鸭跖草叶点霉的致病性与dsRNA及 RAPD类群的相关分析

用随机引物扩增多态性(RAPD)和纤维素法对鸭跖草叶点霉Phyllosticta commelimecola的基因组遗传多样性和dsRNA进行测定,分析遗传多样性和dsRNA与菌株的致病力的关系．结果表明菌株间存在明显的培养类型和致病力分化现象,菌株的致病力从南到北逐渐减弱．选择东北地区有代表性的菌株,进行dsRNA测定和RAPD分析,在所测定的菌株中有10个含有dsRNA,2个不含dsRNA,菌株致病力强弱与dsRNA无相关性．通过RAPD扩增和聚类分析,在类间距离15以下可将12个鸭跖草叶点霉分为5个类群．辽宁省和吉林省的菌株亲缘关系较近,黑龙江省的菌株与辽宁、吉林的菌株亲缘关系远,同一RAPD类群中表现出相似的致病力．     

RNA‐based biocontrol compounds: current status and perspectives to reach the market

Facing current climate challenges and drastically reduced chemical options for plant protection, the exploitation of RNA interference (RNAi) as an agricultural biotechnology tool has unveiled possible new solutions to the global problems of agricultural losses caused by pests and other biotic and abiotic stresses. While the use of RNAi as a tool in agriculture is still limited to a few transgenic crops, and only adopted in restricted parts of the world, scientists and industry are already seeking innovations in leveraging and exploiting the potential of RNAi in the form of RNA‐based biocontrol compounds for external applications. Here, we highlight the expanding research and development pipeline, commercial landscape and regulatory environment surrounding the pursuit of RNA‐based biocontrol compounds with improved environmental profiles. The commitments of well‐established agrochemical companies to invest in research endeavours and the role of start‐up companies are crucial for the successful development of practical applications for these compounds. Additionally, the availability of standardized guidelines to tackle regulatory ambiguities surrounding RNA‐based biocontrol compounds will help to facilitate the entire commercialization process. Finally, communication to create awareness and public acceptance will be key to the deployment of these compounds. © 2019 Society of Chemical Industry     

Molecular mechanisms influencing efficiency of RNA interference in insects

RNA interference (RNAi) is an endogenous, sequence‐specific gene‐silencing mechanism elicited by small RNA molecules. RNAi is a powerful reverse genetic tool, and is currently being utilized for managing insects and viruses. Widespread implementation of RNAi‐based pest management strategies is currently hindered by inefficient and highly variable results when different insect species, strains, developmental stages, tissues, and genes are targeted. Mechanistic studies have shown that double‐stranded ribonucleases (dsRNases), endosomal entrapment, deficient function of the core machinery, and inadequate immune stimulation contribute to limited RNAi efficiency. However, a comprehensive understanding of the molecular mechanisms limiting RNAi efficiency remains elusive. Recent advances in dsRNA stability in physiological tissues, dsRNA internalization into cells, the composition and function of the core RNAi machinery, as well as small‐interfering RNA/double‐stranded RNA amplification and spreading mechanisms are reviewed to establish a global understanding of the obstacles impeding wider understanding of RNAi mechanisms in insects. © 2018 Society of Chemical Industry     

RNA干扰技术的应用现状及研究进展

RNA干扰是指外源双链RNA进入细胞以后引起的与其同源mRNA特异性降解的现象,它参与真核生物抵抗病毒侵染,阻断转座子的异常活动,调控基因表达,并广泛存在于生物界。通过介绍RNA干扰技术的发现过程,探讨该技术的作用机理和研究进展,进而提出该技术的未来发展方向。     

草鱼呼肠孤病毒的研究进展

草鱼呼肠孤病毒(GCRV)是呼肠孤病毒科、水生呼肠孤病毒属的病毒中毒力最强的病毒,严重危害着我国淡水渔业的养殖。基因组由11条分节段dsRNA组成,编码11种结构多肽。GCRV能够合成内源性RNA聚合酶,在体外转录。总结了近20年来我国在GCRV形态结构、理化特性、培养特性、分子生物学以及预防治疗等方面的研究成果,提出了GCRV的重要酶类作为RNA干涉靶基因的思路。由于中和效价最高的蛋白基因M6在毒株中具有相对保守性,初步确认了基因工程亚单位疫苗开发的可行性。     

柔嫩艾美耳球虫病毒的鉴定

鸡球虫病是由艾美耳属（Eimeria）的一种单细胞寄生性原虫引起的严重危害养禽业发展的重要疾病之一,遍及世界各地。感染鸡的艾美耳属球虫中,国外已报道在巨型艾美耳球虫（E.maxima）,堆型艾美耳球虫（E.acervu-lina）,毒害艾美耳球虫（E.necatrix）和布氏艾美耳球虫（E.brunetti）中发现了病毒粒子或病毒RNA。但是作为危害最严重的柔嫩艾美耳球虫是否有病毒感染,国内外迄今尚无报道。本研究首次在柔嫩艾美耳球虫中发现病毒并对其进行了鉴定。通过核酸分析、RNA依赖的RNA聚合酶（RDRP）活性和电镜形态观察对病毒进行鉴定。核酸酶的敏感性试验结果表明在柔嫩艾美耳球虫总核酸电泳图谱上观察到的大小分别为1.4、2.4和3.6kb的3条病毒带均不能被DNA酶（100mg/L）降解,但可被RNA酶（1.0mg/L）降解,表明这些核酸为RNA。另外,这些核酸不能被高盐浓度（0.3mol/L NaCl）RNase A（10mg/L）降解,但可被低盐浓度（0.015mol/L NaCl）RNase A（10mg/L）降解,并且采用α-32P标记的UTP掺入法测得该病毒样核酸具有RNA依赖RNA聚合酶（RDRP）活性,表明这些核酸为双链RNA（dsRNA）。利用蔗糖梯度离心和透射电镜技术对柔嫩艾美耳球虫病毒进行了分离和鉴定。电镜负染观察到柔嫩艾美耳球虫病毒粒子外观呈球形,二十面体,无囊膜,直径38nm。     

纳米载导RNAi技术在害虫防治中的研究和应用

每年重大病虫害的流行和暴发都会对农业产生重大的影响,进而造成严重的经济损失,随着化学试剂的施用其抗药性也逐渐增强,因此探索新的绿色、安全、高效的害虫防治技术具有十分重要的意义。RNAi作为一种研究基因功能的工具,在开发新的害虫防治策略方面显示出巨大的潜力,但RNAi的效率受限于核酸酶的降解作用,为了改善dsRNA的有效递送,研发了纳米载导技术且被广泛应用于害虫防治。同时,基于工程菌高效合成靶向昆虫dsRNA的技术为田间推广提供了可行性及技术支撑。本文主要对RNAi技术及纳米载导RNAi技术在害虫防治中的研究和应用进行了总结和归纳。     

猴轮状病毒(SA11)RT-PCR检测方法的建立

根据具有高度保守性的编码VP7轮状病毒糖蛋白的基因9序列设计引物，分别进行RT-PCR和NT-PCR扩增。结果：引物Beg9／End9、Beg9-1／End9-1及引物RVG9／aET3、RVG9／aET3-1分别能在一次RT-PCR扩增中出现预期的1062bp和374bp扩增带；引物RvGg／aET3、RVG9／aET3-1在NT-PCR扩增中均能出现预期的374bp扩增带；验证了猴轮状病毒SA11属于血清型3；引物RVG9／aET3进行敏感试验能检测到0．5pg的轮状病毒(SA11)dsDNA。     

寄生线虫RNA干扰研究进展

基因的RNA干扰（RNA interference,RNAi）技术是一个功能强大的基因组学研究工具。机体内存在特定的双链RNA（dsRNA）对基因的表达有干扰作用,可以引起基因沉默现象。通过外源dsRNA对寄生线虫进行RNA干扰研究,有助于了解不同发育期的特征现象和特定基因的生物学功能。此外,寄生线虫的RNA干扰研究对RNA干扰技术本身的发展也有较大促进作用。作者总结了近几年来RNA干扰技术在寄生线虫方面的最新研究进展,以期为相关研究工作提供参考。