RNA interference in Nilaparvata lugens (Homoptera: Delphacidae) based on dsRNA ingestion

BACKGROUND: An efficient and convenient RNA interference (RNAi) technique involving double‐stranded RNA (dsRNA) ingestion is useful for gene function studies of non‐model insects. RESULTS: Three dsRNAs targeting different sites within a gene encoding vacuolar ATP synthase subunit E (V‐ATPase‐E, 21E01) were synthesised for RNAi in Nilaparvata lugens. dsRNA was found to be stable in 0.1 g mL^?1 sucrose solution, but unstable in artificial fodder. Therefore, dsRNAs were orally delivered into N. lugens in 0.1 g mL^?1 sucrose solution. RNAi was induced by all three of the dsRNAs at 0.05 µg µL^?1 in N. lugens. Time dynamics analysis of gene silencing indicated that significant suppression of the target gene began as early as 2 days after ingestion of ds2‐21E01 and ds3‐21E01. However, significant repressive effects were recorded up to 10 days after exposure to ds1‐21E01. The maximum reduction in target gene mRNA was observed after 10 days of treatment, with suppression ratios induced by ds1‐21E01, ds2‐21E01 and ds3‐21E01 of 41, 55 and 48% respectively. CONCLUSION: An efficient and convenient RNAi technique involving dsRNA ingestion has been successfully developed for N. lugens. This will be a useful tool for further functional genomic investigation in this organism. Copyright © 2011 Society of Chemical Industry     

Ingested RNA interference for managing the populations of the Colorado potato beetle, Leptinotarsa decemlineata

BACKGROUND: RNA interference (RNAi) is a breakthrough technology for conducting functional genomics studies and also as a potential tool for crop protection against insect pests. The major challenge for efficient pest control using RNAi in the field is the development of efficient and reliable methods for production and delivery of double‐stranded RNA (dsRNA). In this paper, the potential of feeding dsRNA expressed in bacteria or synthesized in vitro to manage populations of Colorado potato beetle, Leptinotarsa decemlineata (Say) (CPB), was investigated. RESULTS: Feeding RNAi successfully triggered the silencing of all five target genes tested and caused significant mortality and reduced body weight gain in the treated beetles. This study provides the first example of an effective RNAi response in insects after feeding dsRNA produced in bacteria. CONCLUSION: These results suggest that the efficient induction of RNAi using bacteria to deliver dsRNA is a possible method for management of CPB. This could be also a promising bioassay approach for genome‐wide screens to identify effective target genes for use as novel RNAi‐based insecticides. Copyright © 2010 Society of Chemical Industry     

象耳豆根结线虫果胶酸裂解酶基因的克隆及其RNAi 效应分析

 从象耳豆根结线虫中首次克隆了果胶酸裂解酶基因Me-pel-1,该基因cDNA 的开放阅读框(ORF)长813 bp,编码270 个氨基酸,具有果胶酸裂解酶第3 家族的4 个保守域特征。ME-PEL-1 与南方根结线虫的果胶酸裂解酶MI-PEL-1 和爪哇根结线虫的果胶酸裂解酶MJ-PEL-1 相似性最高,且系统进化树显示ME-PEL-1 也与它们最为接近。利用RNAi 技术对象耳豆根结线虫2 龄幼虫的Me-pel-1 进行沉默,结果发现Me-pel-1 基因被干扰后,象耳豆根结线虫2 龄幼虫对番茄的侵染率显著降低。该结果表明Me-pel-1 基因在象耳豆根结线虫侵染寄主的过程中发挥重要作用。     

昆虫对双酰胺类杀虫剂抗性机制研究进展

双酰胺类杀虫剂是以昆虫鱼尼丁受体为作用靶标的新型杀虫剂,由于其作用机制独特,对多种鳞翅目害虫具有良好的防治效果而得到广泛应用。但已经有多种害虫的田间种群对该类药剂产生了抗性,甚至导致田间防治失败。本文在综述昆虫对双酰胺类杀虫剂抗性现状的基础上,重点总结了抗性机制方面的最新研究进展,并对今后的研究方向进行了展望,以期为进一步深入研究双酰胺类杀虫剂的抗性机制提供借鉴。     

RNAi在植物寄生线虫功能基因组研究中的应用

最近,南方根结线虫（Meloidogyne incognita）和北方根结线虫（M. hapla）的基因组测序工作已经完成,而大豆孢囊线虫（Heterodera glycines）和马铃薯白线虫(Globodera pallida)的基因组测序工作也在进行之中。截至2008年,Nematode.net数据库中已经有近500 000个表达序列标签ESTs序列和1 200 000个来自30种线虫的基因组序列。如何从众多的序列中筛选出有实际应用价值的信息是线虫学研究面临的一大挑战。对于缺乏有效转化系统的植物寄生线虫来说,RNAi无疑是研究植物寄生线虫基因功能的有力工具。本文介绍了RNAi及其在植物寄生线虫基因功能研究中的应用。     

爪哇根结线虫Mj-1-1基因的克隆、鉴定及RNAi表型分析

根据爪哇根结线虫食道腺内表达的EST序列,结合反式剪接序列SL1,从爪哇根结线虫中克隆了一个假定寄生基因Mj-1-1(KU358725),该基因的c DNA全长为573 bp,包含450 bp的开放阅读框(ORF),编码149个氨基酸。DNA全长为740 bp,包含2个内含子,长度分别为20 bp和111 bp。NCBI BLASTn比对表明,Mj-1-1与南方根结线虫的M.incognita zk1236.5(JQ284068)基因相似性最高,为97%。原位杂交表明Mj-1-1基因在爪哇根结线虫的背食道腺中表达,qRT-PCR结果表明Mj-1-1基因在爪哇根结线虫寄生性3龄幼虫阶段表达量最高。对爪哇根结线虫侵染前2龄幼虫的Mj-1-1基因进行沉默,调查发现,沉默Mj-1-1后,爪哇根结线虫对番茄的侵染力显著下降,表明Mj-1-1对爪哇根结线虫侵染和寄生具有重要的调控作用。     

Genotyping of acetylcholinesterase in insects

To investigate the genotyping criteria for the insect acetylcholinesterase gene (ace), we cloned two types of ace genes in domestic (Bombyx mori) and wild silkworm (Bombyxmandarina) through RT-PCR. The cloned genes were named Bm-ace1, Bm-ace2, Bmm-ace1 and Bmm-ace2, respectively. The ORFs of Bm-ace1 and Bmm-ace1 contained 2025 base pairs, encoding 683 amino acid residues (AA’s). The predicted protein has a molecular weight (MW) of 76.955 kDa and an isoelectric point (pI) of 6.36. The Bm-ace2 and Bm-ace2 genes contained 1917 bp nucleotides, encoding 638 AA’s. The predicted protein has a MW of 71.675 kDa and a pI of 5.49. Both ace1 and ace2 contain signature domains of acetylcholinesterases. Homology analysis of 18 NCBI downloaded insect AChEs peptide sequences and the 4 AChEs deducted in this study revealed that type 1 and type 2 insect AChEs had significant differences. Type 2 sequence is more conserved than type 1. Near the active centers of both types of AChEs, 48 strictly conserved AA’s (336-384) are present, and homology of these two peptide fragments was only 54.16%. Meanwhile, at AA positions 280-297, type 1 and type 2 AChEs both have conserved sequences with the similarity of the two being 52.94%. In type 2 AChEs, a uniquely conserved peptide sequence is found at positions 226-239 (QHLRVRHHQDKPL). We propose to use the above mentioned three conserved regions as criteria for insect acetylcholinesterases gene genotyping. This will benefit the genotyping of other acetylcholinesterase genes and the study of their functions.     

Molecular cytogenetics to characterize mechanisms of gene duplication in pesticide resistance

Recent advances in molecular cytogenetics empower construction of physical maps to illustrate the precise position of genetic loci on the chromosomes. Such maps provide visible information about the position of DNA sequences, including the distribution of repetitive sequences on the chromosomes. This is an important step toward unraveling the genetic mechanisms implicated in chromosomal aberrations (e.g., gene duplication). In response to stress, such as pesticide selection, duplicated genes provide an immediate adaptive advantage to organisms that overcome unfavorable conditions. Although the significance of gene duplication as one of the important events driving genetic diversity has been reported, the precise mechanisms of gene duplication that contribute to pesticide resistance, especially to herbicides, are elusive. With particular reference to pesticide resistance, we discuss the prospects of application of molecular cytogenetic tools to uncover mechanism(s) of gene duplication, and illustrate hypothetical models that predict the evolutionary basis of gene duplication. The cytogenetic basis of duplicated genes, their stability, as well as the magnitude of selection pressure, can determine the dynamics of the genetic locus (loci) conferring pesticide resistance not only at the population level, but also at the individual level. © 2017 Society of Chemical Industry     

siRNA对烟草原生质体中TMV RNA积累的干扰作用研究(英文)

 RNA干扰被认为是转录后基因沉默的一种机制。RNA干扰通过小干扰RNA特异性降解目标mRNA来沉默基因表达。本文以烟草花叶病毒126kD蛋白为靶蛋白,在原生质体水平上研究了小干扰RNA对病毒侵染的干扰和抑制作用。ELISA和Northern杂交的实验结果表明,共转染小干扰RNA和TMV的原生质体内检测到较低的病毒含量。在枯斑寄主上,叶片接种小干扰RNA和TMV共转染原生质体后,与对照叶片相比,仅有很少量的病斑产生。这说明,小干扰RNA能够在原生质体水平对病毒起到干扰和抑制作用。因此认为,烟草原生质体系统有利于快速和定量分析小干扰RNA介导对植物病毒的抑制作用。     

昆虫免疫及五种重要入侵昆虫免疫机制研究进展

我国入侵昆虫种类繁多、为害范围广,在植物检疫与入侵生物学领域备受关注。昆虫免疫是指昆虫识别"自己"和"异己"成分、破坏或排斥外来有害物质,从而维持自身健康并延长寿命的反应机制,在入侵昆虫的传入、定殖和为害过程中发挥着重要作用。本文综述了模式昆虫黑腹果蝇Drosophila melanogaster的免疫机制,并在此基础上分析了5种代表性入侵昆虫红棕象甲Rhynchophorus ferrugineus(Oliver)、烟粉虱Bemisia tabaci(Gennadius)、意大利蜜蜂Apis mellifera lingustica Spinola、日本龟蜡蚧Ceroplastes japonicus Green和舞毒蛾Lymantria dispar(L.)的免疫机制以及该机制在其生物入侵过程中发挥的作用,提出了加强重要入侵昆虫免疫机制研究的展望,一方面要加强研究更多种入侵昆虫的免疫机制,另一方面应将关注点逐步从免疫现象的发现深入至分子机制。