目的:基于 ompK36基因GD突变，建立一种快速检测产肺炎克雷伯菌碳青霉烯酶的肺炎克雷伯菌（KPC-Kp）亚胺培南最低抑菌浓度（MIC）的方法。 方法:方法学建立。收集丽水市中心医院2011年3月至2019年12月的非重复肺炎克雷伯菌258株，PCR扩增膜孔蛋白o mpK36基因以及碳青霉烯酶基因 blaKPC、 blaNDM、 blaIMP、 blaOXA-48并测序确认，微量肉汤稀释法检测菌株亚胺培南MIC值，建立基因型与MIC值的对应模式。基于该模式，设计建立实时荧光聚合酶链反应（RT-PCR）快速检测MIC值的方法。利用丽水市疾控中心2017—2019年收集的159株非重复肺炎克雷伯菌进一步验证。四格表计算敏感度、特异度， Kappa检验比较RT-PCR法与肉汤稀释法结果的一致性。 结果:258株肺炎克雷伯菌中，109株未携带碳青霉烯酶基因，65株携带 ompK36基因GD突变，127株携带 blaKPC，15株携带 blaNDM，9株携带 blaIMP，未检测到 blaOXA-48。以肉汤稀释法为标准，肺炎克雷伯菌耐药基因与亚胺培南MIC值存在3种对应模式：4种碳青霉烯酶基因均阴性时，MIC≤1 mg/L，敏感度为100%（107/107），特异度为98.4%（125/127）； blaKPC阳性而 ompK36基因GD突变阴性时，4 mg/L≤MIC≤16 mg/L，敏感度为88.2%（60/68），特异度为98.8%（164/166）； blaKPC和 ompK36基因GD突变双阳性时，MIC≥32 mg/L，敏感度为96.6%（57/59），特异度为96.6%（169/175）。RT-PCR法可准确检测 blaKPC、 blaNDM、 blaIMP、 blaOXA-48基因；在产酶菌株中o mpK36基因GD突变的RT-PCR结果与测序法100%一致。以丽水市疾控中心来源的159株肺炎克雷伯菌为样本，以肉汤稀释法为参照，RT-PCR检测亚胺培南MIC值在3种模式下敏感度和特异度均大于95%， Kappa值为0.971。 结论:基于 ompK36基因GD突变建立的RT-PCR法有助于快速判断KPC-Kp的亚胺培南MIC值范围。

Aluminum(Al)toxicity can seriously restrict crop production on acidic soils,which comprise 40％of the world's potentially arable land.The zinc finger transcription factor STOP1 has a conserved and essential function in mediating plant Al resistance.Al stress induces STOP1 accumulation via post-transcriptional regulatory mechanisms.However,the upstream signaling pathway involved in Al-triggered STOP1 accu-mulation remains unclear.Here,we report that the MEKK1-MKK1/2-MPK4 cascade positively regulates STOP1 phosphorylation and stability.Mutations of MEKK1,MKK1I2,or MPK4 lead to decreased STOP1 stability and Al resistance.Al stress induces the kinase activity of MPK4,which interacts with and phos-phorylates STOP1.The phosphorylation of STOP1 reduces its interaction with the F-box protein RAE1 that mediates STOP1 degradation,thereby leading to enhanced STOP1 stability and Al resistance.Taken together,our results suggest that the MEKK1-MKK1/2-MPK4 cascade is important for Al signaling and con-fers Al resistance through phosphorylation-mediated enhancement of STOP1 accumulation in Arabidopsis.

Seed dormancy is nature's strategic pause in the plant life cycle,a purposeful interlude during which a seed,poised on the cusp of potential growth,bides its time in a state of quiescence.This period of dormancy is a crucial adaptation,allowing the seed to withstand unfavorable environmental conditions and syn-chronize germination with optimal circumstances for growth and survival(Née et al.,2017).Dormancy is orchestrated by a complex interplay of genetic,physiological,and environmental factors,including phytohormones like abscisic acid and gibberellins(GAs),as well as specific regulators like DELAY OF GERMINATION1 and others(Née et al.,2017;Liu et al.,2020).

Seeds establish dormancy to delay germination until the arrival of a favorable growing season.In this study,we identify a fate switch comprised of the MKK3-MPK7 kinase cascade and the ethylene response factor ERF4 that is responsible for the seed state transition from dormancy to germination.We show that dormancy-breaking factors activate the MKK3-MPK7 module,which affects the expression of some α-EX-PANSIN(EXPA)genes to control seed dormancy.Furthermore,we identify a direct downstream substrate of this module,ERF4,which suppresses the expression of these EXPAs by directly binding to the GCC boxes in their exon regions.The activated MKK3-MPK7 module phosphorylates ERF4,leading to its rapid degradation and thereby releasing its inhibitory effect on the expression of these EXPAs.Collectively,our work identifies a signaling chain consisting of protein phosphorylation,degradation,and gene transcrip-tion,by which the germination promoters within the embryo sense and are activated by germination signals from ambient conditions.

Mitogen-activated protein kinase(MAPK)cascades play important roles in disease resistance in model plant species.However,the functions of MAPK signaling pathways in crop disease resistance are largely unknown.Here we report the function of HvMKK1-HvMPK4-HvWRKY1 module in barley immune system.HvMPK4 is identified to play a negative role in barley immune response against Bgh,as virus-induced gene silencing of HvMPK4 results in enhanced disease resistance whilst stably overexpressing HvMPK4 leads to super-susceptibility to Bgh infection.Furthermore,the barley MAPK kinase HvMKK1 is found to specifically interact with HvMPK4,and the activated HvMKK1DD variant specifically phosphorylates HvMPK4 in vitro.Moreover,the transcription factor HvWRKY1 is identified to be a downstream target of HvMPK4 and phosphorylated by HvMPK4 in vitro in the presence of HvMKK1DD.Phosphorylation assay coupled with mutagenesis analyses identifies S122,T284,and S347 in HvWRKY1 as the major residues phosphorylated by HvMPK4.HvWRKY1 is phosphorylated in barley at the early stages of Bgh infection,which enhances its suppression on barley immunity likely due to enhanced DNA-binding and transcriptional repression activity.Our data suggest that the HvMKK1-HvMPK4 kinase pair acts upstream of HvWRKY1 to negatively regulate barley immunity against powdery mildew.

目的 探究萸连丸(黄连、吴茱萸)对葡聚糖硫酸钠(DSS)诱导的溃疡性结肠炎小鼠脾脏记忆性滤泡辅助性T细胞(Memory follicular T helper cell,mTfh)的调节作用.方法 将 40 只BALB/c小鼠随机分为正常组、模型组、萸连丸组(0.5 g·kg-1)和美沙拉嗪组(0.3 g·kg-1),每组 10 只.采用3%DSS溶液自由饮用 7d诱导溃疡性结肠炎小鼠模型.实验过程每天记录小鼠的精神状态、粪便性状、便血及体质量情况;测量小鼠结肠长度、结肠质量,计算结肠质量指数;采用HE染色法观察结肠组织病理形态变化;ELISA法测定结肠组织中白细胞介素 6(IL-6)、IL-15 的表达水平;流式细胞术测定脾脏组织中mTfh细胞亚群的表达情况;Western Blot法测定结肠组织中Roquin-1、AMPK-α、p-AMPK-α蛋白表达水平.结果 与正常组比较,模型组小鼠的体质量显著下降(P＜0.01),结肠长度显著缩短(P＜0.01),结肠质量明显增加(P＜0.05),结肠质量指数显著升高(P＜0.01),结肠组织病理损伤严重;结肠组织中的促炎细胞因子IL-6、IL-15 表达水平显著升高(P＜0.01);脾脏组织中CD4+CCR7-CXCR5+CD62L+、CD4+CCR7+CXCR5+GL7+、CD4+CCR7-CXCR5+GL7+细胞表达水平显著升高(P＜0.01),而 CD4+CCR7+CXCR5+CD62L+细胞表达水平显著降低(P＜0.01);结肠组织中 Roquin-1、AMPK-α、p-AMPK-α蛋白表达水平明显降低(P＜0.05).与模型组比较,萸连丸组及美沙拉嗪组小鼠的体质量明显上升(P＜0.05),结肠长度显著延长(P＜0.01),结肠质量明显降低(P＜0.05),结肠质量指数显著下降(P＜0.01),结肠组织病理损伤得到较明显改善;结肠组织中的IL-6、IL-15 表达水平显著降低(P＜0.01);脾脏组织中CD4+CCR7-CXCR5+CD62L+、CD4+CCR7+CXCR5+GL7+、CD4+CCR7-CXCR5+GL7+细胞表达水平显著降低(P＜0.01),而 CD4+CCR7+CXCR5+CD62L+细胞表达水平显著升高(P＜0.01);结肠组织中 Roquin-1、AMPK-α、p-AMPK-α蛋白表达水平明显升高(P＜0.05,P＜0.01).结论 萸连丸对DSS诱导的小鼠溃疡性结肠炎具有治疗作用,能够改善结肠组织病理损伤,可能与其激活Roquin-1/AMPK-α信号通路,下调炎性细胞因子IL-6、IL-15 表达,调控mTfh细胞亚群稳态有关.

目的 为解析党参NBS-LRR(Nucleotide-binding site and leucine-rich repeat)抗病基因家族,探究党参抗根腐病机制,从而解决党参根腐病害难题,促进党参育种及产业发展.方法 基于党参响应根腐病病原菌的转录组数据,通过运用生物信息学方法对党参NBS-LRR家族基因进行理化性质、基因结构、系统发育、表达模式及互作网络分析.结果 成功鉴定到88个党参NBS-LRR家族基因,包括N、NL、CN、CNL、TN、TNL、PN共7种类型,分别有50、14、1、14、4、3、2个基因.结果表明,党参CNL及TNL类基因结构比较保守;党参CNL亚家族基因在进化过程中发生扩增;党参NBS-LRR家族基因在尖孢镰刀菌(Fusarium oxysporum)侵染条件下存在时间表达模式差异,且侵染前期(6-24 h)高表达的基因DN64786c1g6、DN64786c1g5、DN48234c0g2、DN54844c1g2、DN59747c0g3、DN56071c1g8、DN64591c1g1、DN48464c1g1、DN59886c0g1在调控党参抗病过程中发挥重要作用.其中党参的抗病蛋白DN54844c1g2可能与GLR家族互作,进而通过调节Ca2+内流参与免疫调控;DN64786c1g5可能与CYTC-1和CYTC-2互作,进而通过参与氧化还原反应参与党参响应根腐病过程;DN59747c0g3可能与MPK3互作,进而通过参与MAP信号级联、磷酸化WRKY转录因子以及参与超敏反应(HR),在党参响应根腐病过程中发挥重要作用.结论 党参NBS-LRR家族基因的鉴定及表达分析对于探究党参抗根腐病机制、发掘基因功能具有重要意义.

Mitogen-activated protein kinase(MAPK)cas-cades play pivotal roles in plant defense against phytopathogens downstream of im-mune receptor complexes.The amplitude and duration of MAPK activation must be strictly controlled,but the underlying mechanism re-mains unclear.Here,we identified Arabidopsis CPL1(C-terminal domain phosphatase-like 1)as a negative regulator of microbe-associated molecular pattern(MAMP)-triggered immunity via a forward-genetic screen.Disruption of CPL1 significantly enhanced plant resistance to Pseudomonas pathogens induced by the bac-terial peptide flg22.Furthermore,flg22-induced MPK3/MPK4/MPK6 phosphorylation was dra-matically elevated in cpl1 mutants but severely impaired in CPL1 overexpression lines,sug-gesting that CPL1 might interfere with flg22-induced MAPK activation.Indeed,CPL1 di-rectly interacted with MPK3 and MPK6,as well as the upstream MKK4 and MKK5.A firefly luciferase-based complementation assay in-dicated that the interaction between MKK4/MKK5 and MPK3/MPK6 was significantly re-duced in the presence of CPL1.These results suggest that CPL1 plays a novel regulatory role in suppressing MAMP-induced MAPK cascade activation and MAMP-triggered immunity to bacterial pathogens.

目的 探索姥鲛烷诱导的系统性红斑狼疮(SLE)模型小鼠肾小球系膜细胞过表达AMP激活蛋白激酶(AMPK)对系膜细胞增生、基质合成和炎性因子分泌的影响.方法 小鼠分为对照组和模型组,姥鲛烷诱导建立小鼠SLE模型.ELISA检测血清自身抗体水平;分离培养对照组和模型组小鼠原代肾小球系膜细胞,模型组小鼠原代肾小球系膜细胞分为模型组、空载体组和过表达组.pEGFP-C1-vector和pEGFP-C1-AMPK分别转染空载组和过表达组细胞.Western blot法检测AMPK证明其过表达情况.AMPK过表达后,采用流式细胞术检测系膜细胞的凋亡情况,Western blot法检测细胞周期蛋白D1(cyclin D1)、增殖细胞核抗原(PCNA)、P21、P27、纤连蛋白(FN)和Ⅳ型胶原蛋白(CoB)、白细胞介素6(IL-6)、IL-1β蛋白水平.结果 模型组小鼠血清抗dsDNA IgG、抗dsDNA IgM、抗Sm IgG、抗Sm IgM水平明显增加,说明建模成功.在SLE模型小鼠肾小球系膜细胞过表达AMPK后,系膜细胞凋亡率明显增加、cyclin D1、PCNA和P21蛋白水平降低、P27蛋白水平增加.FN和Col4及IL-6和IL-1β蛋白水平明显降低.结论 过表达AMPK抑制姥鲛烷诱导的狼疮鼠中肾小球系膜细胞增生,基质合成和炎症反应.

目的:观察番石榴苷(guaijaverin,GAN)对油酸诱导的大鼠H4ⅡE细胞脂质沉积的作用,并从AdipoR-1/A MPK信号通路探讨其作用机制.方法:用0.2 mmol/L的油酸诱导大鼠H4ⅡE细胞脂质沉积模型,采用0.3 mM的番石榴苷干预24小时,采用GPO· DAOS评估细胞甘油三酯的含量,以DCTM法检测总蛋白含量,以甘油三酯/蛋白含量评估脂质沉积的情况;同时采用western blotting法检测肝细胞内乙酰辅酶A羧化酶(Acetyl-CoA arboxylase,ACC)以及其各自磷酸化蛋白表达水平;以实时荧光定量PCR法检测细胞AdipoR-1mRNA含量.结果:番石榴苷具有减少肝细胞内脂质沉积、提高AMPK、ACC磷酸化水平、提高AdipoR-1 mRNA相对表达量的作用,且其减少肝细胞内脂质沉积作用可被AMPK阻滞剂Compound C所逆转.结论:番石榴苷可改善油酸诱导的大鼠H4ⅡE细胞内脂质沉积,其作用机制与通过激活AdipoR-1/AMPK信号通路有关.