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Construction of cytomegalovirus promoter-driven gene expression system in Laodelphax striatellus

Insect Science 2024年第3期31卷 720-732页

作者： Xiaohui Cheng Wan Zhao Guohua Liang Hong Lu Yumei Fu Yiming Li Feng Cui State key laboratory of Integrated management of Pest insects and Rodents Insttute of ZoologyChinese Academy of SciencesBejingChina University of Chinese Academy of Sciences BejingChina School of Life Sciences Hebei UniversityBaodingHebei China key laboratory of Animal Ecology and Conservation Biology Institute of ZoologyChinese Academy of SciencesBejingChina Institute of Wetland Ecology&Clone Ecologyl Zhejiang Provincial key laboratory of plant Evolutionary Ecology and Conservation Taizhou UniversityTaizhouZhejiangChina

The small brown planthopper(SBPH,Laodelphax striatellus)is a significant rice pest,responsible for transmitting rice stripe virus(RSV)in a persistent and propagative *** is one of the most detrimental rice viruses,causing rice stripe disease,which results in considerable loss of rice grain *** RNA interference and gene knockout techniques have enabled gene downregulation in SBPH,no system currently exists for the overexpression of endogenous or exogenous ***,the development of a protein expression system for SBPH is imperative to serve as a technical foundation for pest control and gene function *** study aimed to construct an expression vector using the promoter of the constitutive-expressed tubulin gene of SBPH,and promoter of human cytomegalovirus(CMV).Fluorescence experiments demonstrated that both tubulin and CMV promoter could drive green fluorescent protein(GFP)expression in SBPH,and could also facilitate the expression of a nucleocapsid protein(NP)-GFP fusion protein containing viral NP with comparable *** expression vector optimization,we have identified that the 3 tandem CMV promoters display a significantly higher promoter activity compared with both the 2 tandem CMV promoters and the single CMV *** addition,the incorporation of Star polycation nanoparticles significantly enhanced the expression efficiency in *** results provide a promising technical platform for investigating gene functions in SBPH.

关键词： cytomegalovirus promoter gene expression system nanoparticle small brown planthopper

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Influence of Y151 F mutation in loop B on the agonist potency in insect nicotinic acetylcholine receptor

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Insect Science 2009年第5期16卷 365-369页

作者： Feng Song Yi-Xi Zhang Xiang-Mei Yao Ze-Wen Liu key laboratory of monitoring and management of plant disease and insects Ministry of Agriculture Nanjing Agricultural UniversityNanjing China

Nicotinic acetylcholine receptors （nAChRs） are ligand-gated ion channels, which mediate fast cholinergic synaptic transmission in insect and vertebrate nervous systems. The nAChR agonist-binding site is present at the interface of adjacent subunits and is formed by loops A-C present in α subunits together with loops D-F present in either non-α subunits or homomer-forming α subunits. Although Y151 in loop B has been identified as important in agonist binding, various residues at the 151-site are found among vertebrate and invertebrate nAChR α subunits, such as F 151. In Xenopus oocytes expressing Nlα1 or Nlα1^Y151F plus rat β2, Y151F mutation was found to significantly change the rate of receptor desensitization and altered the pharmacological properties of acetylcholine, but not imidacloprid, including the decrease Of Imax, the increase of EC50 （the concentration causing 50% of the maximum response） and the fast time-constant of decay （τf）. By comparisons of residue structure, the hydroxyl group in the side chain of Y151 was thought to be important in the interaction between Nlα1/β2 nAChRs and acetylcholine, and the phenyl group to be important between Nlα1/β2 nAChRs and imidacloprid.

关键词： acetylcholine agonist potency imidacloprid nicotinic acetylcholine receptor Nilaparvata lugens Xenopus oocytes

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Biological Activities of Purified HarpinXoo and HarpinXoo Detection in Transgenic plants Using Its Polyclonal Antibody

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Acta Biochimica et Biophysica Sinica 2005年第10期37卷 713-718页

作者： Ming LI Min SHAO Xu-Zhong LU Jin-Sheng WANG key laboratory of monitoring and management of plant diseases and insects,Ministry of Agriculture,Nanjing Agricultural University,Nanjing 210095,China key laboratory of monitoring and management of plant diseases and insects Ministry of Agriculture Nanjing Agricultural University Nanjing 210095 China

Many harpins have been found in plant pathogen bacteria that can elicit disease and insectresistance in plants,and promote plant *** this work,we overexpressed and purified Xanthomonasoryzae *** harpin,harpinXoo,in Escherichia coli BL21/*** was fused to the C-terminus of glutathione S-transferase （GST） and purified using the Bulk GST purification module and thrombincleavage capture *** harpinXoo protein was sensitive to protease K and stable to heat treatment,andcould not induce a hypersensitive response after treatment with various plant metabolic inhibitors;thesecharacteristics were similar to harpinEa of Erwinia *** purified harpinXoo showed a similar abilityto induce tobacco mosaic virus resistance in tobacco as *** antibody worked well in detecting thepurified *** in the total protein of *** BL21/pGEX-hpal and an hpal transgenic rice.

关键词： harpinXoo glutathione S-transferase (GST) biological activity polyclonal antibody

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An active piggyBac-like element in Macdunnoughia crassisigna

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Insect Science 2008年第6期15卷 521-528页

作者： Min Wu Zhi-Chan Sun Chun-Lin Hu Gu-Feng Zhang Zhao-Jun Han key laboratory of monitoring and management of plant disease and insects Ministry of Agriculture Nanjing Agricultural University Nanjing Tongzhou Station of plant Protection Tongzhou Jiangsu Province China

In this paper, a highly conservedpiggyBac-like sequence, designated as McrPLE was cloned from a lepidopteran insect, Macdunnoughia crassisigna. It is 2 472 bp long in full length with a single open reading frame and encodes a 595 amino acid transposase. It shares identical terminal and sub-terminal repeats with T. ni IFP2 and is flanked by the typical TTAA target-site duplications. Alignment and phylogenetic analysis revealed that McrPLE had greater than 99.5% identity and appeared to be the closest one in phylogeny to IFP2 among the PLEs so far found in various species. Plasmid-based excision and transposition assay proved it was mobile in cell culture. Otherwise, McrPLE element and all other highly conserved IFP2 sequences reported previously were found to share three common nucleotide substitutions. This suggests that the original IFP2 may be a related variant of a predecessor element that became widespread. The existence of nearly identical piggyBac sequence in reproductively isolated species was thought also a strong indication of horizontal transmission, which raises important considerations for the stability and practical use ofpiggyBac transformation vectors.

关键词： piggyBac-like element (PLE) Macdunnoughia crassisigna transposon

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Analysis of sleep in individual Drosophila melanogaster reveals a self-regulatory role for cuticular hydrocarbons pheromones

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Insect Science 2022年第6期29卷 1737-1746页

作者： Xiaoyu Wu Su Wang Xianguo Zhao Jing Wen Yahong Li Zhangwu Zhao Juan Du Department of Entomology MOA Key Lab of Pest Monitoring and Green ManagementCollege of Plant ProtectionChina Agricultural UniversityBeijingChina

It is well established that pheromones are used by insects to transmit infor-mation between ***,research has revealed that individual insects can be both the sender and the receiver of some pheromonal *** is therefore interesting to consider whether the pheromonal state of an individual insect can exert an effect on *** this study,we monitored the sleep activity of single flies exhibiting a mutation that leads to pheromonal deficiency and found that cuticular hydrocarbons(CHs)exerted self-regulatory effects on the amount of sleep experienced by these *** identify the physiological significance of this mechanism,we compared the amounts of sleep in in-dividual young flies and individual old flies(flies are known to sleep less as they get older)and compared this data with young and old flies exhibiting mutations that lead to CH reception *** differences in the amount of sleep experienced by young and old mutant flies were significantly lower than those of the control *** data show that hydrocarbon signals produced by the cuticle in Drosophila can be self-perceived and regulate the amount of sleep acquired in a maturation-dependent manner.

关键词： cuticular hydrocarbons pheromone sleep

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Tebufenozide resistance is associated with sex-linked inheritance in Plutella xylostella

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Insect Science 2015年第2期22卷 235-242页

作者： Guang-Chun Cao Zhao-Jun Han Entomology Department/key laboratory of monitoring and management of plant diseases and insects Ministry of Agriculture NanjingAgricultural University Nanjing State key laboratory for Biology of plant disease and Insect Pests Institute of Plant ProtectionChinese Academy of Agricultural Sciences Beijing China

The diamondback moth （DBM）, Plutella xylostella （L.）, is a major pest of cruciferous crops. Tebufenozide, a novel nonsteroidal ecdysone agonist, exhibits good efficacy and has played an increasingly important role in the control of Lepidopteran pests in China. For its resistance management, the genetic basis of tebufenozide resistance was studied using a laboratory selected resistant strain of DBM （resistant ratio, RR = 268）. A series of crosses with laboratory susceptible and resistant strains revealed that tebufenozide resistance in this pest was partially biased toward female heredity, with a large difference in RR for F1 （RR = 29） and rF1 progeny （RR = 147）. The dominance calculated for these 2 cross progeny was -0.788 and 0.09, respectively. Further analysis showed that the susceptible male and female larvae were similar in theft sensitivity to tebufenozide, but the resistant female larvae showed significantly higher resistance than the resistant male larvae. The heredity of tebufenozide resistance in DBM might be linked with the W sex chromosome, which suggested that DBM has the ability to develop high levels of resistance to tebufenozide. This is the first report of sex-linked inheritance oftebufenozide resistance in P xylostella （L.）.

关键词： insecticide resistance Plutella xylostella sex linkage tebufenozide

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Characterization of Xanthomonas citri *** from China based on spoligotyping

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Horticultural plant Journal 2022年第6期8卷 727-736页

作者： Wenting Li Zhenxi Li Jiaquan Huang Meirong Xu Zheng Zheng Xiaoling Deng Guangdong Province key laboratory of Microbial Signals and disease Control College of Plant ProtectionSouth China Agricultural UniversityGuangzhou 510642China

Xanthomonas citri ***(Xcc),a gram-negative bacterium,is the causal agent of citrus canker,one of the most devastating diseases threatening the citrus industry *** the diversity and population structure of Xcc is a prerequisite for disease epidemiological monitoring and effective disease *** characterization of the clustered regularly interspaced short palindromic repeats(CRISPR)/cas(CRISPR-associated proteins genes)system with a highly variable repeat number among species provides a new molecular typing method for bacterial genetic *** this study,we performed systematic in silico analyses of 28 Xcc genomes and identified a credible CRISPR/cas in Xcc *** analysis of CRISPR polymorphisms(repeat number and spacer types)in 129 Xcc A strains collected from six provinces in China identified 15 types of CRISPR arrays with 25 *** analysis of Xcc strains based on the CRISPR locus produced a more reliable and accurate typing result compared to the commonly used *** addition,seven associated cas genes—cas1,cas2,cas3,cas4,cas5,cas7(csd2),and cas8(csd1)—were found located adjacent to the CRISPR *** results showed>99%similarity of seven cas genes among Xcc *** analysis of spacer sequences showed that six spacers had possible phage/prophage *** characterization of the CRISPR/cas system among Xcc strains provided an updated strain typing method for Xcc diversity analysis and yielded a panoramic view of CRISPR evolution for further studies of Xcc-phage interactions.

关键词： Xanthomonas citri pv.citri CRISPR Diversity Spacers Typing method

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Using a Suppression Subtractive Library-Based Approach to Identify Non-Heading Chinese Cabbage Genes Up-Regulated in Early Response to Elicitor PB90

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Agricultural Sciences in China 2008年第3期7卷 303-313页

作者： GAN Yun-zhe ZHANG Zheng-guang WANG Yuan-chao ZHENG Xiao-bo key Lab of monitoring and management of plant disease and insects Ministry of Agriculture/College of Plant Protection Nanjing Agricultural University Nanjing 210095 P.R.China

monitoring expression at the transcriptional level is the first essential step for the functional analysis of plant genes. Genes-encoding proteins directly involved in early response to elicitor constitute only a small fraction of all the genes affected by elicitor. Transcriptional responses to various elicitors have been extensively studied in different plants including Nicotiana and Arabidopsis thaliana; however, corresponding data aren＇t available for non-heading Chinese cabbage. To address this problem, we describe a suppression subtractive library-based approach to isolate the plant＇s ESTs up-regulated in the early induction/execution of the HR induced by elicitor PB90 from Phytophthora boehmeriae. According to their putative identification in BLAST searches against the three genome databases, 70 up-regulated genes were classified into 9 parts： some aspect of primary ＇metabolism＇ or ＇energy＇ production; ＇protein synthesis＇ or ＇protein fate＇; cellular communication/signal transduction mechanism; cell fates including Beclin, SPT1, and SPT2; HLA-B and AGO1 which participate in transcription; cellular transport and hypothetical proteins or proteins for which a function has yet to be determined. Seven selected genes such as Beclin, thioredoxin, HLA-B, MAP3K, SPT1, SPT2, and AGO1 were up-regulated induced by PB90, suggesting that the genes may play an important role in PB90-triggered HR.

关键词： PB90 non-heading Chinese cabbage suppression subtractive hybridization up-regulated hypersensitive response

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Amplification and function analysis of N6-adenine-specific DNA methyltransferase gene in Nilaparvata lugens

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Journal of Integrative Agriculture 2016年第3期15卷 591-599页

作者： ZHANG Jiao XING Yan-ru HOU Bo-feng YUAN Zhu-ting LI Yao JIE Wen-cai SUN Yang LI Fei Department of Entomology College of Plant Protection Nanjing Agricultural UniversityKey Laboratory of Monitoring and Management of Plant Diseases and Insects Ministry of Agriculture key Lab of Agricultural Entomology Ministry of AgricultureInstitute of Insect Sciences Zhejiang University

Methylation of the N6 position of adenine, termed N6-methyladenine, protects DNA from restriction endonucleases via the host-specific restriction-modification system. N6-methyladenine was discovered and has been well studied in bacteria. N6-adenine-specific DNA methyltransferase（N6AMT） is the main enzyme catalyzing the methylation of the adenine base and knowledge of this enzyme was mainly derived from work in prokaryotic models. However, large-scale gene discovery at the genome level in many model organisms indicated that the N6AMT gene also exists in eukaryotes, such as humans, mice, fruit flies and plants. Here, we cloned a N6AMT gene from Nilaparvata lugens（Nlu-N6AMT） and amplified its fulllength transcript. Then, we carried out a systematic investigation of N6AMT in 33 publically available insect genomes, indicating that all studied insects had N6AMT. Genomic structure analysis showed that insect N6AMT has short introns compared with the mammalian homologs. Domain and phylogenetic analysis indicated that insect N6AMT had a conserved N6-adenine Mlase domain that is specific to catalyze the adenine methylation. Nlu-N6AMT was highly expressed in the adult female. We knocked down Nlu-N6AMT by feeding ds RNA from the second instar nymph to adult female, inducing retard development of adult female. In all, we provide the first genome-wide analysis of N6AMT in insects and presented the experimental evidence that N6AMT might have important functions in reproductive development and ovary maturation.

关键词： N6-adenine-specific DNA methyltransferase(N6AMT) Nilaparvata lugens expression RNA interference gene structure

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Gene cloning and expression of aminopeptidase N and cadherin from midgut of the rice stem borer, Chilo suppressalis

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Insect Science 2010年第5期17卷 393-399页

作者： Hong-Kun Yu Hao Ghen Yong-Jun Zhang Kong-Ming Wu Ge-Mei Liang Ze-Wen Liu Yu-Yuan Guo State key laboratory for Biology of plant diseases and Insect Pests Institute of Plant Protection Chinese Academy of Agricultural Sciences Beijing key laboratory of monitoring and management of Crop diseases and Pest insects Ministry of Agriculture College of Plant Protection Nanjing Agricultural University Nanjing China

The rice stem borer, Chilo suppressalis Walker is one of the most important insect pests on rice in Asia, north Africa and southern Europe. Transgenic Bt rice has been developed in the laboratory with good resistance to this pest and other Lepidopteran insects, which will provide a possible alternative tool for this pest control. The full-length cDNAs encoding an aminopeptidase N （CsAPN） and a cadherin （CsCad） were cloned from C. suppressalis. CsAPN showed common features of, and high identities to, other insect APNs in its deduced amino acid sequence. Although a full-length cDNA encoding cadherin-like protein has been reported in GenBank, the newly isolated cadherin here （CsCad） showed some differences in its amino acid sequence, especially at the 7th cadherin repeat region （CR7）, which indicated the newly isolated CsCad might be another allele. CsAPN and CsCad were successfully expressed in insect Tn cells, and the blot analysis showed these two proteins could bind Bt toxin CrylAb. The results will provide valuable information for the studies of toxin mode of action and the possible toxin resistance mechanisms in this pest.

关键词： aminopeptidase N cadherin Chilo suppressalis heterologous expression,Tn cells

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