RNA interference by feeding in Paramecium.

RNA interference can be induced very efficiently by feeding the ciliate Paramecium with bacteria engineered to express double-stranded RNA, opening the possibility of large-scale functional screening in this unicell.     

Targeting genes in living mammals by RNA interference

More than a decade has passed since the discovery of RNA interference (RNAi), an eukaryotic sequence-specific degradation of mRNA induced by complementary double-stranded RNA (dsRNA). RNAi became a common tool for controlled down-regulation of gene expression in cultured cells, as well as in various model organisms. This review summarizes RNAi-based tools for silencing genes in living mammals, which include: (i) transgenic RNAi strategies, where RNAi is triggered by a transgene transmitted through the germline and (ii) approaches, where an RNAi trigger is delivered into an adult animal.     

Induction of RNA interference genes by double-stranded RNA; implications for susceptibility to RNA interference

Gene silencing by RNA interference (RNAi) can be a useful reverse genetics tool in eukaryotes. However, some species appear refractory to RNAi. To study the role of the differential expression of RNAi proteins in RNAi, we isolated partial dicer-2, argonaute-2 translin, vasa intronic gene (VIG) and tudor staphylococcus/micrococcal nuclease (TSN) genes from the tobacco hornworm, Manduca sexta, a well-studied insect model which we have found to be variably sensitive to RNAi. We found that the RNAi gene, translin, was expressed at minimal levels in M. sexta tissue and that there is a specific, dose-dependent upregulation of dicer-2 and argonaute-2 expression in response to injection with dsRNA, but no upregulation of the other genes tested. Upregulation of gene expression was rapid and transient. In order to prolong the upregulation we introduced multiple doses of dsRNA, resulting in multiple peaks of dicer-2 gene expression. Our results have implications for the design of RNAi experiments and may help to explain differences in the sensitivity of eukaryotic organisms to RNAi.     

桔小实蝇成虫诱杀试验初报

目前 ,国内外在监测和防治桔小实蝇Bactrocera (Bactrocera)dorsalis (Hendel)方面 ,主要采用两类引诱剂 ,即化学合成引诱剂和食物诱饵。化学合成引诱剂如 :性引诱剂甲基丁香酚(methyleugenol) ;食物诱饵如 :水解蛋白 (又称蛋白诱饵 ) ,二者可混合有机磷杀虫剂马拉硫磷置于专用诱捕器 (Steine和Mcphail)进行田间诱杀。各种综合防治措施中 ,诱杀桔小实蝇成虫 ,与化学药剂保果相比 ,具有经济、安全、无公害的优点 ,能达到控制田间虫口密度 ,减轻为害 ,因而得到了广泛应用。方法主要是田间悬挂性引诱剂 (methyleugenol)诱捕器诱杀雄虫 ,以及…     

Mobile genes and RNA interference

Current views on the role of RNA interference in controlling the expression and transposition of mobile genes in the eukaryotic genome are considered in connection with a recollected work resulting in the discovery of retrotransposons.     

橘小实蝇复合体分类学研究进展

橘小实蝇复合体Bactroceradorsaliscomplex是以橘小实蝇命名的实蝇类群,已知75种,广泛分布于亚洲、澳洲及环太平洋周边地区。文章概述基于形态学和遗传学特征基础上的橘小实蝇复合体的分类鉴定及系统发育研究进展,介绍近年来分子生物学技术在橘小实蝇复合体分类及系统发育研究中的应用情况,展望系统发育和行为学研究是橘小实蝇复合体未来研究的重点领域。     

Silencing of essential genes by RNA interference in Haemonchus contortus

In this study we assessed three technologies for silencing gene expression by RNA interference (RNAi) in the sheep parasitic nematode Haemonchus contortus. We chose as targets five genes that are essential in Caenorhabditis elegans (mitr-1, pat-12, vha-19, glf-1 and noah-1), orthologues of which are present and expressed in H. contortus, plus four genes previously tested by RNAi in H. contortus (ubiquitin, tubulin, paramyosin, tropomyosin). To introduce double-stranded RNA (dsRNA) into the nematodes we tested (1) feeding free-living stages of H. contortus with Escherichia coli that express dsRNA targetting the test genes; (2) electroporation of dsRNA into H. contortus eggs or larvae; and (3) soaking adult H. contortus in dsRNA. For each gene tested we observed reduced levels of mRNA in the treated nematodes, except for some electroporation conditions. We did not observe any phenotypic changes in the worms in the electroporation or dsRNA soaking experiments. The feeding method, however, elicited observable changes in the development and viability of larvae for five of the eight genes tested, including the 'essential' genes, Hc-pat-12, Hc-vha-19 and Hc-glf-1. We recommend the E. coli feeding method for RNAi in H. contortus and provide recommendations for future research directions for RNAi in this species.     

RNA interference of two acetylcholinesterase genes in Plutella xylostella reveals their different functions

Acetylcholinesterase (AChE, EC 3.1.1.7) is an important enzyme with a typical function of degrading the neurotransmitter acetylcholine. Although two ace genes were reported in Plutella xylostella, their function differences remain largely unknown. The chemically synthesized siRNAs (si‐Pxace1 and si‐Pxace2) were injected into the second instar larvae to knock down Pxace1 and Pxace2, either respectively or simultaneously. The mRNA abundance of Pxace1 and Pxace2 was significantly reduced to 7–33.5% of the control levels at 72 h after siRNA injection. The AChE activities were significantly decreased at 96 h after treatment. Silencing of Pxace1 or Pxace2 resulted in mortality of 33.9 and 22.9%, respectively. The survivors in siRNA‐treated groups had apparent growth inhibition such as reduction in larvae weights and lengths, malformation and motor retardation. Knockdown of Pxace1 apparently affected more on larvae growth than that of Pxace2, suggesting that Pxace1 had more important roles than Pxace2. Both Pxace1 and Pxace2 genes might have atypical functions in regulating larvae growth and motor ability.     

Influence of various stressors on the expression of core genes of the small interfering RNA pathway in the oriental fruit fly,Bactrocera dorsalis

RNA interference (RNAi)‐based technology has emerged as a potential tool for controlling insect pests, however, previous studies found that the efficiency of RNAi in Bactrocera dorsalis was variable. In nature, insects often meet various challenges, such as pathogen infections, extreme temperatures, lack of nutrition and heavy metals. To better understand the association of the stressors with efficiency of RNAi, in the current study we tested the expression of three core genes, dicer2 (Bddcr2), r2d2 (Bdr2d2) and argonaute2 (Bdago2), of the small interfering RNA (siRNA) pathway of B. dorsalis upon various stressors. Our results showed that all three genes were upregulated by the infection of invertebrate iridescent virus 6, which suggested a function of the siRNA pathway against viral infection. The loading of FeCl₃ could also increase the expression of Bddcr2. The treatments of Escherichia coli, extremely high (40°C) and low (0°C) temperatures, as well as starvation, could negatively influence the expression of Bddcr2 and/or Bdago2. In total, our results showed that various stressors could influence the expression of core components of B. dorsalis siRNA pathway. This highlights further speculation on the RNAi efficiency upon these stressors. Considering the complexity and variation of RNAi efficiency in different conditions, these results provide initial aspects in possible environmental stressors to influence the activity of the siRNA pathway, but the real impact of RNAi efficiency posed by these stressors requires further studies.     

Coexpression of Escherichia coli RNase III in silkworm cells improves the efficiency of RNA interference induced by long hairpin dsRNAs

Abstract Long hairpin dsRNA transcribed from chromosomal DNA can induce RNA interference in Bombyx mori cells, although its gene silencing efficiency is lower than that of exogenously introduced double‐stranded RNAs (dsRNAs). To solve this problem, we monitored the nuclear cytoplasmic translocation of the transcribed hairpin dsRNA and analyzed the processing efficiency into mature small interfering RNA (siRNA). Northern blot analysis revealed that the transcribed hairpin dsRNAs were spliced and transported into the cytoplasm, but were not effectively diced into siRNAs. Interestingly, RNAi with hairpin dsRNAs from genome‐integrated IR transgene was stimulated by the coexpression of Escherichia coli RNase III, although this exogenous enzyme seemed to bring about nonspecific cleavage of cellular mRNA.     

橘小实蝇植物源引诱活性物质的生物测定

植物源引诱物质可显著提高橘小实蝇Bactrocera dorsalis(Hendel)蛋白饵剂的应用效果,本文测试了柑橘、番木瓜、芒果、番石榴、杨桃的叶片浸提物、果实以及已知植物次生物质对橘小实蝇的引诱效果。植物浸提物的生物测定结果表明,杨桃的二氯甲烷浸提物的引诱效果最佳,平均引诱率为20.83%,对于不同的萃取剂而言,二氯甲烷、乙醇浸提物效果明显优于石油醚,三者浸提的平均引诱率分别为15.67%、15.17%和10.50%;对于不同寄主植物而言,柑橘浸提物的效果最佳,3种有机溶剂浸提物的平均引诱率为18.33%,其中石油醚、二氯甲烷和乙醇浸提物的引诱率分别为20.00%、15.83%和19.17%,其他植物浸提物的引诱效果为杨桃>番木瓜>芒果>番石榴,3种有机溶剂浸提物的平均引诱率分别为17.22%、11.67%、11.11%和10.56%。寄主果实的生物测定结果表明,柑橘的平均引诱率为65.83%,其中雌虫和雄虫的引诱率分别为61.67%和70.00%,明显高于杨桃、番石榴和番木瓜,三者的引诱率分别为31.67%、31.67%和21.67%,与柑橘引诱率差异显著(P<0.05)。已知植物次生物质的生物测定结果表明,甲基丁香酚的引诱效果最佳,平均引诱率为45.00%,其中雄虫和雌虫的引诱率分别为86.67%和3.33%;乙酸乙酯和番石榴香精的引诱效果次之,平均引诱率分别为32.50%和28.33%,其中雌虫的引诱率均为36.67%;杨桃香精和柠檬酸的引诱效果较差,平均引诱率分别为25.00%和26.67%。     

橘小实蝇快速检疫鉴定方法

采用形态学观察与PCR技术相结合的方法,针对台湾水果中截获的可疑橘小实蝇Bactroceradorsalis(Hendel)幼虫和蛹进行快速鉴定。采用异硫氰酸胍方法快速提取昆虫基因组DNA,并根据特异性引物,采用PCR技术从截获的可疑橘小实蝇幼虫和蛹样本中均扩增得到224 bp的特异性条带,经测序比较发现,扩增产物序列与橘小实蝇目的基因序列完全相同,由此证明可疑样本为橘小实蝇的幼虫和蛹。该方法解决了橘小实蝇幼虫和蛹等未成熟虫态的快速检疫难题,大大缩短了检测周期,值得口岸检验检疫借鉴应用。     

多杀菌素和荧光桃红B对橘小实蝇取食的影响

40mg/kg多杀菌素(spinosad)或1000、3000mg/kg荧光桃红B(Phloxine-B)均不影响3日龄橘小实蝇雌Bactrocera dorsalis(Hendel)、雄虫对蔗糖的取食量(P>0.05);与蒸馏水对照和马拉硫磷对照相比,40mg/kg多杀菌素或1000mg/kg荧光桃红B对雌、雄虫吐食率也无显著影响(P>0.05),但3000mg/kg荧光桃红B对雌、雄虫吐食率有极显著影响(P<0.01)。40mg/kg多杀菌素或1000mg/kg荧光桃红B对6日龄橘小实蝇雌、雄虫对蔗糖的取食量与蒸馏水对照无差异(P>0.05);3000mg/kg荧光桃红B对雌虫的取食量与蒸馏水对照有显著差异(P<0.05),与马拉硫磷对照无差异(P>0.05),对雄虫的取食量与蒸馏水对照无显著差异(P>0.05),与马拉硫磷对照有显著差异(P<0.05)。与蒸馏水、马拉硫磷相对照,40mg/kg多杀菌素或1000mg/kg荧光桃红B对雌、雄虫的吐食率均无显著影响(P>0.05),而3000mg/kg的荧光桃红B对雌、雄虫的吐食率均有极显著影响(P<0.01)。     

利用微卫星标记初步分析橘小实蝇4个地理种群的遗传多态性

橘小实蝇Bactrocera dorsalis(Bactrocera)(Hendel)是我国重要的检疫性害虫,对果蔬生产及其国际贸易造成很大影响。作者运用微卫星分子标记技术,用6对微卫星引物对采自我国福建、广东、云南3个地区及邻近国越南橘小实蝇种群间的遗传关系进行了初步分析,结果显示上述4个橘小实蝇地理种群间存在一定的遗传差异,地理隔离是造成这一差异的主要原因。     

橘小实蝇、瓜实蝇和南亚果实蝇人工饲料的优化

在温度25²8℃、相对湿度70%28℃、相对湿度70%⁷5%和光照周期L∶D=14∶10条件下进行了人工大量饲养橘小实蝇Bactrocera dorsalis(Hendel)和瓜实蝇B.cucurbitae(Coquillett)成虫人工饲料配方的筛选试验。结果显示,1∶2重量比例混合的蔗糖和啤酒酵母是饲养这2种果实蝇的最佳成虫人工饲料,用其饲养的单雌产卵量、产卵期和孵化率分别为424.1675%和光照周期L∶D=14∶10条件下进行了人工大量饲养橘小实蝇Bactrocera dorsalis(Hendel)和瓜实蝇B.cucurbitae(Coquillett)成虫人工饲料配方的筛选试验。结果显示,1∶2重量比例混合的蔗糖和啤酒酵母是饲养这2种果实蝇的最佳成虫人工饲料,用其饲养的单雌产卵量、产卵期和孵化率分别为424.16⁴45.75粒,30.90445.75粒,30.90³1.87 d,74.60%31.87 d,74.60%⁷5.40%。同时,对18种由不同配方配制成的幼虫人工饲料饲养橘小实蝇、瓜实蝇和南亚果实蝇B.tau(Walker)的效果进行了比较。结果表明,玉米和麦麸作为饲料的介质优于麦片和麦麸。橘小实蝇幼虫人工饲料的优化配方为:玉米+麦麸(125 g+25 g),蔗糖25 g,啤酒酵母25 g,对羟基苯甲酸甲酯0.9 g,1 mol/L盐酸4 mL,纸巾4 g,自来水300 mL;用其幼虫人工饲料饲养该虫的生物学参数包括子代孵化率、化蛹率、羽化率和平均蛹重分别为81.17%±0.05%,96.41%±0.02%,94.85%±0.01%与(19.40±0.08)mg。而瓜实蝇和南亚果实蝇幼虫人工饲料的优化配方为:玉米+麦麸(100 g+50 g),蔗糖30 g,啤酒酵母25 g,以及一定量的其他组分(同上);用其饲料饲养这2种果实蝇的相关参数:子代孵化率、化蛹率、羽化率和平均蛹重分别为78.50%±0.04%与76.96%±0.12%,95.73%±0.03%与94.69%±0.02%,94.57%±0.02%与95.82%±0.03%,(18.62±0.23)mg与(22.83±1.38)mg。试验证实,优化后的成、幼虫人工饲料具有饲养效果好、方法简便,配方材料来源广泛和价格低廉等优点,可用于室内人工大量饲养上述3种果实蝇属害虫。     

Tobamovirus-resistant tobacco generated by RNA interference directed against host genes

Two homologous Nicotiana tabacum genes NtTOM1 and NtTOM3 have been identified. These genes encode polypeptides with amino acid sequence similarity to Arabidopsis thaliana TOM1 and TOM3, which function in parallel to support tobamovirus multiplication. Simultaneous RNA interference against NtTOM1 and NtTOM3 in N. tabacum resulted in nearly complete inhibition of the multiplication of Tomato mosaic virus and other tobamoviruses, but did not affect plant growth or the ability of Cucumber mosaic virus to multiply. As TOM1 and TOM3 homologues are present in a variety of plant species, their inhibition via RNA interference should constitute a useful method for generating tobamovirus-resistant plants.     

长尾潜蝇茧蜂对橘小实蝇幼虫的寄生效能

以橘小实蝇幼虫为寄主,在(25±1)℃,RH(70±10)%,光照周期14L∶10D的条件下,研究了在不同密度的寄生蜂与寄主条件下的长尾潜蝇茧蜂寄生效能。结果表明:不同密度的橘小实蝇幼虫对长尾潜蝇茧蜂的寄生作用有明显的影响,其寄生效应符合功能反应模型HollingⅡ型,方程为Na=0.7291No/(1 0.02362No)。长尾潜蝇茧蜂自身密度对寄生作用也存在干扰效应,用Hassell-Varley模型可表示为a=0.1993P-0.2966。