水稻条纹病毒山东济宁分离物RSV-SD-JN2分子变异分析

为从分子水平探讨强致病性水稻条纹病毒山东济宁分离物RSV-SD-JN2的变异及其致病性,采用RT-PCR技术,克隆RSV-SD-JN2的RNA3、RNA4区段cDNA。结果显示,RSV-SD-JN2的RNA3和RNA4核苷酸序列长度分别为2487、2157bp;RNA3中,NS3基因长为636bp,基因间隔区(IR)为725bp,CP基因为969bp;RNA4中,SP基因长为537bp,基因间隔区(IR)为654bp,NSvc4基因为861bp。不同时期、不同区域和不同致病性的RSV分离物的序列比较发现,RNA3、RNA4序列5′和3′末端非编码区具有高度保守性,仅存在个别碱基的差异;基因编码区保守性较高,核苷酸和氨基酸序列同源性分别在93%和97%以上,且大部分碱基变异为无意义变异;基因间隔区(IR)易于变异。RSV的分子变异与其地理分布具有密切的关系。RNA4的IR的变异导致RNA二级结构——发夹结构的稳定性提高是病毒致病性增强的重要原因。     

转录后基因沉默

 转录后基因沉默(PTGS)普遍存在于生物界,如植物的共抑制、源于病原的RNA介导的病毒抗性、真菌的基因沉默和动物的RNA干扰等。这类现象有许多共同特点,如都是以向细胞内引入核酸(转基因、双链RNA或病毒RNA)为诱因,依赖RNA的RNA聚合酶(RdRP)活性与基因沉默密切相关,在发生基因沉默的细胞中大多存在特定长度的小分子RNA (21~25 nt),PTGS导致细胞质内mRNA的特异性降解,不同生物的PTGS相关基因及产物具有很高的相似性,基因沉默能够在细胞间传播并能以表型遗传的方式传递给下一代等。这说明各种生物的转录后基因沉默可能有相似的遗传起源,是一种抵抗外来核酸(如病毒和转座子)入侵的共同的防御机制     

植物激活蛋白对烟草花叶病毒RNA复制及外壳蛋白合成的抑制作用

研究了植物激活蛋白对烟草花叶病毒RNA及外壳蛋白的抑制作用。结果表明，烟草经植物激活蛋白处理后，植株体内烟草花叶病毒RNA含量比对照降低28％；用ELISA和Western方法检测烟草花叶病毒外壳蛋白的含量，处理分别比对照减少20．7％和25．0％。植物激活蛋白显著干扰烟草花叶病毒外壳蛋白的体外聚合过程，22—37％条件下其干扰作用达显著水平。     

基于RNA干扰的生物农药的发展现状与展望

RNA干扰又称转录后基因沉默,是一种能有效沉默或抑制目标基因表达的新兴基因工程技术。基于RNA干扰的生物农药被认为是未来植保领域的颠覆性技术,将极大改变人类防治农业病、虫、草等有害生物的思路和策略。本文我们简单回顾了RNA干扰的基本作用机制和发展历程,全面总结了RNAi生物农药的研究水平和应用现状,深入分析了RNAi生物农药发展面临的机遇和挑战,以及未来的发展前景。以期为我国RNAi生物农药的研发提供参考。     

RNAi‐mediated plant protection against aphids

Aphids (Aphididae) are major agricultural pests that cause significant yield losses of crop plants each year by inflicting damage both through the direct effects of feeding and by vectoring harmful plant viruses. Expression of double‐stranded RNA (dsRNA) directed against suitable insect target genes in transgenic plants has been shown to give protection against pests through plant‐mediated RNA interference (RNAi). Thus, as a potential alternative and effective strategy for insect pest management in agricultural practice, plant‐mediated RNAi for aphid control has received close attention in recent years. In this review, the mechanism of RNAi in insects and the so far explored effective RNAi target genes in aphids, their potential applications in the development of transgenic plants for aphid control and the major challenges in this regard are reviewed, and the future prospects of using plant‐mediated RNAi for aphid control are discussed. This review is intended to be a helpful insight into the generation of aphid‐resistant plants through plant‐mediated RNAi strategy. © 2016 Society of Chemical Industry     

PCR方法鉴别大家白蚁和台湾乳白蚁

本文分析了大家白蚁(Coptotermes curvignathus Holmgren)和台湾乳白蚁(C.formosamus Shiraki)形态特征的相似性,并根据GenBank中两者12S rRNA基因序列设计了特异性引物for1、cur2和通用引物com3、com4,通过PCR方法鉴别确认了未知样品为台湾乳白蚁.该方法具有快速、简便、样品量少、准确等优点,适合用于大家白蚁和台湾乳白蚁的鉴别.     

两种类型黄瓜花叶病毒卫星RNA的序列、结构以及对辅助病毒的致病性调控分析

黄瓜花叶病毒(Cucumber mosaic virus,CMV)卫星RNA(satellite RNA,satRNA)通常影响CMV的致病性。本研究设计了检测satCMV的简并引物,通过RT-PCR从山东省泰安市3种自然发病寄主中检测到satCMV,发现了两类长度有明显差异的satCMV:白菜与萝卜中克隆的satCMV为339个核苷酸,二者的核苷酸一致率为99.41%;烟草中克隆的satCMV为383个核苷酸,与白菜和萝卜中satCMV的核苷酸序列一致率均为71.06%。系统进化分析表明这两类长度有明显差异的satCMV亲缘关系较远。两种类型的satCMV与CMV-Fny混合接种对CMV致病症状的调控存在明显差异。利用m Fold软件分析了两类不同长度的CMV卫星RNA基因组及其互补链的RNA结构特征,表明两类sat CMV与CMV互作的活跃度以及核心结构域存在差异。两类satCMV全基因组的克隆以及结构分析为研究不同类型satCMV与CMV的互作及其对CMV致病性的影响奠定了基础。     

烟草丛顶病毒RNA依赖RNA聚合酶(RdRp)介导的体外复制体系

通过重叠PCR扩增得到烟草丛顶病毒(Tobacco bushy top virus,TBTV)中国分离物RdRp的编码序列,构建以pMALC2X为基础载体的原核表达载体pMAL-TB-RdRp。0.5 mM IPTG诱导可特异性表达分子量约为120 kDa的MBP-RdRp融合蛋白。温度梯度实验显示,18℃下诱导表达的MBP-RdRp融合蛋白的可溶性比例较高,约17%;经亲和层析纯化的MBPRdRp可特异性识别TBTV正链和负链的3'末端序列,催化体外复制;对正负链的3'末端的体外复制效率存在差别,识别负链3'末端的体外复制效率明显高于正链3'末端。本研究创建的TBTV RdRp介导的体外复制体系为进一步研究TBTV基因组复制调控奠定了基础。     

Leonotis nepetaefolia: An important plant virus reservoir in central Mexico

The presence of viruses in the weedLeonotis nepetaefolia in central México is reported from two field surveys.L. nepetaefolia, with viral-like symptoms such as mosaic, leaf deformation and calico, was observed growing next to cultivated fields in the Valley of Atlixco, Puebla, an important agricultural region in Mexico. The viruses harbored by this plant were characterized biologically, serologically and by molecular methods. The viruses detected wereAlfalfa mosaic virus (AMV),Cucumber mosaic virus (CMV), a satellite RNA of CMV (CMV satRNA) andTobacco mosaic virus (TMV). This last one was detected only during the first survey. CMV was the predominant virus found in both surveys, and was associated mostly to mosaic symptom. Phylogenetic analysis based on the coat protein gene sequence of CMV indicated that this isolate belongs to subgroup IA and confirmed that it is a mosaic-inducing isolate, whereas AMV belongs to subgroup II. Finally, CMV satRNA was found to be a non-necrogenic ameliorative variant, both by symptomatology and by phylogenetic analysis. Our results suggest thatL. nepetaefolia is a reservoir for several viruses in central Mexico, and given its wide distribution in several parts of the world, its role as a virus reservoir could be more general. http://www.phytoparasitica.org posting Aug. 31, 2005.     

尖孢镰刀菌双链RNA的分离鉴定

 对尖孢镰刀菌10个专化型18个菌株进行分析发现,在尖孢镰刀菌大豆专化型Fusarium oxysporum f.sp.Glycines 1193-31中存在双链RNA,由6种成分组成,其大小分别为3.8kb,3.0kb,2.4kb,0.48kb,0.43kb和0.21kb,该菌株气生菌丝少,形成大量的粉红色分生孢子堆,并发生角变。     

松材线虫mapk基因克隆及RNAi效应分析

促分裂原活化蛋白激酶(Mitogen Activated Protein Kinase)在线虫生长发育过程中具有重要作用。本研究利用RACE方法克隆了松材线虫mapk基因,其基因全长1 744 bp,5'UTR长243 bp,3'UTR长81 bp,含有一个1 098 bp的开放读码框,包括6个外显子和5个内含子,为单拷贝基因,其预测的蛋白序列与其他线虫的序列相似性为77.66%~84.01%。通过体外转录合成341 bp的dsRNA,将松材线虫的幼虫浸泡在1.5 mg·mL-1dsRNA中24 h后,mapk表达量下降约85%,松材线虫的死亡率约为对照的4.2倍,经dsRNA浸泡的成虫,繁殖能力下降约50%,表明mapk基因的沉默可能抑制松材线虫的繁殖能力。     

RNA interference technology in crop protection against arthropod pests,pathogens and nematodes

Scientists have made significant progress in understanding and unraveling several aspects of double‐stranded RNA (dsRNA)‐mediated gene silencing during the last two decades. Now that the RNA interference (RNAi) mechanism is well understood, it is time to consider how to apply the acquired knowledge to agriculture and crop protection. Some RNAi‐based products are already available for farmers and more are expected to reach the market soon. Tailor‐made dsRNA as an active ingredient for biopesticide formulations is considered a raw material that can be used for diverse purposes, from pest control and bee protection against viruses to pesticide resistance management. The RNAi mechanism works at the messenger RNA (mRNA) level, exploiting a sequence‐dependent mode of action, which makes it unique in potency and selectivity compared with conventional agrochemicals. Furthermore, the use of RNAi in crop protection can be achieved by employing plant‐incorporated protectants through plant transformation, but also by non‐transformative strategies such as the use of formulations of sprayable RNAs as direct control agents, resistance factor repressors or developmental disruptors. In this review, RNAi is presented in an agricultural context (discussing products that have been launched on the market or will soon be available), and we go beyond the classical presentation of successful examples of RNAi in pest‐insect control and comprehensively explore its potential for the control of plant pathogens, nematodes and mites, and to fight against diseases and parasites in beneficial insects. Moreover, we also discuss its use as a repressor for the management of pesticide‐resistant weeds and insects. Finally, this review reports on the advances in non‐transformative dsRNA delivery and the production costs of dsRNA, and discusses environmental considerations. © 2017 Society of Chemical Industry