Plant diseases and insect pests threaten the safety of crop production greatly. Traditional methods for pest management are challenged by the problems such as environmental pollution, off-target effects, and resistance of pathogens and insects. New biotechnology-based strategies for pest control are expected to be developed. RNA interference (RNAi) is an endogenous process of gene regulation, which has been widely used to study the gene functions in various organisms. In recent years, RNAi-based pest management has received increasing attention. The effective delivery of the exogenous interference RNA into the targets is a key step in RNAi-mediated plant diseases and pest control. Considerable advances were made on the mechanism of RNAi, and various RNA delivery systems were developed for efficient pest control. Here we review the latest advances on mechanisms and influencing factors of RNA delivery, summarize the strategies of exogenous RNA delivery in RNAi-mediated pest control, and highlight the advantages of nanoparticle complexes in dsRNA delivery.
Animals ; RNA Interference ; Pest Control ; Insecta/genetics* ; RNA, Double-Stranded ; Gene Expression Regulation

OBJECTIVETo construct cyclin D2 (CCND2) short hairpin RNA ( shRNA) plasmid for repressing the expression of CCND2 in human myeloma cell line LP-1,and to detect its effect on the proliferation and apoptosis of LP-1 cell.

METHODSA CCND2 shRNA model was constructed and cloned into plasmid pGensil-2, then the plasmid was transfected into LP-1 cell in vitro. The CCND2 expression cell proliferation, cell cycle and cell apoptosis of the transfected LP-1 cells were studied by RT-PCR, trypanosome staining, flow cytometry and annexin V assay.

RESULTSThe transfection efficiency of LP-1 cell was 34. 2%. In the transfected LP-1 cell CCND2 mRNA expression was reduced significantly, the cell growth was inhibited significantly and the cell cycle was partly arrested in G, phase. The apoptosis rate of the transfected LP-1 cell after 72 h was (25.7+/-4.8)%.

CONCLUSIONThe inhibition of CCND2 in LP-1 cells could inhibit the cell growth and induce cell apoptosis. CCND2 maybe a new therapeutic target.

Apoptosis ; Cell Proliferation ; Cyclin D2 ; Cyclins ; genetics ; Humans ; RNA Interference ; RNA, Double-Stranded ; RNA, Small Interfering ; Transfection ; Tumor Cells, Cultured

OBJECTIVETo study the effect of RNA interference (RNAi) on small heat shock protein (sHSP) Sjp40 of Schistosoma japonicum and its synergistic effect on the expression of SjHSP60, SjHSP70, and SjHSP90 mRNA, and observe the mRNA expression levels of Sjp40, SjHSP60, SjHSP70, and SjHSP90 in different stages of S.japonicum.

METHODSDouble-stranded RNA (dsRNA) of Sjp40 (dsSjp40) and a control dsRNA of green fluorescent protein (dsGFP) were generated by in vitro transcription and transfected into adult worm by immersing the worm in dsRNA solution. The total RNA and proteins were isolated simultaneously from the adult worms using TRIzol reagent 7 days after transfection. The expression levels of Sjp40, SjHSP60, SjHSP70, and SjHSP90 mRNA and the expression level of Sjp40 protein were determined by quantitative real-time PCR (qPCR) and Western blotting, respectively. The mRNA expression of HSPs of S. japonicum in different stages was evaluated by qPCR.

RESULTSCompared with those in the control worms transfected with dsGFP, Sjp40 mRNA level was decreased by 80% in the worms transfected with dsSjp40, and the level of Sjp40 protein showed also a significant decrease. The mRNA expression levels of SjHSP60, SjHSP70, and SjHSP90 did not show an obvious synergism after Sjp40 RNAi. The expression profiles of Sjp40, SjHSP60, SjHSP70, and SjHSP90 showed significant differences in different stages of S. japonicum, and the expression level of Sjp40 mRNA in the egg stage was much higher than that of other HSP genes.

CONCLUSIONdsSjp40-RNAi can induce effective suppression of Sjp40 gene expression at both mRNA and protein levels, but no obvious synergism occurs in the mRNA expressions of SjHSP60, SjHSP70, and SjHSP90.

Animals ; Gene Expression Profiling ; Heat-Shock Proteins, Small ; genetics ; Helminth Proteins ; genetics ; RNA Interference ; RNA, Double-Stranded ; RNA, Messenger ; genetics ; Schistosoma japonicum ; genetics

OBJECTIVETo construct a lentiviral vector of repressor element-1/neuron-restrictive silencer element (RE-1/NRSE) double-stranded RNA (dsRNA).

METHODSThe RE-1/NRSE cDNA containing both sense and antisense oligo DNA fragments of the targeting sequence was synthesized and cloned into the pGC-LV vector. The obtained lentiviral vector containing RE-1/NRSE dsRNA was confirmed by PCR and sequencing. A total of 293T cells were cotransfected with lentiviral vector of L-smNRSE/RE-1, pHelper 1.0, and pHelper 2.0. The titer of virus was measured based on the expression level of green fluorescent protein. The transfection efficiency of green fluorescent protein into rat mesenchymal stem cells was calculated.

RESULTSPCR and DNA sequencing demonstrated that the constructed lentivirus vector of L-smNRSE/RE-1 produced RE-1/NRSE dsRNA.The titer of the concentrated virus was 4x108 TU/m1. The virus was stably transfected into rat mesenchymal stem cells, and the infection efficiency reached 100% when the multiplicity of infection was 80.

CONCLUSIONThe lentivirus vector of RE-1/NRSE dsRNA is successfully constructed.

Animals ; Bone Marrow Cells ; Cells, Cultured ; Genetic Vectors ; Lentivirus ; genetics ; Mesenchymal Stromal Cells ; Plasmids ; genetics ; RNA, Double-Stranded ; genetics ; Rats ; Repressor Proteins ; genetics ; Silencer Elements, Transcriptional ; genetics ; Transfection

RIG-I-like receptors (RLRs) belong to pattern recognition receptors, which perform significant roles in antiviral responses. RLRs can initiate a cascade of signaling transduction that induces the production of type I interferon and activates the interferon signaling pathway, ultimately resulting in antiviral responses. In the course of evolution, viruses have been constantly counteracting host immune systems to facilitate their own survival and replication, and have developed a set of antagonistic strategies. These mainly comprise elusion, disguise and attack strategies to eliminate the activation of RLRs. In virus-infected cells, RLRs recognize viral RNA and then induce antiviral responses. A better understanding of viral antagonistic strategies against RLRs will provide insights into the development of new antiviral medicines. This mini-review concludes that there are three main antagonistic strategies by which RNA viruses can counteract the activation of the RLRs pathway. It aims to provide references and insights for similar studies on viral antagonism in an array of RNA viruses.
DEAD Box Protein 58 ; DEAD-box RNA Helicases ; genetics ; immunology ; Host-Pathogen Interactions ; Humans ; RNA Viruses ; genetics ; immunology ; physiology ; RNA, Double-Stranded ; genetics ; immunology ; RNA, Viral ; genetics ; immunology ; Virus Diseases ; genetics ; immunology ; virology

OBJECTIVETo explore the molecular characteristics and molecular variation of human rotavirus (HRV) strains and to understand the relationship between clinical characteristics and epidemiology of different HRV-VP7 and NSP4.

METHODSDouble-strand RNA of rotavirus extracted from stool samples was used as the template for reverse transcription of gene VP7, which was followed by nested PCR for VP7 typing. NSP4 genes from 22 epidemic strains of human rotavirus isolated in Kunming in 2002 and 2003 were amplified with RT-PCR. Then cDNAs were sequenced and compared with 4 human rotavirus NSP4 (Wa, KUN, AU-1, Hochi)) and 3 animal rotavirus NSP4 (EW, OSU, SA11) available in the GenBank while the epidemic strains of human rotavirus isolated in different areas of China were compared, using the Clustal-mp, DNAssist, MEGA2 software. The G serotype of VP7 was analysed by PCR.

RESULTSSerotype G1 was prevalent in 2002 while serotype G3 was the prevalent in Kumming in 2003. The NSP4 genes from 22 epidemic strains of human rotavirus isolated in Kunming in 2002 and 2003 belonged to Wa with highly conservative amino acid. Samples isolated in the same years but not in the same area shared higher homology. Symptoms associated with heavy diarrhea did not seem to be associated with NSP4 molecular variation (P > 0.05).

CONCLUSIONObvious variations of VP7 typing were seen in the same season, as well as in different areas and years. Due to the stable nature of NSP4, it seem to be a better candidate for vaccine production, than VP7.

China ; DNA, Complementary ; genetics ; DNA, Viral ; Genes, Viral ; Humans ; Polymerase Chain Reaction ; RNA, Double-Stranded ; genetics ; RNA, Viral ; RNA-Directed DNA Polymerase ; Rotavirus ; classification ; genetics ; isolation & purification ; Rotavirus Vaccines ; Sequence Analysis, DNA ; Sequence Homology, Nucleic Acid ; Serotyping

OBJECTIVE: To report the use of Dicer to cleave double-stranded RNA (dsRNAs) into small interference RNAs (D-siRNAs) that can target multiple sites within an mRNA, and to acquire an new method to cure inflammation of the airway and tumor. METHODS: Using RiboMAX Large Scale RNA Production Systems-SP6 and T7 kit were used to transcribe A549 cell COX-2 DNA into RNA (dsRNAs). We mixed dsRNAs with Dicer in the reaction buffer. We recovered siRNAs using RNA Purification Column. RESULTS: Dicer efficiently converted double-stranded RNA of COX-2 into small interference RNAs of 21 approximately 23 bp. CONCLUSION Dicer efficiently converts double-stranded RNA (dsRNA) into small interference RNAs (D-siRNAs of 21 approximately 23 bp).
Base Sequence ; Cell Line, Tumor ; Cyclooxygenase 2 ; genetics ; Humans ; Molecular Sequence Data ; RNA Interference ; RNA, Double-Stranded ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; metabolism ; Ribonuclease III ; metabolism ; Sequence Analysis, DNA

OBJECTIVETo investigate the role of histone acetylation in regulating influenza virus replicative intermediate double-stranded RNA (dsRNA)-induced interleukin-6 (IL-6) expression in A549 cells.

METHODSA549 cells were treated with influenza virus replicative intermediate dsRNA, histone deacetylase (HDAC) inhibitor trichostatin A (TSA), or HADC small interfering RNA (siRNA). The changes in the cellular IL-6 promoter activities were detected by dual-luciferase assay, and IL-6 mRNA and protein expressions in the cells were determined using real-time RT-PCR and ELISA, respectively.

RESULTSInfluenza virus replicative intermediate dsRNA obviously up-regulated IL-6 expression in the cells. HDAC inhibitor TSA significantly enhanced the activity of IL-6 promoter and increased IL-6 mRNA expression in A549 cells, and HDAC3 may play an important role in this process. HDAC inhibitor TSA and DNMT inhibitor DAC showed no synergic effect in regulating IL-6 expression.

CONCLUSIONSInfluenza virus replicative intermediate dsRNA-induced IL-6 expression in A549 cells is regulated by histone acetylation.

Acetylation ; Cell Line, Tumor ; Gene Expression Regulation ; Histone Deacetylase Inhibitors ; pharmacology ; Histones ; metabolism ; Humans ; Interleukin-6 ; metabolism ; Orthomyxoviridae ; genetics ; metabolism ; Promoter Regions, Genetic ; RNA, Double-Stranded ; RNA, Messenger ; genetics ; RNA, Viral

Four dsRNA bands were extracted from Pleurotus ostreatus TD300 by the dsRNA isolation technique with sizes of 8.2 kb, 2.5 kb, 2.1 kb, and 1.1 kb, respectively. Four virus-eliminated methods, i. e. hyphal tips cut (HTC), protoplast regeneration (PR), single spore hybridization (SSH), and frozen and lyophilized (FL), were applied to prepare virus-eliminated strains, and one virus-eliminated strain was selected for each virus-elimination method. The virus-eliminated strains were named as HTC8, PR15, FL01, and SSH11, respectively. There were low concentration of 8.2 kb dsRNA remained in HTC8, as well as low concentration of 8.2 kb and 2.5 kb dsRNA remained in FL01. However, no dsRNA remained in PR15 and SSH11. The hyphal growth rate and laccase activity of the virus-eliminated strains increased, especially HTC8 and PR15, whose hyphal growth rate was higher by 22.73% and 18.18%, and laccase activities higher by 145.83% and 134.38% than that of the original strain, respectively. The conclusion is that hyphal tips cut and protoplast regeneration are suitable to prepare virus-eliminated strains of edible fungi.
Food Microbiology ; Freeze Drying ; Hybridization, Genetic ; Hyphae ; virology ; Pleurotus ; cytology ; genetics ; growth & development ; virology ; Protoplasts ; virology ; RNA, Double-Stranded ; analysis ; isolation & purification ; RNA, Fungal ; analysis ; isolation & purification ; Spores, Fungal ; genetics ; virology ; Viruses ; isolation & purification

OBJECTIVETo silence the expression of alpha-synuclein in MN9D dopaminergic cells using vector mediated RNA interference (RNAi) and examined its effects on cell proliferation and viability.

METHODSWe identified two 19-nucleotide stretches within the coding region of the alpha-synuclein gene and designed three sets of oligonucleotides to generate double-stranded (ds) oligos. The ds oligos were inserted into the pENTR/H1/TO vector and transfected into MN9D dopaminergic cells. alpha-Synuclein expression was detected by RT-PCR, real-time PCR, immunocytochemistry staining and Western blot. In addition, we measured cell proliferation using growth curves and cell viability by 3-(4, 5)-dimethylthiahiazo (-z-y1)-3, 5-di- phenytetrazoliumromide (MTT).

RESULTSThe mRNA and protein levels of alpha-synuclein gene were significantly down-regulated in pSH2/alpha-SYN-transfected cells compared with control MN9D and pSH/CON-transfected MN9D cells, while pSH1/alpha-SYN-transfected cells showed no significant difference. Silencing alpha-synuclein expression does not affect cell proliferation but may decrease cell viability.

CONCLUSIONOur results demonstrated pSH2/alpha-SYN is an effective small interfering RNA (siRNA) sequence and potent silencing of mouse alpha-synuclein expression in MN9D cells by vector-based RNAi, which provides the tools for studying the normal function of alpha-synuclein and examining its role in Parkinson's disease (PD) pathogenesis. alpha-Synuclein may be important for the viability of MN9D cells, and loss of alpha-synuclein may induce cell injury directly or indirectly.

Animals ; Cell Line ; Cell Proliferation ; Cell Survival ; drug effects ; genetics ; Down-Regulation ; drug effects ; genetics ; Gene Silencing ; Genetic Vectors ; genetics ; Hybridomas ; Mice ; Mice, Inbred C57BL ; Nerve Degeneration ; genetics ; metabolism ; Neurons ; drug effects ; metabolism ; pathology ; Oligonucleotides ; genetics ; Parkinson Disease ; genetics ; metabolism ; Plasmids ; genetics ; RNA Interference ; RNA, Double-Stranded ; genetics ; pharmacology ; RNA, Small Interfering ; genetics ; Transfection ; methods ; alpha-Synuclein ; genetics ; metabolism