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Characterization of carotenoid biosynthetic pathway genes in the pea aphid(Acyrthosiphon pisum)revealed by heterologous complementation and RNA interference assays

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Insect Science 2022年第3期29卷 645-656页

作者： Bi-Yue Ding Xiu-Cheng Xie Feng Shang Guy Smagghe Jin-Zhi Niu Jin-Jun Wang Key Laboratory of Entomology and Pest Control Engineering College of Plant ProtectionSouthwest UniversityChongqingChina International Joint Laboratory of China-Belgium on Sustainable Crop Pest ControlAcademy of Agricultural SciencesSouthwest UniversityChongqingChina Department of Plants and Crops Faculty of Bioscience EngineeringGhent UniversityGhentBelgium

Carotenoids are involved in many essential physiological functions and are produced from geranylgeranyl pyrophosphate through synthase,desaturase,and cyclase activities.In the pea aphid(Acyrthosiphon pisum),the duplication of carotenoid biosynthetic genes,including carotenoid synthases/cyclases(ApCscA-C)and desaturases(ApCdeA-D),through horizontal gene transfer from fungi has been detected,and ApCdeB has known dehydrogenation functions.However,whether other genes contribute to aphid carotenoid biosynthesis,and its specific regulatory pathway,remains unclear.In the current study,functional analyses of seven genes were performed using heterologous complementation and RNA interference assays.The bifunctional enzymes ApCscA-C were responsible for the synthase of phytoene,and ApCscC may also have a cyclase activity.ApCdeA,ApCdeC,and ApCdeD had diverse dehydrogenation functions.ApCdeA catalyzed the enzymatic conversion of phytoene to neurosporene(three-step product),ApCdeC catalyzed the enzymatic conversion of phytoene to ζ-carotene(two-step product),and ApCdeD catalyzed the enzymatic conversion of phytoene to lycopene(four-step product).Silencing of ApCscs reduced the expression levels of ApCdes,and silencing these carotenoid biosynthetic genes reduced the α-,β-,and γ-carotene levels,as well as the total carotenoid level.The results suggest that these genes were activated and led to carotenoid biosynthesis in the pea aphid.

关键词： carotenoid biosynthesis heterologous complementary pea aphid RNA interference

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Control of a sap-sucking insect pest by plastidmediated RNA interference

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Molecular Plant 2022年第7期15卷 1176-1191页

作者： Yi Dong Mengting Wu Qi Zhang Jinqiu Fu F.Vanessa Loiacono Yong Yang Zican Wang Shengchun Li Ling Chang Ralph Bock Jiang Zhang State Key Laboratory of Biocatalysis and Enzyme Engineering Hubei Hongshan LaboratorySchool of Life SciencesHubei UniversityWuhan 430062China Max-Planck-Institut fur Molekulare Pflanzenphysiologie Am Mühlenberg 1D-14476 Potsdam-GolmGermany

Expression of double-stranded rnas in plastids offers great potential for the efficient control of chewing insects.However,many insect pests do not consume plant tissue but rather feed on the host plant by sucking sap from the vascular system.Whether or not plastid-mediated RNA interference(rnai)can be employed to control sap-sucking insects is unknown.Here,we show that five species of sap-sucking hemipteran insects acquire plastid rna upon feeding on plants.We generated both nuclear transgenic and transplastomic tobacco plants expressing double-stranded rnas targeting the MpDhc64C gene,a newly identified efficient target gene of rnai whose silencing causes lethality to the green peach aphid Myzus persicae.In a whole-plant bioassay,transplastomic plants exhibited significant resistance to aphids,as evidenced by reduced insect survival,impaired fecundity,and decreased weight of survivors.The protective effect was comparable with that conferred by the best-performing nuclear transgenic plants.We found that the proportion of aphids on mature leaves of transplastomic plants was significantly lower compared with that of nuclear transgenic plants.When aphids were allowed to infest.only the mature leaves,transplastomic plants grew significantly faster and were overall better protected from the pest compared with nuclear transgenic plants.When monitored by electrical-penetration-graph analyses and aphid avoidance response experiments,the insects displayed remarkable alterations in feeding behavior,which was different in nuclear transgenic and transplastomic plants,likely reflecting specific avoidance strategies to toxic rna molecules.Taken together,our study demonstrates that plastid-mediated rnai provides an efficient strategy for controlling at least some sap-sucking insect pests,even though there is most likely no or only very little chloroplast rna in the sap.

关键词： plastid transformation RNA interference pest control sap-sucking insect aphid hemiptera

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Identification of Rab family genes and functional analyses of LmRab5 and LmRab11A in the development and RNA interference of Locusta migratoria

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Insect Science 2022年第2期29卷 320-332页

作者： Mureed Abbas Yun-He Fan Xue-Kai Shi Lu Gao Yan-Li Wang Tao Li Anastasia M.W.Cooper Kristopher Silver Kun Yan Zhu Jian-Zhen Zhang Institute of Applied Biology Shanxi UniversityTaiyuanChina College of Life Science Shanxi UniversityTaiyuanChina Department of Entomology Kansas State UniversityManhattanKansasUSA

Rab proteins constitute the largest family of small GTPases,which play pivotal roles in intracellular membrane trafficking in all eukaryotes.A number of Rab genes have been identified in eukaryotes;however,very little information about these genes has been reported in insects.In the current study,for the first time we identified and characterized 27 Rab family genes from Locusta migratoria.Phylogenetic analysis and comparison of domain architecture indicated that Rab family genes are highly conserved among insect species.Tissue-dependent expression profiles indicated that expression of Rab genes was highest in the ovary,except for LmRab3,which was most highly expressed in hemolymph.The biological function of each Rab gene was investigated using RNA interference(rnai).Double-stranded rna targeting each Rab gene was injected into the hemocoel of nymphs and revealed that suppression of two Rab genes(LmRab5 and LmRab11A)caused 100%mortality.In addition,nymphs injected with dsLmRab5 exhibited severe phenotypic defects in the gastric caeca and midgut,while dsLmRab11A arrested the molting process.We then applied the rnai of rnai technique to test if silencing either of these two genes would affect the suppression of the lethal giant larvae(LmLgl)reporter gene and found that suppression of LmRab5 diminished the rnai efficiency of LmLgl,whereas suppression of LmRab11A enhanced rnai efficiency of LmLgl.These results indicate that Rab genes contribute differently to rnai efficiency in different tissues.Our study provides a foundation for further functional investigations of Rab genes and their contributions to rnai efficiency in L.migratoria.

关键词： gene expression patterns Locusta migratoria Rab family genes RNA interference rnai efficiency

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RNA interference of a trehalose-6-phosphate synthase gene reveals its roles in the biosynthesis of chitin and lipids in Heortia vitessoides(Lepidoptera:Crambidae)

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Insect Science 2020年第2期27卷 212-223页

作者： Jing-Xiang Chen Zi-Hao Lyu Chun-Yan Wang Jie Cheng Tong Lin College of Forestry and Landscape Architecture South China Agricultural UniversityGuangzhouChina

Trehalose 6-phosphate synthase(TPS),an enzyme that hydrolyzes two glucose molecules to yield trchalose,plays a pivotal role in various physiological processes.In this study,we cloned the trehalose-6-phosphate synthase gene(HvTPS)and investigated its expression patterns in various tssues and d:velopmental stages in Heortia vitessoides Moore(Lepidoptera:Crambidac).HvTPS was highly expressed in the fat body and after pupation or before molting.We knocked down TPS in H.vitessoides by RNA interference and found that 3.0μg of dsHvTPS resulted in optimal interference at 24 h and 36 h post-injection and caused a sharp decline in the survival rate during the 5th instar larval-pupal stage and obviously abnormal or lethal phenotypes.Additionally.compared to the controls,TPS activity and trehalose contents were significantly lower and the glucose content was significantly higher 24 h or 36 h after injection with 3.0μg of dsHIvTPS.Furthermore,the silencing of HvTPS suppressed the cxpression of six key genecs in the chitin biosynthesis pathway and one key gene related to lipid catabolism.The expression levels of two genes associated with lipid biosynthesis were upregulated.These results strongly suggest that HvTPS is essential for the normal growth and development of H.vitessoides and provide a reference for further studies of the utility of key genes involved in chitin and lipid biosynthesis for controlling insect development.

关键词： chitin biosynthesis Heortia vitessoides Moore lipid biosynthesis metamor-phosis RNA interference trehalose-6-phosphate synthase

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Effect of RNA interference Targeting STAT3 Gene Combined with Ultrasonic Irradiation and SonoVue Microbubbles on Proliferation and Apoptosis in Keratinocytes of Psoriatic Lesions

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Chinese Medical Journal 2018年第17期131卷 2097-2104页

作者： Li-Wei Ran Hao Wang Dong Lan Hong-Xia Jia Si-Si Yu Department of DermatolocIy Jinqxi Campus Beijing Chaoyang Hospital Capital Medical University Beijing 100043 China Department of Dermatology The Second Affiliated Hospital of Xi'an Jiaotong University Xi'an Shaanxi 710004 China

Background： Signal transducer and activator of transcription 3 （STAT3） was strongly expressed and activated in psoriatic keratinocytes （KCs） and correlated with the severity of psoriasis. The study aimed to investigate the effects of STAT3 small interfering rna （sirna） combined with ultrasonic irradiation and SonoVue microbubbles on the proliferation and apoptosis in KCs of psoriatic lesions and the relative mechanisms. Methods： Psoriatic KCs were transfected under four experimental conditions：（1） STA T3 sirna carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles （LUS group）; （2） STA T3 sirna only carried by Lipofectamine 3000 （L group）; （3） the negative control ofsirna carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles （sirna-NC）; （4） not treated as Blank. Cell Counting Kit-8 assay was used to evaluate the cell proliferation. Cell cycle analysis was detected with cycle test Plus DNA reagent kit associated with flow cytometer. FITC Annexin V apoptosis detection kit associated with flow cytometer was applied for apoptosis analysis. Fluo calcium indicator associated with flow cytometer was used to analyze intracellular free calcium concentration （[Ca^2＋）. The expressions of cyclin D1 and Bcl-xL were detected both at the mrna level by real-time reverse transcription-polymerase chain reaction （RT-PCR） and at the protein level by Western blotting. The obtained data were statistically evaluated by two-way analysis of variance. Results： STAT3 sirna inhibited the growth of KCs in a time-dependent manner showing the highest proliferation inhibition in LUS group with proliferation ratio of 45.38% ± 5.85% at 72h （P 〈 0.05 vs. L group, sirna-NC, or Blank）. STAT3 sirna induced an altered cell cycle distribution of KCs showing the highest increases in G2/M-phase population up to 18.06% ± 0.36% in LUS group （P 〈 0.05 vs. L group, sirna-NC, or Blank）. ST,4 T3 sirna induced late apoptosis of KCs with the highest late apoptosis percentage of 22.87% ± 1

关键词： Apoptosis Cell Proliferation Keratinocytes Microbubbles Psoriasis RNA interference Small Interfering rna STAT3 Transcription Factor

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Metal-organic frameworks nanoswitch:Toward photo-controllable endo/lysosomal rupture and release for enhanced cancer RNA interference

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Nano Research 2020年第1期13卷 238-245页

作者： Gan Lin Yang Zhang Long Zhang Junqing Wang Ye Tian Wen Cai Shangui Tang Chengchao Chu JiaJing Zhou Peng Mi Xiaoyuan Chen Gang Liu State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics&Center for Molecular Imaging and Translational Medicine School of Public HealthXiamen UniversityXiamen361102China The Department of Chemical Engineering The University of MelbourneParkvilleVictoria3010Australia School of Chemical and Biomedical Engineering Nanyang Technological UniversitySingapore637457Singapore State Key Laboratory of Biotherapy and Cancer Center West China HospitalCollaborative Innovation Center for BiotherapySichuan UniversityChengdu610041China Laboratory of Molecular Imaging and Nanomedicine National Institute of Biomedical Imaging and BioengineeringNational Institutes of HealthBethesdaMD20892USA

Endo/lysosomal escape and gene release are two critical bottlenecks in gene delivery.Herein,a novel photo-controllable metal-organic frameworks(MOFs)nanoswitch is rationally designed for enhancing small interfering rna(sirna)delivery.One single laser triggers the“off-to-on”switching of MOFs nanocomplexes,inducing significant sirna release accompanied by rapid MOFs dissociation into protonatable 2-methylimidazalo and osmotic rupturing Zn2+ions,which cooperatively contribute to remarkable endo/lysosomal rupture(∼90%).The simultaneous endo/lysosomal rupture and release enable a high spatio-temporal control on RNA interference for effective cancer therapy.Notably,the“off-to-on”switching also activates fluorescence recovery for real-time monitoring sirna delivery.The nanoswitch could easily be extended to deliver other therapeutic agents(e.g.,DNA,protein,anticancer drug)for overcoming endo/lysosomal entrapment.

关键词： metal-organic frameworks endo/lysosomal rupture and release photo-controllable nanoswitch RNA interference cancer therapy

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Characterization of the essential role of bone morphogenetic protein 9 (BMP9) in osteogenic differentiation of mesenchymal stem cells (MSCs) through RNA interference

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Genes & Diseases 2018年第2期5卷 172-184页

作者： Shujuan Yan Ruyi Zhang Ke Wu Jing Cui Shifeng Huang Xiaojuan Ji Liping An Chengfu Yuan Cheng Gong Linghuan Zhang Wei Liu Yixiao Feng Bo Zhang Zhengyu Dai Yi Shen Xi Wang Wenping Luo Bo Liu Rex C.Haydon Michael J.Lee Russell R.Reid Jennifer Moriatis Wolf Qiong Shi Hue H.Luu Tong-Chuan He Yaguang Weng Ministry of Education Key Laboratory of Diagnostic Medicine and School of Laboratory Medicine Chongqing Medical UniversityChongqing 400016China Molecular Oncology Laboratory Department of Orthopaedic Surgery and Rehabilitation MedicineThe University of Chicago Medical CenterChicagoIL 60637USA The School of Pharmacy and the Affiliated Hospitals of Chongqing Medical University Chongqing 400016China Key Laboratory of Orthopaedic Surgery of Gansu Province and the Department of Orthopaedic Surgery The Second Hospital of Lanzhou UniversityLanzhou730030China Department of Biochemistry and Molecular Biology China Three Gorges University School of MedicineYichang 443002China Department of Surgery The Affiliated Zhongnan Hospital of Wuhan UniversityWuhan 430071China Department of Orthopaedic Surgery Chongqing Hospital of Traditional Chinese MedicineChongqing 400021China Department of Orthopaedic Surgery Xiangya Second Hospital of Central South UniversityChangsha 410011China Department of Surgery Laboratory of Craniofacial Biology and DevelopmentSection of Plastic SurgeryThe University of Chicago Medical CenterChicagoIL 60637USA

Mesenchymal stem cells(MSCs)are multipotent stem cells and capable of differentiating into multiple cell types including osteoblastic,chondrogenic and adipogenic lineages.We previously identified BMP9 as one of the most potent BMPs that induce osteoblastic differentiation of MSCs although exact molecular mechanism through which BMP9 regulates osteogenic differentiation remains to be fully understood.Here,we seek to develop a recombinant adenovirus system to optimally silence mouse BMP9 and then characterize the important role of BMP9 in osteogenic differentiation of MSCs.Using two different sirna bioinformatic prediction programs,we design five sirnas targeting mouse BMP9(or simB9),which are expressed under the control of the converging H1 and U6 promoters in recombinant adenovirus vectors.We demonstrate that two of the five sirnas,simB9-4 and simB9-7,exhibit the highest efficiency on silencing exogenous mouse BMP9 in MSCs.Furthermore,simB9-4 and simB9-7 act synergistically in inhibiting BMP9-induced expression of osteogenic markers,matrix mineralization and ectopic bone formation from MSCs.Thus,our findings demonstrate the important role of BMP9 in osteogenic differentiation of MSCs.The characterized simB9 sirnas may be used as an important tool to investigate the molecular mechanism behind BMP9 osteogenic signaling.Our results also indicate that recombinant adenovirus-mediated expression of sirnas is efficient and sustained,and thus may be used as an effective delivery vehicle of sirna therapeutics.

关键词： BMP9 Bone formation Mesenchymal stem cells Osteogenic differentiation RNA interference Recombinant adenovirus sirna

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RNA interference targeting ω-secalin genes differentially affects the processing quality in a wheat T1BL·1RS translocation line

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The Crop Journal 2021年第2期9卷 456-464页

作者： Shuo Zhou Cuimian Zhang He Zhao Mengyu Lyu Fushuang Dong Yongwei Liu Fan Yang Haibo Wang Jianfang Chai Hebei Province Plant Genetic Engineering Center Institute of Genetics and PhysiologyHebei Academy of Agriculture and Forestry SciencesShijiazhuang 050051HebeiChina

Wheat-rye T1BL·1RS translocation lines are widely used,especially in China,but their processing quality is generally poor.An interfering expression vector targeting theω-secalin genes was constructed with the 1Bx7 seed-specific promoter.Biolistic-mediated genetic transformation of the wheat cultivar KN199 carrying the T1BL·1RS translocation generated 10 transgenic lines.Two representative transgenic lines,8-2 and 13-7,were selected for analysis.Compared with the control,the two transformants showed an up to 4.5-fold decrease in totalω-secalins and various levels of decrease inω-gliadins,γ-gliadins,and low-molecular-weight glutenins.A decrease in high molecular weight(HMW)glutenin 1Bx7 was detected only in 8-2,owing possibly to promoter methylation.Increased levels ofα-gliadins were observed in both transformants,but increased levels of HMW glutenins were observed only in 13-7.Line 13-7 showed increases in gluten index,Zeleny sedimentation value,stabilization time,and maximum resistance.Its bread volume was 849.6 mL,an 11.9%increase over that of the control.Line 8-2 showed decreases in these parameters,but its total cake-making quality score was 88,an 17.3%increase over that of the control.The study demonstrates that the same rnai construct may produce different effects on wheat processing quality and highlights the influence of the vector promoter in RNA interference.

关键词： T1BL·1RS Quality improvement RNA interference Secalin Triticum aestivum

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Prediction of effective RNA interference targets and pathway-related genes in lepidopteran insects by rna sequencing analysis

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Insect Science 2018年第3期25卷 356-367页

作者： Ruo-Bing Guan Hai-Chao Li Xue-Xia Miao Key Laboratory of Insect Developmental and Evolutionary Biology Institute of Plant Physiology and Ecology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200032 China

When using RNA interference （rnai） to study gene functions in Lepidoptera insects, we discovered that some genes could not be suppressed; instead, their expression levels could be up-regulated by double-stranded rna （dsrna）. To predict which genes could be easily silenced, we treated the Asian corn borer （Ostriniafilrnacalis） with dsGFP （green fluorescent protein） and dsMLP （muscle lim protein）. A transcriptome sequence analysis was conducted using the cDNAs 6 h after treatment with dsrna. The results indicated that 160 genes were up-regulated and 44 genes were down-regulated by the two dsrnas. Then, 50 co-up-regulated, 25 co-down-regulated and 43 unaffected genes were selected to determine their rnai responses. All the 25 down-regulated genes were knocked down by their corresponding dsrna. However, several of the up-regulated and unaffected genes were up-regulated when treated with their corresponding dsrnas instead of being knocked down. The genes up-regulated by the dsGFP treatment may be involved in insect immune responses or the rnai pathway. When the immune-related genes were excluded, only seven genes were induced by dsGFP, including ago-2 and dicer-2. These results not only provide a reference for efficient rnai target predications, but also provide some potential rnai pathway-related genes for further study.

关键词： Ostrinia furnacalis RNA interference rnai targets rna-seq

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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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