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Knockout of three aminopeptidase n genes does not affect susceptibility of Helicoverpa armigera larvae to Bacillus thuringiensis Cry1A and Cry2A toxins

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Insect Science 2020年第3期27卷 440-448页

作者： Jing Wang Ya-Yun Zuo Ling-Li Li Hui Wang Shao-Yan Liu Yi-Hua Yang Yi-Dong Wu College of Plant Protection Nanjing Agricultural UniversityNanjingChina

Bacillus thuringiensis(Bt)insecticidal toxins have been globally utilized for control of agricultural insects through spraying or transgenic crops.Binding of Bt toxins to special receptors on midgut epithelial cells of target insects is a key step in the mode of action.Previous studies suggested aminopeptidase n1(APn1)as a receptor or putative receptor in several lepidopteran insects including Helicoverpa armigera through evidence from RnA interefence‐based gene silencing approaches.In the current study we tested the role of APns in the mode of action of Bt toxins using clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR‐associated protein 9‐mediated gene knockout.Three APn genes(HaAPn1,HaAPn2 and HaAPn5)were individually knocked out in a susceptible strain(SCD)of H.armigera to establish three homozygous knockout strains.Qualitative in vitro binding studies indicated binding of Cry1Ac or Cry2Ab to midgut brush border membrane vesicles was not obviously affected by APn knockout.Bioassay results showed that none of the three knockouts had significant changes in susceptibility to Cry1A or Cry2A toxins when compared with the SCD strain.This suggests that the three HaAPn genes we tested may not be critical in the mode of action of Cry1A or Cry2A toxins in H.armigera.

关键词： aminopeptidase n Bacillus thuringiensis CRISPR/Cas9 Helicoverpa armigera receptor

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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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Real-time identification of gut microbiota with aminopeptidase n using an activable nIR fluorescent probe

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Chinese Chemical Letters 2021年第10期32卷 3053-3056页

作者： Lei Feng Zhenhao Tian Ming Zhang Xin He Xiangge Tian Zhenlong Yu Xiaochi Ma Chao Wang Dalian Key Laboratory of Metabolic Target Characterization and Traditional Chinese Medicine Intervention College of PharmacyAcademy of Integrative MedicineThe National&Local Joint Engineering Research Center for Drug Development of Neurodegenerative DiseaseDalian Medical UniversityDalian 116044China School of Life Sciences Northwestern Polytechnical UniversityXuzhou 221004China Jiangsu Key Laboratory of new Drug Research and Clinical Pharmacy Xuzhou Medical UniversityXi’an 710072China

A nIR fluorescent probe(DDAA) derived from fluorophore DDAO with alanine as the recognition group was developed for sensing aminopeptidase n(APn) in gut microbiota.Using DDAA as the real-time guidance tool for the fluorescence imaging of intestinal microorganism,target bacteria and saccharomycete possessing active APn were identified successfully from human feces.

关键词： aminopeptidase n Fluorescent probe Gut microbiota Bacteria Fungi

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Cloning and Sequencing of a Gene Encoding aminopeptidase n in the Midgut of Helicoverpa armigera(Hubner)

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Agricultural Sciences in China 2003年第7期2卷 760-767页

作者： WAnG Gui-rong, LIAnG Ge-mei, WU Kong-ming and GUO Yu-yuan(State Key Laboratory of Plant Disease and Insect Pests , Institute of Plant Protection , Chinese Academy of Agricultural Sciences , Beijing 100094 , P.R.China) State Key Laboratory of Plant Disease and Insect Pests Institute of Plant Protection Chinese Academy of Agricultural Sciences Beijing 100094 P. R. China

A cDnA encoding aminopeptidase n was cloned by degenerated PCR combined with RACE technique in this paper. The full-length of APn-Harm is 3 043 bp. Open reading frame is 2 856 bp in length, encoding 951 amino acid residues. Its predicted molecular weight and isoelectric point are 108. 3 kDa and 5.29, respectively. This deduced amino acid sequence shares some common structural features with aminopeptidase n from several moth species, including the consensus zinc-binding motif HEXXHX18E and the GAMEn motif common to gluzincin aminopeptidases. The first 20 amino acid residues at n-termini is hydrophobic transmembrane helix. The sequence of APn-Harm was deposited in GenBank and the accession number is AY181026.

关键词： Helicoverpa armigera aminopeptidase n Gene cloning

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Genome-wide identification and comparative analysis of Cry toxin receptor families in 7 insect species with a focus on Spodoptera litura

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Insect Science 2022年第3期29卷 783-800页

作者： Qilin Li Mengge Li Mengyao Zhu Jielai Zhong Liang Wen Jie Zhang Ruonan Zhang Qiang Gao Xiao-Qiang Yu Yuzhen Lu Guangdong Provincial Key Laboratory of Insect Developmental Biology and Applied Technology Institute of Insect Science and TechnologySchool of Life SciencesSouth China Normal UniversityGuangzhou510631China College of Biology Hunan UniversityChangsha410082China

Cadherin,aminopeptidase n(APn)and alkaline phosphatase(ALP)have been characterized as Cry receptors.In this study,comparative genomic analysis of the 3 receptor families was performed in 7 insects.ALPs and APns are divided into three and eight clades in phylogenetic trees,respectively.ALPs in clade 3 and APns in clade 1 contain multiple paralogs within each species and most paralogs are located closely in chromosomes.Drosophila melanogaster has expanded APns in clade 5 and were lowly expressed in midgut.Cadherins are divided into 16 clades;they may diverge before holometabolous insect speciation except for BtR and Cad89D-like clades.Eight insects from different orders containing BtR orthologs are sensitive to Cryl A or Cry3A,while five species without BtR are insensitive to both toxins.Most APns in clade 1,several ALPs in clade 3,BtR and Cad89D-like genes were highly or moderately expressed in larval midgut of Spodoptera litura and the other six species,and several members in these clades have been identi-•fied as Cry receptors.Expressions of putative 5.litura Cry receptors in the midgut after exposing to Bt toxins were also analyzed.

关键词： alkaline phosphatase aminopeptidase n Bacillus thuringiensis cadherin Cry toxin

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Possible contribution of aminopeptidase n(APn/CD13)to Migration and Invasion of Human Osteosarcoma Cell Lines

Possible contribution of Aminopeptidase N(APN/CD13)to Migrat...

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2014年浙江省骨科学学术年会

作者： Wenqing Liang Guojian Xu Dong Weng Minghua Xie Yu Qian Department of Orthopaedics Shaoxing People’s Hospital

Osteosarcoma is the most common primary malignancy of the bone cancers.aminopeptidase n(APn,CD13),a Zn+2-dependent ectopeptidase localized on the cell surface,is widely considered to influence the invasion mechanism.This study explores the potential involvement of APn in migration and invasion of human osteosarcoma cells in vitro using inhibitors and activators of APn.Cells treated with APn inhibitor bestatin displayed decreased migration and invasion in a Boyden chamber transwell assay.Western blotting revealed reduced levels of MAPK and PI3 K pathway proteins,reduced phosphorylation of p-38,ERK1/2 and JnK and decreased levels of nF κB.Bestatin treatment also lowered APn,Matrix Metalloproteinase(MMP)-2 and-9 enzymatic activity and their mRnA expression.Reduced MMP-2 and-9 protein levels were also observed.By comparison,cells treated with cytokine Interleukin-6(IL-6),a stimulator of APn,displayed increased migration and invasion.Western blotting revealed increased levels of MAPK and PI3 K pathway proteins,phosphorylated p-38,ERK1/2 and JnK,and nF κB.IL-6treatment also increased APn and MMP-2 and-9 enzymatic activity.An increase of APn,MMP-2 and-9 mRnA levels,and MMP-2 and-9 protein levels was also observed.Together these experiments reveal potential enzymatic and signalling roles for APn in osteosarcoma and establish a starting point for an in-depth analysis of the role of APn in regulating invasiveness.A deeper knowledge about the regulatory mechanisms of APn may contribute to the development of anti—metastatic therapies.

关键词： Osteosarcoma Metastasis aminopeptidase n Matrix Metalloproteinase

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沉默APn对胶质瘤细胞U251的增殖、侵袭和凋亡的影响及其作用机制

沉默APN对胶质瘤细胞U251的增殖、侵袭和凋亡的影响及其作用机制

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作者：董云朋重庆医科大学

学位级别：硕士

目的:探究aminopeptidase n(APn)对胶质瘤细胞U251增殖能力、迁移侵袭能力和凋亡等生物学行为的影响及可能的作用机制。方法:通过RnA干扰技术沉默胶质瘤细胞U251中APn的表达后,然后通过相适应的功能实验方法来检测沉默后胶质瘤U251的生... 详细信息

目的:探究aminopeptidase n(APn)对胶质瘤细胞U251增殖能力、迁移侵袭能力和凋亡等生物学行为的影响及可能的作用机制。方法:通过RnA干扰技术沉默胶质瘤细胞U251中APn的表达后,然后通过相适应的功能实验方法来检测沉默后胶质瘤U251的生物学功能。运用RT-qPCR的方法检测沉默胶质瘤细胞U251中APn前后的mRnA表达水平的变化,采用Western blot方法来检测APn蛋白表达水平的变化,分别在基因和蛋白水平来检测胶质瘤细胞U251中APn的表达水平和RnA干扰的效果;接下来用Cell Counting Kit 8(CCK-8)试剂检测胶质瘤细胞U251 Blank组、nC-siRnA组和APn-siRnA组的细胞增殖活性;采用流式细胞仪检测沉默胶质瘤细胞U251细胞中APn前后的细胞凋亡率和细胞周期分布;Transwell迁移实验和细胞划痕实验用来检测各组胶质瘤细胞U251的迁移能力;Transwell实验用来检测不同组胶质瘤细胞U251的侵袭能力;采用Western blot检测RnA干扰后和空白对照组的STAT3信号通路蛋白t-STAT3、p-STAT3、Bcl-2、MMP-2、PCnA和Cyclin D1的蛋白表达水平。结果:与空白对照组和阴性对照组相比,沉默U251中APn的表达后APn在基因和蛋白表达水平下降(PnA干扰达到了理想的效果;沉默APn后U251的增殖活性明显下降(PnC-siRnA组和APn-siRnA组细胞的S期比例分别为(33.72±4.79)%、(29.28±4.31)%和(8.3±2.88%);G/M期比例分别为(10.35±4.85)%、(9.2±1.42)%和(7±1.61)%;G/G期比例分别为(55.93±9.57)%、(61.52±3.29)%和(84.7±2.66)%,与空白对照组相比APn-siRnA组的S期比例明显减低(pnC-siRnA组和APn-siRnA组的总体凋亡率分别为(4.89±3.38)%、(2.72±2.55)%和(30.58±9.51)%,APn-siRnA组的凋亡率比空白组明显增加,差距具有统计学意义(pnC-siRnA组和APn-siRnA组的迁移率分别为(60±1.73)%、(55±4.58)%和(27±4.58)%,APn-siRnA组的运动能力比空白组明显减弱,差距具有统计学意义(pnswell的方法来检测不同组U251的迁移能力的变化,我们发现空白对照组、nC-siRnA组和APn-siRnA组每个视野细胞分别35±4个、34±5个和22±2个,与空白对照组相比APn-siRnA组的细胞显著减少,差距有统计学意义(pnswell侵袭方法检测U251侵袭能力的实验中发现空白对照组、nC-siRnA组和APn-siRnA组每个视野细胞分别33±3个、31±3个和15±2个,与空白对照组相比APn-siRnA组的细胞显著减少,差距有统计学意义(pn Blot探究沉默胶质瘤细胞U251中APn后STAT3信号通路蛋白表达的变化发现与空白对照组相比APn-siRnA组的p-STAT3、Bcl-2、MMP-2、PCnA和Cyclin D1的蛋白表达水平明显降低差异有统计学意义(pn可抑制胶质瘤细胞U251细胞增殖能力、迁移能力和侵袭侵袭,并能诱导凋亡,STAT3信号传导途径是APn影响胶质瘤细胞U251生物学活性的作用机制之一。

关键词：胶质瘤 aminopeptidase n RnA干扰 STAT3

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Identification of Px APn5 serve as a functional receptor of Cry2Ab in Plutella xylostella

Identification of Px APN5 serve as a functional receptor of ...

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中国生物化学与分子生物学会2016年全国学术会议

作者： Yu-Jing Zhu Lian Xu Zhi-Zhen Pan Qing-Xi Chen Bo Liu Agricultural Bio-Resources Research Institute Fujian Academy of Agricultural Sciences State Key Laboratory of Cellular Stress Biology Key Laboratory of the Ministry of Education for Coastal and Wetland Ecosystems School of Life Sciences Xiamen University

Insecticidal activities of Cry toxins relied on their interaction with midgut receptors inlaying in the brush border membrane vesicles(BBMV)of target insects.In present study,we demonstrated that an aminopeptidase n(Px APn5)served as a functiona l receptor of Cry2Ab in Plutella xylostella.Silence of Px APn5 in P.xylostella larvae greatly reduced their

关键词： Bacillus thuringiensis Cry2Ab toxin Plutella xylostella aminopeptidase n RnAi ligand blot

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