC89,获得了多种转基因植株。室内抗病性检测发现:不同茎环比例的hpRNA介导的病毒抗性效率不同;茎环比例为4:1、2:1和1:1时效率较高,抗性植株的比例达60%左右;随着环长度的逐渐增加,抗性植株的比例逐渐降低;当茎环比例为1:8时,抗性植株的比例仅为9.52%。Southern blot分析结果表明:外源基因已整合于烟草的基因组中,且转基因植株的抗病性与转基因的拷贝数之间无明显的相关性。Northern blot分析结果表明:目的片段转录产物的积累量与植株的抗病性呈负相关,证明所获得的抗病性是RNA介导的。     

马铃薯Y病毒CP基因5'端和3'端反向重复结构介导的抗病性研究

 本研究克隆了来源于马铃薯Y病毒坏死株系(PVY^N)外壳蛋白(CP)基因(PVY^N CP)5'端和3'端的反向重复cDNA序列,并构建了植物表达载体pROKⅡ-5'IR和pROKⅡ-3'IR,利用农杆菌介导的叶盘法转化烟草NC89,分别获得166和126株转基因烟草。抗病性试验表明,转化PVY^N CP基因3'端茎50 bp(环50 bp) hpRNA(hairpin RNA)的烟草抗病率达69%,而转化PVY^N CP基因5'端相同片段长度的hpRNA的烟草却全部发病。Southern blot结果表明,转基因植株的抗病性与转基因的拷贝数之间无明显的相关性;Northern blot结果表明,目的片段转录产物的积累量与植株的抗病性呈负相关,证明所获得的抗病性是RNA介导的抗病性。研究结果证明PVY^N CP基因的3'端比5'端更能有效地诱发基因沉默,并且hpRNA茎部的长度可减少到50 bp。该结果为探索利用CP基因的最短长度和有效部位的基因片段来培育多抗病毒转基因植物提供了依据。     

Transgenic resistance to cucumber mosaic virus in tomato: blocking of long-distance movement of the virus in lines harboring a defective viral replicase gene

ABSTRACT Tomato breeding lines were transformed with a defective replicase gene from RNA 2 of cucumber mosaic virus (CMV). A total of 63 transformants from five tomato genotypes were evaluated for resistance to CMV strains. The responses of R1 transgenic offspring fit into three categories: fully susceptible lines (44%), fully resistant lines (8%), and an intermediate-type mixture of susceptible and resistant seedlings in variable proportions (48%). Further characterization of the response of two highly resistant lines was performed by mechanical inoculation, aphid transmission, or grafting experiments. No virus was detected in noninoculated leaves from these lines, although a low level of virus accumulated initially in the inoculated leaf. The homozygous R2 plants and further generations that were evaluated (up to R5) showed resistance to the Fny-CMV strain, two Israeli isolates tentatively classified as subgroup IA, and K-CMV (a representative of subgroup IB). These lines were partially resistant to LS-CMV (a representative of subgroup II) when a high-virus-titer inoculum was used. Expression of the viral transgene was verified in these lines; however, the expected translation product was not detectable. In grafting experiments, we demonstrated that CMV virions were blocked in their ability to move from infected rootstocks of nontransformed tomato or tobacco into the transgenic scions. Interestingly, virions could not move through a transgenic intersection into the upper scion. These results provide an additional indication that replicase-mediated resistance affects long-distance movement.     

利用RNAi 介导的抗病性获得抗2 种花生病毒的转基因烟草

 分别以花生条纹病毒红安株系(PStV-Hongan)外壳蛋白基因(CP),花生矮化病毒轻型株系(PSV-Mi)和花生黄瓜花叶病毒(CMV-CA)复制酶基因2a为模板,通过PCR 方法分别得到PStV-CP 5′ 端,PSV-Mi 和CMV-CA 2a3′ 端150 bp 的片段,3 种片段混合物为模板经PCR 拼接得到450 bp 的片段,此拼接片段通过Gateway 系统重组至植物表达载体pK7GW1WG2,得到含反向重复拼接片段的植物表达载体pK450。冻融法导入根癌农杆菌(Agrobacterium tumefaciens)菌株GV3101 后,叶盘法转化本生烟(Nicotiana benthamiana),经PCR 检测,获得转基因植株。对T1 代转基因植株分别人工接种3 种病毒,66. 7% 的植株表现对PStV 免疫,9% 的植株表现对CMV-CA 的恢复抗性,全部植株对PSV 感病。siRNA 的Northern blot 结果表明,所有转基因烟草植株都含有病毒特异siRNA,siRNA 含量随接种后时间延长而衰减。     

核基质结合区对马铃薯Y病毒全长非翻译CP基因介导抗病性的影响

 为探索核基质结合区(matrix attachment regions,MARs)对RNA介导的病毒抗性的影响,我们将从烟草中克隆到的核基质结合区TM2构建在包含马铃薯Y病毒全长非翻译CP基因的植物表达载体pRPVYCPN的表达盒的两侧,构建了植物表达载体pRTM2CPNTM2。采用农杆菌介导基因转化法,将表达载体pRPVYCPN和pRTM2CPNTM2转入烟草品种NC89中,分别获得了144株和344株转基因烟草。抗病性检测发现,核基质结合区的存在能明显提高RNA介导抗性的产生效率。在含MARs转基因植株中,抗病植株的比率为15.1%,而不含核基质结合区的转基因植株的抗病比率则为8.3%。这一研究结果对抗病毒植物的分子育种和转基因表达调控有指导意义。