Hippocampal gene expression in a rat model of depression after electroacupuncture at the Baihui and Yintang acupoints

Preliminary basic research and clinical findings have demonstrated that electroacupuncture ther- apy exhibits positive effects in ameliorating depression. However, most studies of the underlying mechanism are at the single gene level; there are few reports regarding the mechanism at the whole-genome level. Using a rat genomic gene-chip, we profiled hippocampal gene expression changes in rats after electroacupuncture therapy. Electroacupuncture therapy alleviated depres- sion-related manifestations in the model rats. Using gene-chip analysis, we demonstrated that electroacupuncture at Baihui （DU20） and Yintang （EX-HN3） regulates the expression of 21 genes. Real-time PCR showed that the genes Vgf, lgf2, Trnp32, Loc500373, Hifla, Folrl, Nrnb, and Rtn were upregulated or downregulated in depression and that their expression tended to nor- malize after electroacupuncture therapy. These results indicate that electroacupuncture at Baihui and Yintang modulates depression by regulating the expression of particular genes.

Detections of mefA, ermB, and mphA Macrolides Resistant Genes in Bacteria Isolated from Covid-19 Patients from Selected Health Facilities in Ibadan, Nigeria

Background: COVID-19 is a disease caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Epidemiological data indicated that bacterial complications in COVID-19 would decrease clearance rate of the infecting agent and increase mortality rate. Macrolides such as Azithromycin are usually administered to COVID-19 patients as palliative treatments. Currently, a considerable number of bacterial strains have developed resistance to various antibiotics, especially macrolides. Resistance is reported to be due to possession of mefA, ermB, and mphA genes by Gram positive and Gram negative bacteria. Therefore, this study determined antibiotic resistance patterns and identify mefA, ermB and mphA macrolide-resistant genes in bacterial pathogens isolated from COVID-19 cases in Ibadan, Nigeria. Methods: 400 Nasopharyngeal samples were collected from symptomatic cases before antibiotic medication;structured questionnaires were administered to collect socio-demographic data of participants. Samples were cultured on Blood, Chocolate, MacConkey and Mannitol salt agar at 37°C for 48 hrs. Bacterial identification was performed using VITEK 2.0 ID cards and API 20E for Gram positive and negative bacteria respectively. Antibiotic Susceptibility Testing was performed using Kirby Bauer disc diffusion methods and VITEK 2.0 AST card kits. DNA of multidrug resistant bacterial isolates was extracted;resistant genes were determined using a polymerase chain reaction with specific primers. Amplified genes were detected using agarose gel electrophoresis. Results: 240 (60%) had bacterial growth and 97 (22.2%) yielded no growth. From the 240 bacterial isolates, 38 (15.83%) were multi-drug resistant including resistance to macrolides (Azithromycin) 20 (52.63%) of which were positive for either mefA or ermB, and none (0.0%) possess mphA gene;14 (36.8%) isolates had mefA gene, 10 (26.3%) isolates carried ermB gene. Conclusion: Multi-drug bacterial resistance including macrolides and quinolones was detected. Only mefA and ermB genes were detected in the bacterial isolates, especially in Gram positive organisms. The detection of mefA and ermB genes in the MDR bacterial isolates raised concern on the use of azithromycin as palliative treatment for COVID-19 symptomatic patients.

The role of FZR1 in tumorigenesis: Focus on cell-cycle control

Fizzy-related protein homolog 1 (FZR1) mainly functions as a specific activator of the anaphase-promotingcomplex/cyclosome (APC/C) in the cell cycle and controls the G0 and G1 phases of the cell cycle. We highlightrecent work that has studied the role of FZR1 in tumorigenesis, growth, differentiation, and genome stability throughcell-cycle control. We summarize the current state of knowledge regarding FZR1 structure, function, and the distinctways of APC/C dysregulation in solid tumors and hematologic malignancies. We also discuss novel approaches fortargeting the FZR1 as a cancer therapy and research area for future work.

三氧化二砷对急性髓系白血病细胞HL-60 Cdc20及Mad2的影响

目的:探讨三氧化二砷(As_2O_3)对急性髓系白血病细胞HL-60增殖过程中Cdc20及Mad2的影响。方法:不同浓度As_2O_3处理HL-60细胞24、48、72 h后CCK-8法检测细胞增殖情况;Real-Time PCR及Western blot技术检测不同浓度As_2O_3作用于HL-60细胞48 h后Mad2和Cdc20 mRNA及蛋白表达情况。结果:As_2O_3能显著抑制HL-60细胞增殖,并具有良好的时间剂量相关性。RT-PCR和Western blot检测结果表明,As_2O_34、8、10μmol/L作用于HL-60细胞后,Mad2表达上调,Cdc20表达下降(P<0.05)。结论:As_2O_3对人急性髓系白血病HL-60细胞增殖具有较明显的抑制作用,其机制可能与上调Mad2表达和下调Cdc20表达有关。

Expression of CDC20, TOP2A in Esophageal Phosphorus Carcinoma and Its Relationship with Survival of Esophageal Carcinoma

Objective:To study the expression of CDC20,TOP2A in esophageal squamous cell carcinoma and its relationship with survival of esophageal carcinoma.Methods:65 patients with esophageal squamous cell carcinoma from January 2016 to June 2018 were selected by computer random selection.All patients were treated with radical operation.The CDC20,TOP2A expression of the patients was examined.At the same time,the relationship between 3-year survival rate and CDC20,TOP2A was analyzed by follow-up investigation.Results:the CDC20,TOP2A expression level of cancer tissue group was higher than that of adjacent tissue group and normal tissue group(P<0.05),and the CDC20,TOP2A expression level of adjacent tissue group was higher than that of normal tissue group(P<0.05).There was no significant difference in sex,age,tumor size,tumor location and CDC20,TOP2A expression level in patients with esophageal squamous cell carcinoma,P>0.05;there were differences between groups(P P>0.05)and positive proportion in TNM stage,lymphatic metastasis,invasion factor and CDC20,TOP2A expression level;there were differences in tumor differentiation,5-year survival rate and CDC20,TOP2A expression level(P P>0.05),and showed inverse proportion relationship.Conclusion:metastasis,recurrence and prognosis of esophageal squamous cell carcinoma are related to the level of CDC20,TOP2A expression.These two indexes can effectively evaluate the pathological situation of esophageal cancer and provide an important reference for the prognosis of esophageal squamous cell carcinoma patients.

CDC20通过稳定NLRP3的表达促进食管癌细胞增殖的研究

背景与目的:食管癌(esophageal carcinoma,ESCA)是死亡率较高的恶性肿瘤之一,其发生、发展的机制不明。CDC20被认为具有癌基因功能,其表达失调与肿瘤的发生、发展密切相关。CDC20在多种肿瘤中表达升高,敲低CDC20能够抑制肿瘤细胞增殖。NLRP3是炎症小体的主要成分之一,炎症小体失调与肿瘤的发生、发展也密切相关。本研究旨在探究CDC20是否通过NLRP3促进ESCA细胞增殖,同时分析其调控机制。方法:通过癌症基因组图谱(The Cancer Genome Atlas,TCGA)数据库和GTEx公共数据库分析ESCA患者中CDC20和NLRP3基因的表达水平。收集新乡医学院第一附属医院收治的80例ESCA患者的临床病理学资料和组织,通过免疫组织化学染色检测NLRP3在ESCA患者中的蛋白表达水平。本研究通过新乡医学院第一附属医院伦理委员会的审批(编号:EC-021-137)。通过短发夹RNA(short hairpin RNA,shRNA)技术检测敲低CDC20和NLRP3基因后对食管鳞状细胞癌细胞系EC9706和KYSE150增殖能力的影响。通过免疫共沉淀(Co-immunoprecipitation,Co-IP)、蛋白酶体抑制剂和泛素化实验检测CDC20是否与NLRP3相互作用,以及阐明CDC20是否通过泛素化途径调控NLRP3表达。结果:TCGA数据库分析结果显示,ESCA组织中CDC20和NLRP3 mRNA表达水平明显高于癌旁组织。免疫组织化学结果也进一步显示,与癌旁组织相比,CDC20、NLRP3在ESCA组织中蛋白表达水平升高。敲低CDC20和NLRP3基因可抑制ESCA细胞增殖。Co-IP、蛋白酶体抑制剂和泛素化实验证实CDC20通过NLRP3的LRR区与NLRP3相互作用,且CDC20通过促进NLRP3泛素化稳定其表达。结论:CDC20和NLRP3在ESCA癌组织中表达上调,且CDC20通过泛素化NLRP3稳定其表达,从而促进ESCA细胞增殖,提示CDC20和NLRP3可能是ESCA潜在的诊断靶向标志物。

HCC组织CDC20表达及其与发病机制的关系研究

目的肝细胞癌(HCC)是世界上最常见的肝恶性肿瘤之一.HCC的病因和潜在的分子事件尚不清楚.我们应用综合生物信息学技术识别与HCC相关的关键致病基因细胞分裂周期20(CDC20),并揭示其潜在的分子机制.方法从Gene Expression Omnibus(GEO)数据库下载GSE36376基因表达谱,共433个样本,其中HCC组织240例,正常肝组织193例,用综合生物信息学方法进行深入分析.应用RStudio中的R软件筛选HCC和正常肝组织差异表达基因(DEG),从STRING数据库中构建DEG的蛋白质-蛋白质相互作用(PPI)网络,应用Metascape在线工具进行富集分析,采用MCODE法对Hub基因进行鉴定.结果筛选GSE36376数据集得到706个DEG,其中共鉴定出554个下调基因和152个上调基因;从PPI网络中鉴定出DEG关系基因,显著上调基因是UBE2C、CDC20、COL4A1、NUSAP1、HSP90AB1和CD-KN3;Metascape富集分析表明,在GO生物过程中,主要集中在小分子分解代谢过程、一元羧酸代谢过程、类固醇代谢过程、辅因子代谢过程、药物分解代谢过程、初级酒精代谢过程、对有毒物质的反应、有机阴离子转运、单糖代谢过程、芳香族氨基酸家族代谢过程、解毒作用、伯醇分解代谢过程和有机环状化合物分解代谢过程等;在KEGG信号通路中,主要集中在碳代谢、脂肪酸降解、补体和凝血级联、色氨酸代谢和过氧物酶体等过程;MCODE分析显示Hub基因是UBE2C、CDC20和HSP90AB1.结论应用综合生物信息学方法筛选出的CDC20基因是HCC发生发展的关键基因,对HCC的早期诊断和预防具有临床意义,为HCC的防治提供了有效的靶点.

基于生物信息学数据库分析CDC20基因在膀胱癌中的表达及临床意义

目的:探讨膀胱癌中CDC20基因的表达及其临床预后意义。方法:使用Oncomine数据库和GEPIA数据库分析CDC20基因在膀胱癌中的表达水平。采用Kaplan-Meier Plotter分析CDC20表达水平与膀胱癌病人总体生存时间的关系,采用LinkedOmics数据库分析CDC20基因表达与膀胱癌病人年龄及临床病理特征的相关性,通过STRING数据库绘制CDC20有关蛋白网络图,分析蛋白富集的生理过程。结果:膀胱癌组织中CDC20基因的表达水平明显高于正常膀胱组织(P<0.01)。Kaplan-Meier生存分析显示,CDC20高表达的膀胱癌病人生存时间低于CDC20低表达的膀胱癌病人(P<0.05)。STRING数据库分析显示,与CDC20相关的蛋白有FBXO5、CCNB1、AURKA等,主要富集在细胞周期、卵母细胞减数分裂、孕酮介导的卵母细胞成熟、泛素介导的蛋白水解等生理过程。LinkedOmics数据库分析显示,CDC20的表达与病人年龄、人种均有相关关系(P<0.05和P<0.01),与TNM分期及病理分期均无明显相关关系(P>0.05)。结论:CDC20基因在膀胱癌中高表达,且与膀胱癌病人预后负相关。

CDC20在子宫内膜癌中的表达及意义

目的探讨细胞分裂周期蛋白20(cell division cycle 20,CDC20)在子宫内膜癌中的表达及意义。方法选取2020年1月—2021年3月于齐齐哈尔医学院附属第三医院妇产科收治入院的96例子宫内膜癌患者为研究对象,采用免疫组化法和实时荧光定量聚合酶链反应法检测CDC20水平。根据子宫内膜癌组织中CDC20表达水平将其分为CDC20高表达组及CDC20低表达组,对比两组临床病理特征及分析子宫内膜癌患者CDC20表达情况的影响因素分析。结果96例子宫内膜癌组织中CDC20高表达69例,低表达27例。两组年龄、体质指数、分娩次数、分娩方式比较,差异无统计学意义(P>0.05);两组组织学分型、国际妇产科联盟(Federation International of Gynecology and Obstetrics,FIGO)分期、病理分级、CDC20mRNA及CDC20蛋白评分比较,差异有统计学意义(P<0.05)。经Cox回归分析显示,子宫内膜癌患者CDC20表达情况的影响因素是FIGO分期(OR=2.604)、病理分级(OR=1.559)、CDC20mRNA(OR=4.198)、CDC20蛋白(OR=2.265)。结论CDC20在子宫内膜癌中呈高表达,FIGO分期、病理分级、CDC20mRNA、CDC20蛋白是子宫内膜癌患者CDC20表达情况的影响因素。

CDC20调控Wnt/β-catenin通路在肿瘤发生发展中的作用研究进展

CDC20的高表达与肿瘤的侵袭、转移、耐药和免疫浸润以及自噬密切相关。β-catenin是Wnt/β-catenin信号通路中的关键蛋白,CDC20通过靶向调控Wnt/β-catenin信号通路影响肿瘤患者的化疗耐药、复发以及预后不良。本文针对这一相关的作用机制进行综述。

肺腺癌关键基因筛选及CDC20在肺腺癌中的表达

目的:筛选肺腺癌的关键基因,揭示肺腺癌潜在的分子机制。方法:利用3个肺腺癌相关基因芯片数据集,共包括156例肺腺癌和106例正常肺组织,进行肺腺癌潜在关键基因的筛选。结果:通过芯片数据分析得到341个重叠差异表达基因(differentially expressed gene,DEG),DEG主要富集的生物学过程是细胞外基质组织、调控化学激酶介导的信号通路、细胞对转化生长因子刺激的反应、细胞外基质组装、调控新生血管生成等。通过蛋白互作网络(protein protein interaction,PPI)筛选出10个关键基因,其中8个基因AURKA(aurora kinase A)、CDC20(cell division cycle 20)、CDH5(cadherin 5)、COL1α1(collagenⅠ,α1)、EDN1(endothelin 1)、MMP9(matrix metalloprotein 9)、PECAM1(platelet endothelial cell adhesion molecule 1)、SPP1(secreted phosphoprotein 1)与肺腺癌预后相关。其中CDC20与肺腺癌预后相关性最高,亚组分析发现,CDC20在肺腺癌各年龄段患者中的表达均明显高于正常组,CDC20在一期肺腺癌中表达低于二期、三期、四期,CDC20在腺泡型、实性为主型、混合型、透明细胞型表达高于其他亚型,CDC20在吸烟的肺腺癌患者中的表达低于不吸烟者。免疫组化检测显示CDC20在肺腺癌组织中表达明显高于正常肺组织,且腺泡型、实体型表达高于其他亚型。结论:CDC20在肺腺癌中高表达,且其高表达与肺腺癌的不良预后显著相关。CDC20有可能成为肺腺癌治疗的新靶点。

Gene-activated matrix harboring a miR20a-expressing plasmid promotes rat cranial bone augmentation

Gene-activated matrix(GAM)has a potential usefulness in bone engineering as an alternate strategy for the lasting release of osteogenic proteins but efficient methods to generate non-viral GAM remain to be established.In this study,we investigated whether an atelocollagen-based GAM containing naked-plasmid(p)DNAs encoding microRNA(miR)20a,which may promote osteogenesis in vivo via multiple pathways associated with the osteogenic differentiation of mesenchymal stem/progenitor cells(MSCs),facilitates rat cranial bone augmentation.First,we confirmed the osteoblastic differentiation functions of generated pDNA encoding miR20a(pmiR20a)in vitro,and its transfection regulated the expression of several of target genes,such as Bambi1 and PPARc,in rat bone marrow MSCs and induced the increased expression of BMP4.Then,when GAMs fabricated by mixing 100 ll of 2%bovine atelocollagen,20mg b-TCP granules and 0.5mg(3.3 lg/ll)AcGFP plasmid-vectors encoding miR20a were transplanted to rat cranial bone surface,the promoted vertical bone augmentation was clearly recognized up to 8 weeks after transplantation,as were upregulation of VEGFs and BMP4 expressions at the early stages of transplantation.Thus,GAM-based miR delivery may provide an alternative non-viral approach by improving transgene efficacy via a small sequence that can regulate the multiple pathways.

miR-9-5p对肺腺癌细胞增殖和侵袭迁移能力的调控作用及其机制

目的观察微小RNA(miR)-9-5p对肺腺癌细胞增殖和侵袭迁移能力的调控作用,并基于痉挛性截瘫20(SPG20)基因及Notch信号通路探讨相关机制。方法培养肺腺癌细胞株A549并分为抑制物组、阴性对照组和未转染组,抑制物组和阴性对照组分别转染miR-9-5p抑制物和阴性对照,未转染组不进行转染;采用MTT法检测细胞增殖能力,Transwell小室实验检测细胞侵袭能力,划痕愈合实验检测细胞迁移能力,Western blotting法检测Notch信号通路相关蛋白[E-钙黏蛋白(E-cadherin)、Notch1、Snail和基质金属蛋白酶2(MMP-2)]。采用TargetScan(http://www.targetscan.org/vert_80/)预测miR-9-5p与SPG20基因结合位点,并用双荧光素酶报告基因分析进行验证;检测肺腺癌组织和细胞中的miR-9-5p、SPG20 mRNA和蛋白;将A549细胞分为抑制物组、阴性对照组和未转染组,采用RT-PCR法检测各组细胞中的SPG20 mRNA。结果培养12、24、36、48 h时miR-9-5p抑制物组细胞增殖能力低于阴性对照组和未转染组(P均<0.05)。抑制物组穿模细胞数和细胞迁移距离低于阴性对照组和为转染组(P均<0.05)。抑制物组Notch1、Snail、MMP-2蛋白表达低于阴性对照组和未转染组,E-cadherin蛋白表达高于阴性对照组和未转染组(P均<0.05)。SPG20基因存在连续的可以与miR-9-5p互补的核苷酸序列;共转染SPG20-WT和miR-9-5p质粒的A549细胞相对荧光素酶活性低于其他细胞(P均<0.05)。肺腺癌组织和细胞中miR-9-5p高表达、SPG20mRNA和蛋白低表达(P均<0.05),肺腺癌组织中miR-9-5p与SPG20 mRNA表达呈负相关(r=-0.914,P<0.05)。抑制物组SPG20 mRNA表达高于阴性对照组和未转染组(P均<0.05)。结论miR-9-5p能够抑制肺腺癌细胞的增殖能力及侵袭、迁移能力,作用机制可能与负性调节SPG20基因表达及调控Notch信号通路有关。

RNA联合干扰对肝癌细胞CDC20和HPSE表达的影响

目的探讨RNA联合干扰(RNAico)对肝癌细胞株Hepa1-6中丝裂原调控子20(CDC20)和乙酰肝素酶(HPSE)表达的抑制作用。方法设计针对小鼠肝癌细胞Hepa1-6 CDC20和HPSE mRNA的小干扰RNA,以阳离子脂质体包埋后转染鼠肝癌细胞Hepa1-6,即RNAico;转染48 h后,采用RT-PCR和Re-al-Time PCR测定CDC20、HPSE mRNA表达的变化。结果联合干扰组CDC20和HPSE mRNA表达明显抑制,与其他各组相比,在统计学上有显著性差异(P<0.05)。结论 RNAico可同时显著抑制小鼠肝癌细胞Hepa1-6中CDC20和HPSE mRNA的表达。

CDC20在细胞分裂周期中的作用

CDC20是细胞周期相关蛋白之一。在细胞分裂周期中,CDC20是纺锤体组装检查点的靶向物和有丝分裂后期促进复合体的正调控因子,在引导细胞周期中某些蛋白质的泛素化降解和确保染色体正常分离的过程中起着重要的作用。

过表达cdc20基因对绒山羊卵母细胞体外成熟的影响

为研究过表达cdc20基因对绒山羊卵母细胞体外成熟的影响,构建了Myc-cdc20载体,在体外转录成cRNA后显微注射到绒山羊卵母细胞中,观察其对绒山羊卵母细胞体外成熟过程的影响。结果表明,在对照组卵母细胞体外培养的过程中,卵母细胞培养到8 h后发生GVBD,18 h后进入MⅠ期,直到24 h后,约75%的绒山羊卵母细胞排出第一极体。卵母细胞在GV期时显微注射g Myc-cdc20 cRNA,培养10 h后,通过Western Blotting的方法检测到了MYC-CDC20融合蛋白的表达。但统计结果显示,试验组卵母细胞各阶段发育时间和成熟率与对照并无显著差异。因此,单独过表达cdc20基因不影响绒山羊卵母细胞体外成熟过程。

超表达Cdc20基因不影响牛卵母细胞第一极体排出

CDC20(cell division cycle 20)是后期促进复合物(anaphase-promoting complex,APC)的共激活剂之一,也是纺锤体组装检查点(spindle assembly checkpoint,SAC)的靶点,在细胞周期调控中扮演重要角色.为探讨Cdc20在第一极体排出(first polar body extrusion,PBE I)中的作用,Cdc20基因被成功克隆并构建了真核表达载体pCdc20-Venus,随后用T7 Mmessage Mmachine Kit(Ambion)以线性化pCdc20-Venus为模板体外转录(in vitro transcription)获得带帽的Cdc20-Venus mRNA,将Ccdc20-Venus mRNA显微注射到体外培养的牛卵母细胞胞质中进行超量表达.结果表明,真核表达载体pCdc20-Venus转染HeLa细胞后能够正常表达,绿色荧光在细胞核周围呈弥散状分布;将Cdc20-Venus mRNA注射到牛卵母细胞胞质后,胞质内有绿色荧光出现.Cdc20-Venus mRNA注射组卵母细胞的PBE I率(48.9%)与Venus mRNA注射组卵母细胞的PBE I率(50.9%)和未注射组卵母细胞的PBE I率相比均没有显著差异(P>0.05).通过Cdc20在牛卵母细胞内的超量表达,结果显示,超量表达Cdc20基因对牛卵母细胞PBE I没有显著影响(P>0.05).

细胞分裂周期蛋白73基因缺失抑制粟酒裂殖酵母细胞的有性生殖和有丝分裂

cdc73基因编码粟酒裂殖酵母(Schizosaccharomyces Pombe)RNA聚合酶Ⅱ辅助因子Cdc73,参与G_(2)期检查点激活并调控细胞周期。但cdc73基因缺失对细胞有丝分裂动力学的调控尚不清楚。本研究采用活细胞成像、荧光蛋白标记技术探究粟酒裂殖酵母cdc73基因缺失后对细胞有性生殖和细胞有丝分裂中微管、肌动蛋白、线粒体和组蛋白动力学的影响。结果表明:在有性生殖中,cdc73基因缺失会导致子囊孢子长度增加14.23%,产4个孢子数量的细胞减少64.08%;活细胞成像结果分析发现,在有丝分裂中,分裂后期微管伸长的长度缩短11.21%,伸长时间减少17.39%;肌动蛋白环形成和收缩速率分别降低33.33%和26.09%,形成和收缩时间分别延长58.00%和40.38%;同时,肌动蛋白环、线粒体和组蛋白表达量都增加。本研究揭示了cdc73基因缺失可抑制有丝分裂中纺锤体的伸长,延缓肌动蛋白环的形成与收缩,为进一步探寻Cdc73蛋白在细胞分裂中参与调控微管和肌动蛋白动力学功能提供了一定的科学依据。

Host Interferon-Stimulated Gene 20 Inhibits Pseudorabies Virus Proliferation

Host interferon-stimulated gene 20(ISG20)exerts antiviral effects on viruses by degrading viral RNA or by enhancing IFN signaling.Here,we examined the role of ISG20 during pseudorabies virus(PRV)proliferation.We found that ISG20 modulates PRV replication by enhancing IFN signaling.Further,ISG20 expression was upregulated following PRV infection and poly(I:C)treatment.Ectopic expression of ISG20 inhibited PRV proliferation in PK15 cells,whereas knockdown of ISG20 promoted PRV proliferation.In addition,ISG20 expression upregulated IFN-βexpression and enhanced IFN downstream signaling during PRV infection.Notably,PRV UL24 suppressed the transcription of ISG20,thus antagonizing its antiviral effect.Further domain mapping analysis showed that the N terminus(amino acids 1-90)of UL24 was responsible for the inhibition of ISG20 transcription.Collectively,these findings characterize the role of ISG20 in suppressing PRV replication and increase the understanding of host-PRV interplay.

朱砂叶螨β-N-乙酰己糖胺酶基因TecHEX3的克隆及在蜕皮发育中的作用

【目的】本研究旨在探明朱砂叶螨Tetranychus cinnabarinus几丁质降解通路中的关键酶β-N-乙酰己糖胺酶基因(TecHEX3)在螨蜕皮发育中的功能,为开发新型安全的防治害螨生物农药奠定基础。【方法】利用PCR克隆朱砂叶螨TecHEX3,并进行生物信息学分析。利用RT-qPCR检测TecHEX3在朱砂叶螨不同发育阶段(卵、幼螨、若螨和成螨)以及饲喂蜕皮激素20-羟基蜕皮酮(20-hydroxyecdysone,20E)的若螨中的表达量。通过饲喂法进行朱砂叶螨雌成螨和若螨TecHEX3的RNAi,分析沉默效率,统计并观察朱砂叶螨若螨的死亡率和致死表型。【结果】成功克隆了朱砂叶螨TecHEX3(GenBank登录号:OR413561),其编码蛋白具有典型的20糖苷水解酶家族结构特征;系统发育树表明TecHEX3与二斑叶螨T.urticae TuHEX3的亲缘性最高且均属于β-N-乙酰己糖胺酶Ⅳ型。TecHEX3在朱砂叶螨不同发育阶段都有表达,在成螨期表达量最高;饲喂500 ng/μL的20E对TecHEX3的表达有最好的诱导效果。RNAi结果表明饲喂ds TecHEX3时朱砂叶螨若螨TecHEX3表达量较对照组(饲喂ds EGFP)显著下调81%;沉默TecHEX3导致朱砂叶螨蜕皮困难或蜕皮后形态异常,致死率达40.58%。【结论】TecHEX3参与朱砂叶螨几丁质降解过程,并在朱砂叶螨的蜕皮发育过程中发挥重要作用。