【目的】制备番鸭丝裂原活化蛋白激酶1(mitogen-activated protein kinase,MAPK1)蛋白多克隆抗体,鉴定其特异性与适用性,并探究MAPK1蛋白在卵泡颗粒细胞及卵泡中的表达定位,为研究其在番鸭卵泡发育与生殖调控中的功能提供试验工具。【...【目的】制备番鸭丝裂原活化蛋白激酶1(mitogen-activated protein kinase,MAPK1)蛋白多克隆抗体,鉴定其特异性与适用性,并探究MAPK1蛋白在卵泡颗粒细胞及卵泡中的表达定位,为研究其在番鸭卵泡发育与生殖调控中的功能提供试验工具。【方法】以番鸭卵巢组织cDNA为模板,通过PCR扩增MAPK1基因并测序,运用生物信息学工具分析MAPK1蛋白理化性质及主要抗原表位,筛选免疫原区段。对免疫原序列进行大肠杆菌密码子优化并人工合成,经同源重组克隆至原核表达载体pET-30a(+),构建重组表达载体pET30a-MAPK1并转化大肠杆菌BL21(DE3)感受态细胞。在HB-PET自诱导培养基中表达、纯化MAPK1重组蛋白,将纯化蛋白与QuickAntibody-Rabbit8W佐剂混合后免疫新西兰白兔制备多克隆抗体。采用ELISA、Western blotting、细胞免疫荧光(CIF)、组织免疫荧光(TIF)和免疫组化(IHC)等方法检测多克隆抗体的效价、特异性与适用性,并以颗粒细胞标志物促卵泡激素受体(FSHR)为参照分析MAPK1表达定位。【结果】成功克隆了番鸭MAPK1基因,大小约1107 bp,编码368个氨基酸。MAPK1蛋白理论分子质量约42 ku。生物信息学预测显示,MAPK1蛋白不含信号肽和跨膜结构;第50—100、200—300和250—350位氨基酸区段亲水性良好,第30—90和200—300氨基酸区段抗原指数较高,第30—70、120—150、200—260及320—360位氨基酸区段的氨基酸表面暴露概率较大。MAPK1蛋白二级结构以α-螺旋为主,包含典型激酶保守结构,活化环呈无规则卷曲状态,主要定位于细胞质和细胞核。综合蛋白特性,最终筛选了第4—364位氨基酸作为候选免疫原区段。成功构建原核重组表达载体pET30-MAPK1,MAPK1重组蛋白以可溶性形式表达,分子质量约48 ku。成功制备了兔抗鸭MAPK1蛋白多克隆抗体,ELISA结果显示该抗体效价达1∶102400;Western blotting、CIF、TIF和IHC结果显示,制备的多克隆抗体可特异识别MAPK1重组蛋白及番鸭卵泡颗粒细胞和卵泡中的内源性MAPK1蛋白。在颗粒细胞中,MAPK1主要定位于卵泡颗粒细胞的细胞质和细胞核;在卵泡中,MAPK1主要分布于卵泡颗粒细胞层,表达定位与FSHR基本一致。【结论】试验成功制备了番鸭MAPK1多克隆抗体,该抗体特异性良好,适用于番鸭卵泡相关细胞与组织样本中MAPK1的免疫学检测。研究结果为MAPK1在卵泡发育及生殖调控中的功能研究提供了技术支撑。展开更多
目的:基于网络药理学方法探讨丹参-牡丹皮配伍治疗膝骨性关节炎(knee osteoarthritis,KOA)的作用机制。方法:在中药系统药理学数据库与分析平台(traditional Chinese medicine systems pharmacology database and analysis platform,TCM...目的:基于网络药理学方法探讨丹参-牡丹皮配伍治疗膝骨性关节炎(knee osteoarthritis,KOA)的作用机制。方法:在中药系统药理学数据库与分析平台(traditional Chinese medicine systems pharmacology database and analysis platform,TCMSP)检索并筛选丹参-牡丹皮活性成分及相关靶点蛋白,利用UniPort数据库进行靶点蛋白名转化。构建丹参-牡丹皮活性成分-靶点网络。在人类基因数据库(the human gene database,GeneCards)、Disgenet数据库、在线人类孟德尔遗传数据库(online mendelian inheritance in man,OMIM)、药物靶标数据库(therapeutic targetdatabase,TTD)、DrugBank数据库检索KOA疾病相关靶点。筛选丹参-牡丹皮作用于KOA的活性成分靶点。利用Cytoscape 3.7.2软件及其插件Bisogenet、CytoNCA对靶点进行交集分析并构建蛋白-蛋白互作(protein-protein interactions,PPI)网络图。将直接作用靶点输入核心靶基因功能注释数据库(the database for annotation visualization and integrated discovery,DAVID)进行基因本体论(gene ontology,GO)功能和京都基因与基因组百科全书(kyoto encyclopedia of genes and genomes,KEGG)通路富集分析。结果:筛选出丹参65个活性成分,牡丹皮11个活性成分。获得丹参靶点蛋白932个,牡丹皮靶点蛋白237个。通过UniPort数据库转化最终获得作用靶点214个。筛选出KOA疾病靶点1207个,得到药物与疾病共同作用靶点97个。IL-6、VEGF、TP53、ESR1、MMP、MYC、CXCL8、FOS、JUN、TNF等可能是丹参-牡丹皮治疗KOA的关键靶点;GO功能富集分析获得349个富集结果,其中包括分子功能、细胞组分、生物过程;KEGG通路富集分析获得90个相关通路。结论:丹参-牡丹皮的有效成分包括槲皮素、木樨草素等,它们通过作用于IL-6、VEGFA、TNF、MMP等靶点,主要介导MAPK信号通路,从而调控软骨细胞的凋亡与退变,减少细胞外基质的降解,抑制骨吸收,介导炎症反应,促进血管新生,加速炎症吸收和代谢分解,最终减缓软骨组织的病损进展,发挥抗KOA的治疗作用。展开更多
促分裂原活化蛋白激酶(Mitogen-Activated Protein Kinase, MAPK)级联途径能够将细胞外刺激传导至细胞内,在植物生长发育和逆境响应中发挥重要作用。为进一步探究大豆MAPK基因在盐胁迫响应中的功能,本研究对大豆MAPK基因家族成员进行了...促分裂原活化蛋白激酶(Mitogen-Activated Protein Kinase, MAPK)级联途径能够将细胞外刺激传导至细胞内,在植物生长发育和逆境响应中发挥重要作用。为进一步探究大豆MAPK基因在盐胁迫响应中的功能,本研究对大豆MAPK基因家族成员进行了系统进化分析、共线性分析、motif分析、基因结构分析、顺式作用元件分析、组织特异性表达分析以及NaCl、NaHCO_(3)、PEG和甘露醇胁迫下的表达分析,旨在解析MAPK基因家族的盐胁迫响应机制。通过比较不同非生物胁迫下的表达模式,证实该基因家族通过差异表达调控网络介导大豆对盐胁迫的适应性响应。结果表明:共筛选出19个大豆MAPK家族成员,分布于12条染色体上,其中16号染色体分布最多。理化性质分析表明,MAPK家族成员氨基酸长度为373~571 aa,分子量为41.03~62.81 kDa。系统进化树分析显示,大豆MAPK家族成员可分为4个亚家族。共线性、motif和基因结构分析表明,各亚家族成员间联系紧密,保守性高。启动子顺式作用元件分析发现,大豆MAPK家族成员启动子中含有多种激素和应激响应元件,推测该家族参与大豆非生物胁迫响应。已有研究证实B、C和D亚家族成员参与植物逆境响应机制,因此本研究未将这3族作为重点。表达模式分析显示,在A亚家族的6个成员中,GmMAPK23-4在NaCl、NaHCO_(3)、PEG和甘露醇胁迫下的表达量变化最为显著。本研究为MAPK基因家族的深入研究提供了理论依据,为进一步理解植物逆境适应性及提升作物耐逆性提供了重要参考。展开更多
The evolutionarily conserved mitogen-activated protein kinase(MAPK)cascades relay extracellular signals into cells,triggering a variety of cellular responses.We previously revealed NtMPK4 as a positive regulator of ni...The evolutionarily conserved mitogen-activated protein kinase(MAPK)cascades relay extracellular signals into cells,triggering a variety of cellular responses.We previously revealed NtMPK4 as a positive regulator of nicotine biosynthesis;however,its upstream regulation remains unclear.Here,we characterized a MAPK cascade,comprising NtMEKK1b,NtMPKK2a,and NtMPK4,that promotes nicotine biosynthesis.This signaling module transduces external cues,including jasmonate and pathogen elicitors such as flg22,into post-translational modifications that enhance transcriptional activity and pathway gene expression.NtMPKK2a physically interacts with and phosphorylates NtMPK4 in vivo,confirming its role as an upstream kinase.RNAi-mediated silencing of NtMPKK2a significantly reduced the expression of nicotine pathway genes and decreased nicotine accumulation,whereas induced-overexpression of NtMPKK2a upregulated nicotine pathway genes and increased nicotine contents in tobacco hairy roots.Overexpression of NtMPKK2a in tobacco cells enhanced the transactivation activity of a NIC2-locus Ethylene Response Factor NtERF221 on Putrescine N-methyltransferase(NtPMT)promotor,further supporting its role in promoting nicotine biosynthesis.Furthermore,we identified NtMEKK1b,a tobacco MEKK that interacts with NtMAPKK2a in yeast cells.Knock-down of NtMEKK1b in transgenic tobacco plants attenuated the expression of nicotine pathway genes and reduced nicotine contents,whereas induced-overexpression of NtMEKK1b upregulated gene expression and nicotine accumulation.Our findings uncover a previously uncharacterized MAPK cascade module,NtMEKK1b-NtMPKK2a-NtMPK4,that regulates nicotine biosynthesis,highlighting the importance of posttranslational regulation in nicotine biosynthesis.展开更多
文摘目的:基于网络药理学方法探讨丹参-牡丹皮配伍治疗膝骨性关节炎(knee osteoarthritis,KOA)的作用机制。方法:在中药系统药理学数据库与分析平台(traditional Chinese medicine systems pharmacology database and analysis platform,TCMSP)检索并筛选丹参-牡丹皮活性成分及相关靶点蛋白,利用UniPort数据库进行靶点蛋白名转化。构建丹参-牡丹皮活性成分-靶点网络。在人类基因数据库(the human gene database,GeneCards)、Disgenet数据库、在线人类孟德尔遗传数据库(online mendelian inheritance in man,OMIM)、药物靶标数据库(therapeutic targetdatabase,TTD)、DrugBank数据库检索KOA疾病相关靶点。筛选丹参-牡丹皮作用于KOA的活性成分靶点。利用Cytoscape 3.7.2软件及其插件Bisogenet、CytoNCA对靶点进行交集分析并构建蛋白-蛋白互作(protein-protein interactions,PPI)网络图。将直接作用靶点输入核心靶基因功能注释数据库(the database for annotation visualization and integrated discovery,DAVID)进行基因本体论(gene ontology,GO)功能和京都基因与基因组百科全书(kyoto encyclopedia of genes and genomes,KEGG)通路富集分析。结果:筛选出丹参65个活性成分,牡丹皮11个活性成分。获得丹参靶点蛋白932个,牡丹皮靶点蛋白237个。通过UniPort数据库转化最终获得作用靶点214个。筛选出KOA疾病靶点1207个,得到药物与疾病共同作用靶点97个。IL-6、VEGF、TP53、ESR1、MMP、MYC、CXCL8、FOS、JUN、TNF等可能是丹参-牡丹皮治疗KOA的关键靶点;GO功能富集分析获得349个富集结果,其中包括分子功能、细胞组分、生物过程;KEGG通路富集分析获得90个相关通路。结论:丹参-牡丹皮的有效成分包括槲皮素、木樨草素等,它们通过作用于IL-6、VEGFA、TNF、MMP等靶点,主要介导MAPK信号通路,从而调控软骨细胞的凋亡与退变,减少细胞外基质的降解,抑制骨吸收,介导炎症反应,促进血管新生,加速炎症吸收和代谢分解,最终减缓软骨组织的病损进展,发挥抗KOA的治疗作用。
基金supported partially by the Kentucky Tobacco Research and Development Center(KTRDC),University of Kentucky.
文摘The evolutionarily conserved mitogen-activated protein kinase(MAPK)cascades relay extracellular signals into cells,triggering a variety of cellular responses.We previously revealed NtMPK4 as a positive regulator of nicotine biosynthesis;however,its upstream regulation remains unclear.Here,we characterized a MAPK cascade,comprising NtMEKK1b,NtMPKK2a,and NtMPK4,that promotes nicotine biosynthesis.This signaling module transduces external cues,including jasmonate and pathogen elicitors such as flg22,into post-translational modifications that enhance transcriptional activity and pathway gene expression.NtMPKK2a physically interacts with and phosphorylates NtMPK4 in vivo,confirming its role as an upstream kinase.RNAi-mediated silencing of NtMPKK2a significantly reduced the expression of nicotine pathway genes and decreased nicotine accumulation,whereas induced-overexpression of NtMPKK2a upregulated nicotine pathway genes and increased nicotine contents in tobacco hairy roots.Overexpression of NtMPKK2a in tobacco cells enhanced the transactivation activity of a NIC2-locus Ethylene Response Factor NtERF221 on Putrescine N-methyltransferase(NtPMT)promotor,further supporting its role in promoting nicotine biosynthesis.Furthermore,we identified NtMEKK1b,a tobacco MEKK that interacts with NtMAPKK2a in yeast cells.Knock-down of NtMEKK1b in transgenic tobacco plants attenuated the expression of nicotine pathway genes and reduced nicotine contents,whereas induced-overexpression of NtMEKK1b upregulated gene expression and nicotine accumulation.Our findings uncover a previously uncharacterized MAPK cascade module,NtMEKK1b-NtMPKK2a-NtMPK4,that regulates nicotine biosynthesis,highlighting the importance of posttranslational regulation in nicotine biosynthesis.
