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Host-induced silencing of MpPar6 confers Myzus persicae resistance in transgenic rape plants

Journal of Integrative Agriculture 2024年第1期23卷 187-194页

作者： Qi Zhang Wenqin Zhan Chao Li Ling Chang Yi Dong Jiang Zhang State Key Laboratory of Biocatalysis and Enzyme Engineering School of Life SciencesHubei UniversityWuhan 430062China Oil Crops Research Institute Chinese Academy of Agricultural SciencesWuhan 430062China Agricultural Genomics Institute at Shenzhen Chinese Academy of Agricultural SciencesShenzhen 518000China

Plant-mediated rna interference(rnai)has emerged as a promising technology for insect control.The green peach aphid,Myzus persicae,feeds on over 400 species of host plants.Brassica napus(rape)is the second most important oilseed crop worldwide.Myzus persicae is highly reproductive and causes severe damage to the rape plants due to its quite flexible life cycle.In this study,we tested the rnai effects of transgenic rape plants on M.persicae.By in vitro feeding M.persicae with artificial diets containing double-stranded rnas(dsrnas)targeting seven aphid genes,we identified a new gene encoding the partitioning-defective protein 6(Par6)as the most potent rnai target.Tissue-and stage-expression analysis of Par6 suggested this gene is highly expressed in the embryo and adult stage of M.persicae.We next generated transgenic rape plants expressing ds Par6 by Agrobacteriummediated transformation and obtained nine independent transgenic lines.Compared to wild-type control plants,transgenic rape lines expressing ds Par6 showed strong resistance to M.persicae.Feeding assays revealed that feeding transgenic rape plants to M.persicae significantly decreased MpPar6 expression and survival rate and impaired fecundity.Furthermore,we showed that the resistance levels to M.persicae are positively correlated with ds Par6 expression levels in transgenic rape plants.Our study demonstrates that transgenic rape plants expressing ds Par6 are efficiently protected from M.persicae.Interfering with the genes involved in embryo development could be the effective rnai targets for controlling aphids and potentially other insect pests.

关键词： oilseed rape pest control aphid double-stranded rna rna interference

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Gene silencing:double-stranded rna mediated mrna degradation and gene inactivation

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Cell Research 2001年第3期11卷 181-186页

作者： TANG WEI, XIAO YAN LUO, VANESSA SANMUELS North Carolina State University, Forest Biotechnology Groap, Raleigh, NC 27695, USA University of North Carolina, Department of Cell and Developmental Biology, ChaPel Hill, NC 27599, USA North Carolina State University Forest Biotechnology Group Raleigh USA Department of Cell and Developmental Biology University of North Carolina Chapel Hill USA

The recent development of gene transfer approaches in plants and animals has revealed that transgene can undergo silencing after integration in the genome. Host genes can also be silenced as a consequence of the presence of a homologous transgene. More and more investigations have demonstrated that double- stranded rna can silence genes by triggering degradation of homologous rna in the cytoplasm and by directing methylation of homologous nuclear DNA sequences. Analyses of Arabidopsis mutants and plant viral suppressors of silencing are unraveling rna-silencing mechanisms and are assessing the role of methy- lation in transcriptional and posttranscriptional gene silencing. This review will focus on double-stranded rna mediated mrna degradation and gene inactivation in plants.

关键词： Gene silencing double-stranded rna methylation homologous rna transgene.

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rna interference and its current application in mammals

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Chinese Medical Journal 2004年第7期117卷 1084-1091页

作者：沈维干 Jiangsu Key Laboratory for Medical Biotechnology

Objective The aim of this review was to assess rna interference (rnai) and its possibility as a potential and powerful tool to develop highly specific double-stranded rna ( dsrna) or small interfering rna (sirna) based gene-silencing therapeutics. Data sources The data used in this review were obtained from the current rnai-related research reports. Study selection dsrna-mediated rnai has recently emerged as a powerful reverse genetic tool to silence, gene expression in multiple organisms. The discovery that synthetic duplexes of 21 nucleotides sirnas trigger gene-specific silencing in mammalian cells has further expanded the utility of rnai in to the mammalian system. Data extraction The currently published papers reporting the discovery and mechanism of rnai phenomena and application of rnai on gene function in mammalian cells were included. Data synthesis Since the recent development of rnai technology in the mammalian system, investigators have used rnai to elucidate gene function, and to develop gene-based therapeutics by delivery exogenous sirna or sirna expressing vector. The general and sequence-specific inhibitory effects of rnai that will be selective, long-term, and systemic to modulate gene targets mentioned in similar reports have caused much concern about its effectiveness in mammals and its eventual use as a therapeutic mordality. Conclusions It is certain that the ability of rnai in mammals to silence specific genes, either when transfected directly as sirnas or when generated from DNA vectors, will undoubtedly accelerate the study of gene function and might also be used as a potentially useful method to develop highly gene-specific therapeutic methods. It is also expected that rnai might one day be used to treat human diseases.

关键词： rna interference post-transcriptional gene silencing double-stranded rna small interfering rna mammalian gene knock down therapeutics

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rna interference targeting ecdysone receptor blocks the larval-pupal transition in Henosepilachna vigintioctopunctata

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Insect Science 2021年第2期28卷 419-429页

作者： Jian-Jian Wu Li-Li Mu Wei-Nan Kang Long-Ji Ze Chen-Hui Shen Lin Jin Ahmad Ali Anjum Guo-Qing Li Education Ministry Key Laboratory of Integrated Management of Crop Diseases and Pests College of Plant ProtectionNanjing Agricultural UniversityNanjingChina

Henosepilachna vigintioctopunctata is a serious insect pest which attacks a large number of nightshades and cucurbits in Asian countries,Brazil and Australia.Prolonged application of traditional pesticides has caused environmental pollution and exerted deleterious effects on human health.Finding new approaches with high target specificity and low environmental contamination has become an urgent task.rna interference(rnai)induced by double-stranded rna(dsrna)is expected to be applicable to managing this pest.Here we evaluated the effects of Escherichia co/Z-expressed dsrnas targeting ecdvsone receptor(EcR)gene via dietary delivery in laboratory and foliar spraying in a greenhouse.The target transcript was successfully knocked down when the 4th-instar larvae had fed on potatofoliage dipped with dsEcR in a laboratory bioassay.Around 85%of the HvEcR rnai larvae remained as prepupae or became abnormal pupae,and failed to emerge into adults.Ingestion of ds£c7?-immersed foliage by the 3rd-instar larvae effectuated a comparable rnai response and brought about more severe defects:all the resultant larvae arrested development,remained as prepupae and finally died.For assay in the greenhouse,a ds£c7?-contained E.coli suspension was directly sprayed to the foliage of greenhouse-growing potato plants and the 3rd-and 4th-instar larvae were transferred to the leaves.High rnai efficacy was obtained and identical rnai phenotypes were observed in treated larvae.In addition,spraying dsEcR reduced leaf damage.Our results indicate a possibility of practical application of dsEcR as an environmentally friendly rna pesticide to control H.vigintioctopunctata larvae.

关键词： bacterially expressed double-stranded rna ecdysis ecdysone receptor Henosepilachna vigintioctopunctata

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rna interference-mediated silencing of a Halloween gene spookier affects nymph performance in the small brown planthopper Laodelphax striatellus

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Insect Science 2015年第2期22卷 191-202页

作者： Shuang Jia Pin-Jun Wan Li-Tao Zhou Li-Li Mu Guo-Qing Li Education Ministry Key Laboratory of Integrated Management of Crop Diseases and Pests College of Plant Protection Nanjing AgriculturalUniversity Nanjing China

Post-embryonic development of insects is highly dependent on ecdysteroid hormone 20-hydroxyecdysone. Halloween gene spookier （spok, cyp307a2） has been documented to be involved in ecdysteroidogenesis in Drosophila melanogaster and Bombyx mori. We describe here the cloning and characterization of Halloween gene spookier （Lsspok, Lscyp307a2） in the small brown planthopper Laodelphax striatellus, a hemipteran insect species. LsSPOK has three insect-conserved P450 motifs, that is, Helix-K, PERF motif and heme-binding domain. Temporal and spatial expression patterns of Lsspok were evaluated by quantitative polymerase chain reaction. Through the fouth- instar and the early fifth-instar stages, Lsspok showed two expression peaks in the second- and fifth-day fourth-instar nymphs, and two troughs in the first-day fourth and fifth in- stars. On day 5 of the fourth-instar nymphs, Lsspok clearly had a high transcript level in the thorax where prothoracic glands were located. Dietary introduction of double-stranded rna of Lsspok in the nymph stage successfully knocked down the target gene, decreased expression level ofecdysone receptor （LsEcR） gene, caused nymphal lethality and delayed development. Ingestion of 20-hydroxyecdysone in Lsspok-dsrna-exposed nymphs did not increase Lsspok expression level, but almost completely rescued the LsEcR mrna level and relieved the negative effects on survival and development. Thus, our data suggest that the ecdysteroidogenic pathway is conserved in insects and LsSPOK is responsible for specific steps in ecdysteroidogenesis in L. striatellus.

关键词： development double-stranded rna ecdysteroidogenesis Laodelphaxstriatellus lethality spookier

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rna interference in Colorado potato beetle(Leptinotarsa decemlineata): A potential strategy for pest control

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Journal of Integrative Agriculture 2020年第2期19卷 428-437页

作者： MA Mei-qi HE Wan-wan XU Shi-jing XU Le-tian ZHANG Jiang State Key Laboratory of Biocatalysis and Enzyme Engineering College of Life SciencesHubei University

Colorado potato beetle(CPB),Leptinotarsa decemlineata,is a notorious destructive pest that mainly feeds on the leaves of potato and several other solanaceous plants.CPB is widely recognized for its adaptation to a remarkable variety of host plants and diverse climates,and its high resistance to insecticides and Bacillus thuringiensis toxins.rna interference(rnai)is a sequence-specific,endogenous gene silencing mechanism evoked by small rna molecules that is used as a robust tool for virus and pest control.rnai has been extensively tested for CPB management by employing various target genes and delivery methods.This article reviews the screening of rnai target genes,efficient rnai delivery systems,and factors affecting rnai efficiency in CPB,which may help understand the mechanisms of rnai and its application in CPB control strategy.

关键词： Colorado potato beetle double-stranded rna pest control rna interference rnai delivery system

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Inhibition of epidermal growth factor receptor expression by rna interference in A549 cells

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Acta Pharmacologica Sinica 2004年第1期 63-69页

作者： Min ZHANG, Xin ZHANG, Chun-xue BAI, Jie CHEN, MinQ WEILung Department of Zhong Shan Hospital, Fudan University, Shanghai 200032, China Department of Medicine, University of Queensland, Brisbane Qld 4032, Australia

AIM: To investigate the biological features of A549 cells in which epidermal growth factor (EGF) receptors expression were suppressed by rna interference (rnai). METHODS: A549 cells were transfected using short small interfering rnas (sirnas) formulated with Lipofectamine 2000. The EGF receptor numbers were determined by Western blotting and flowcytometry. The antiproliferative effects of sequence specific double stranded rna (dsrna) were assessed using cell count, colony assay and scratch assay. The chemosensitivity of transfected cells to cisplatin was measured by MTT. RESULTS: Sequence specific dsrna-EGFR down-regulated EGF receptor expression dramatically. Compared with the control group, dsrna-EGFR reduced the cell number by 85.0 %, decreased the colonies by 63.3 %, inhibited the migration by 87.2 %, and increased the sensitivity of A549 to

关键词： epidermal growth factor receptor rna interference double-stranded rna small interfering rna non-small-cell lung carcinoma

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Molecular cloning and characterization of the putative Halloween gene Phantom from the small brown planthopper Laodelphax striatellus

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Insect Science 2015年第6期22卷 707-718页

作者： Shuang Jia Pin-Jun Wan Guo-Qing Li Education Ministry Key Laboratory of Integrated Management of Crop Diseases and Pests College of Plant Protection Nanjing AgriculturalUniversity Nanjing 210095 China

Ecdysteroid hormone 20-hydroxyecdysone plays fundamental roles in in- sect postembryonic development and reproduction. Several cytochrome P450 mono- oxygenases （CYPs）, encoded by the Halloween genes, have been documented to be involved in ecdysteroidogenesis in representative insects in Diptera, Lepidoptera and Orthoptera. Here the putative Halloween gene Phantom （Phm, cyp306al） from a hemipteran insect species, the small brown planthopper Laodelphax striatellus, was cloned. LsPHM shows five insect conserved P450 motifs, that is, Helix-C, Helix-I, Helix-K, PERF and heme- binding motifs. Temporal and spatial expression patterns of LsPhm were evaluated by quantitative polymerase chain reaction. Through the fourth-instar and the early fifth-instar stages, LsPhm showed two expression peaks in day 2 and days 4-5 fourth-instar nymphs, and three troughs in day 1 and 3 fourth instars and day 1 fifth instars. On day 5 of the fourth-instar nymphs, LsPhm clearly had a high transcript level in the thorax where the prothoracic glands were located. Dietary introduction of double-stranded rna （dsrna） of LsPhm at the nymph stage successfully knocked down the target gene, decreased expres- sion level ofecdysone receptor （LsEcR） gene and caused a higher nymphal mortality rate and delayed development. Ingestion of 20-hydroxyecdysone on LsPhm-dsrna-exposed nymphs did not increase LsPhm expression level, but almost completely rescued the LsEcR mrna level, and relieved the negative effects on survival and development. Thus, our data suggest that the putative LsPhm encodes a ftmctional 25-hydroxylase that catalyzes the biosynthesis of ecdysteroids in L. striatellus.

关键词： double-stranded rna ecdysteroidogenesis Laodelphax striatellus perfor-mance Phantom

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rnai technology for plant protection and its applicatior in wheat

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aBIOTECH 2021年第4期2卷 365-374页

作者： Shaoshuai Liu Shuaifeng Geng Aili Li Yingbo Mao Long Mao Institute of Crop Science Chinese Academy of Agricultural SciencesBeiing 100081China Chinese Academy of Sciences(CAS)Key Laboratory of Insect Developmental and Evolutionary Biology CAS Center for Excellence in Molecular Plant SciencesShanghai Institute of Plant Physiology and EcologyUniversity of CASChinese Academy of SciencesShanghai 200032China Sino-Agro Research Station for Salt Tolerant Crops Yellow River DeltaKenli DistrictDongying 257500ShandongChina

The rnai technology takes advantage of the intrinsic rna interference(rnai)mechanism that exists in nearly all eukaryotes in which target mrnas are degraded or functionally suppressed.Significant progress has been made in recent years where rnai technology is applied to several crops and economic plants for protection against diseases like fungi,pests,and nematode.rnai technology is also applied in controlling pathogen damages in wheat,one of the most important crops in the world.In this review,we first give a brief introduction of the rnai technology and the underneath mechanism.We then review the recent progress of its utilization in crops,particular wheat.Finally,we discuss the existing challenges and prospect future development of this technology in crop protection.

关键词： double-stranded rna Pathogens Pests Nematodes rna interference Small rna Wheat

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Genetic Recoding of Host and Viral rnas by rna Editing

Genetic Recoding of Host and Viral RNAs by RNA Editing

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中国生物化学与分子生物学会第十二届全国会员代表大会暨2...

作者： Charles E.Samuel Department of Molecular Cellular and Developmental BiologyUniversity of California

Genetic recoding by C6-deamination of adenosine(A) to produce inosine(I) in double-stranded (ds) rna structures is an important post-transcriptional mechanism that diversifies the transcriptome and modulates the innate immune interferon response.This rna modification by deamination,known as A-to-I rna editing,is catalyzed by a family of enzymes,adenosine deaminases acting on dsrna(ADARs).The ADAR1 gene codes for two size forms of active deaminase using alternative promoters and alternative splicing: an IFN-inducible p150 ADAR1 that is both cytoplasmic and nuclear and a constitutively expressed p110 ADAR1 that is exclusively nuclear.Suppression of dsrna-triggered induction and action of interferon responses,the cornerstone of innate immunity,is a key and possibly sole function of ADAR1 editing of self(cellular) and non-self(viral) dsrnas.ADAR1 deficiency leads to activation of three cellular sensors of dsrna: the MDA5 RLR receptor,the PKR eIF2α kinase,and the OAS 2’-5’-oligoadenylate synthetase.A-to-I modification of rna is essential for the proper regulation of cellular processes.Dysregulation of A-to-I editing by ADAR1 can have profound consequences,ranging from effects on cell growth and development to autoimmune disorders.ADAR1 regulates sensing of dsrna structures,minimizing auto triggering of innate immune responses under conditions of low concentration of dsrna,yet permitting sensor activation at high concentrations of dsrna as can occur in virus-infected cells.

关键词： adenosine deaminase acting on rna double-stranded rna innate immunity interferon rna editing

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