Purification and some properties of Papaya meleira virus, a novel virus infecting papayas in Brazil

'Meleira', or 'sticky disease', is currently the most damaging papaya disease in the mid-eastern Brazilian growing regions. Consistent disease transmission via latex injection, presence of similar isometric particles in the laticiferous vessels of diseased plants, and detection of double-stranded DNA in naturally and experimentally infected papaya trees suggest that a virus is the causal agent. Conclusive evidence for viral aetiology was previously lacking, mostly because every attempt to purify the putative virus from infected papayas had failed. Following the successful purification and partial characterization of the meleira virus, healthy papaya seedlings injected with purified virus particles later developed typical symptoms of the disease. Negatively stained, isometric, full and 'empty' purified virus particles measured 42 and 38 nm, respectively. The viral genome was a single dsRNA molecule of about 12 kbp. Several capsid proteins, ranging in size from 14·4 to 45 kDa, were consistently revealed by PAGE. Papaya meleira virus (PMeV) appears to represent a novel group of viruses, with no known similar counterpart among known plant-, vertebrate-, invertebrate- or prokaryote-infecting viruses.     

Identification of divergent variants of Grapevine virus A

Eight isolates of Grapevine virus A (GVA), which induced different symptoms in leaves of Nicotiana benthamiana, were recovered from various grapevines. The dsRNA patterns of two isolates, which consistently induced mild vein clearing (referred here as mild isolates of GVA) were similar, but different from those of other isolates of GVA. Analysis based on overall nucleotide (nt) sequence identity in the 3 terminal part of the GVA genome, comprising part of ORF3 (putative movement protein, MP), entire ORF4 (capsid protein, CP), entire ORF5 and part of 3 UTR, revealed that GVA isolates separate into three groups (I, II, III), sharing 91.0–99.8% nt sequence identity within groups and 78.0–89.3% nt sequence identity between groups. Mild isolates of the virus were group III and shared only 78.0–79.6% nt sequence identity with the other isolates. The comparison of predicted amino acid sequences for MP and CP revealed many amino acid alterations, revealing distinct local net charges of these proteins for mild isolates of the virus. Based on both conserved and divergent nt regions in the CP and ORF5, oligonucleotide primers were designed for the simultaneous RT-PCR detection of all GVA isolates and for the specific detection of the most divergent virus variants represented here by mild isolates of the virus.     

用于RNA干扰研究的大肠杆菌表达系统的条件优化

选取在E.coli中对于dsRNA表达比较重要的3个因素,即诱导时间、诱导温度和诱导剂IPTG浓度。利用已经构建好的大肠杆菌E.coli HT115(DE3),并结合正交试验设计研究dsRNA在HT115(DE3)内的最优表达条件。结果表明,工程菌在诱导时间8 h、诱导温度30℃、诱导剂浓度10.0 mmol/L的培养条件下所得到的dsRNA表达量最大。     

RNAi技术防治害虫的研究进展

利用RNAi技术来防治害虫成了人们研究的热点之一。RNAi可分为细胞自主性的和非细胞自主性的。对应用RNAi技术防治害虫来讲,研究的焦点集中在非细胞自主性RNAi,即昆虫能通过取食来内化目标基因的dsRNA。RNAi技术防治害虫的特异性极高,不会出现残留污染问题。可以通过注射、饲喂和转基因dsRNA来进行害虫的防治。本文还介绍了昆虫吸收dsRNA的机制。     

gp64基因相应dsRNA对家蚕核型多角体病毒（BmNPV）增殖的抑制

 【目的】研究gp64基因相对应的多个dsRNA对家蚕核型多角体病毒增殖的抑制效果。【方法】体外合成dsRNA，通过病毒滴度试验、实时定量RT-PCR法研究gp64基因的不同区域、不同长度与RNAi干扰效果的关系。【结果】供试的6个dsRNA的最大抑制效果相当（滴度TCID50的最大抑制差值为4.00左右）；经RNAi的不同时间点的gp64 mRNA 表达水平均明显下调(P<0.01)，其中48 h的gp64 mRNA的表达量约为对照的1/300。【结论】6个dsRNA均能有效地抑制病毒的基因表达和增殖；gp64 ORF第1 390～1 499位点（G3-3）是RNAi的较佳靶位点。     

双链RNA分析技术在鉴定RNA病毒中的应用

ｄｓＲＮＡ技术是以无ＲＮＡ病毒或类似病毒因子侵染的植物组织中不含有容易鉴定的同原大分子的ｄｓＲＮＡ（＞０．１×１０６）存在为前提。在植物体内ｄｓＲＮＡ是ＲＮＡ病毒和类病毒因子存在的迹象，ｄｓＲＮＡ包括ｄｓＲＮＡ病毒的基因或ｓｓＲＮＡ病的复制中间型。ｄｓＲＮＡ在寄主组织相当稳定，可以用物理和化学的方法分离出来。叙述了ｄｓＲＮＡ技术用于植物病毒群、成员、同一病毒的不同株系、感染作物的新病毒及其复合侵染、类病毒、卫星病毒等的鉴定。证明该方法使用广泛、快速准确，不需要贵重设备，在一般实验室中即可进行。该方法有重要应用和学术价值，已引起植物病毒和植物病理学工作者广泛兴趣。     

双链RNA干扰技术在毛状根体系中对何首乌芪合酶基因Fm STS功能研究中的应用

[目的]将双链RNA介导的基因沉默与发根农杆菌瞬间表达渗入相结合,为植物功能基因的研究提供一套有效的方法。[方法]构建含gfp基因、CaMV 35S启动子及基于何首乌中芪合酶Fm STS基因序列设计的双链RNA的干扰重组质粒pBIN-35S-正向-反向-GFP(干扰),并将其与空白表达质粒pBIN-35S-GFP(空白)分别导入野生型发根农杆菌MSU440中,转化何首乌无菌苗的叶片,诱导生成毛状根。[结果]双链RNA介导的芪合酶Fm STS基因表达沉默,空白对照组二苯乙烯苷含量为2.18 mg/g,是双链RNA干扰组(0.65 mg/g)的3倍多。[结论]应用dsRNA介导的RNAi能有效沉默何首乌Fm STS基因,芪合酶Fm STS基因是何首乌中二苯乙烯苷合成代谢的关键酶基因。     

香蕉枯萎病菌myosin-1基因的功能分析及外施dsRNA介导的抗性评估

 香蕉枯萎病是由尖孢镰刀菌古巴专化型(Fusarium oxysporum f. sp. cubense,Foc)引起的香蕉毁灭性土传病害,其中 4号生理小种(Foc4)能感染几乎所有的香蕉品系,危害最严重。SMART在线软件分析myosin-1基因具有肌球蛋白马达蛋白(myosin motor domain, MMD),肌动蛋白尾结构TH1(myosin tail)和 Src家族同源结构域SH3(src homology domain 3),与禾谷镰刀菌中氰烯菌酯靶标基因myosin-5具高度的蛋白同源性,相似性高达83%。利用Split-marker基因重组技术获得Foc4的myosin-1基因敲除突变体,Δmyosin-1突变株丧失了对氰烯菌酯的敏感性,菌丝生长缓慢,产孢量减少且孢子畸形,对香蕉致病力严重下降,证实myosin-1是氰烯菌酯在Foc4中的作用靶标基因。外施靶向myosin-1体外转录的dsRNA,能抑制菌丝的生长,降低菌丝活性;菌丝膨胀扭曲分枝增多,出现典型的球状结构,与氰烯菌酯处理后的表型一致。在盆栽活体人工接种实验中,体外施用dsRNA可以明显抑制枯萎病外部症状的发展,推迟发病时间,赋予寄主抗性,结果说明体外施用dsRNA可以作为新型杀菌剂防治香蕉枯萎病。综上,myosin-1基因作为氰烯菌酯在Foc4中的靶标基因具有高度的序列保守,在调控菌丝生长发育,产孢以及致病力等方面发挥重要作用,而外施dsRNA具有防治香蕉枯萎菌的巨大潜力。     

RNA干扰的研究进展

RNA干扰(RNA interference,RNAi)现象是1998年在对秀丽线虫的研究中发现的.RNAi利用双链RNA(dsRNA)特异性地降解相应序列的mRNA.从而特异性地阻断相应基因的表达.本文介绍了RNA干扰现象的发现、分子机制、生物学意义及其技术的应用发展.