Pollen tube polar growth is a key physiological activity for angiosperms to complete double fertilization, which is highly dependent on the transport of polar substances mediated by secretory vesicles.The exocyst and ...Pollen tube polar growth is a key physiological activity for angiosperms to complete double fertilization, which is highly dependent on the transport of polar substances mediated by secretory vesicles.The exocyst and Sec1/Munc18(SM) proteins are involved in the regulation of the tethering and fusion of vesicles and plasma membranes, but the molecular mechanism by which they regulate pollen tube polar growth is still unclear. In this study, we found that loss of function of SEC1A, a member of the SM protein family in Arabidopsis thaliana, resulted in reducing pollen tube growth and a significant increase in pollen tube width. SEC1A was diffusely distributed in the pollen tube cytoplasm, and was more concentrated at the tip of the pollen tube. Through coimmunoprecipitation-mass spectrometry screening,protein interaction analysis and in vivo microscopy,we found that SEC1A interacted with the exocyst subunit SEC6, and they mutually affected the distribution and secretion rate at the tip of the pollen tube. Meanwhile, the functional loss of SEC1A and SEC6 significantly affected the distribution of the SNARE(soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex member SYP125 at the tip of the pollen tube, and led to the disorder of pollen tube cell wall components. Genetic analysis revealed that the pollen tube-related phenotype of the sec1a sec6 double mutant was significantly enhanced compared with their respective single mutants. Therefore, we speculated that SEC1A and SEC6 cooperatively regulate the fusion of secretory vesicles and plasma membranes in pollen tubes, thereby affecting the length and the width of pollen tubes.展开更多
目的探讨一种小鼠α-(1,2)-岩藻糖基转移酶SEC1基因敲除(SEC1^(-/-))对葡聚糖硫酸钠(dextran sulphate sodium,DSS)诱导的结肠炎小鼠中结肠炎症和肠道菌群的影响。方法采用CRISPR/Cas9技术构建SEC1^(-/-)C57BL/6小鼠,通过连续口服3%葡...目的探讨一种小鼠α-(1,2)-岩藻糖基转移酶SEC1基因敲除(SEC1^(-/-))对葡聚糖硫酸钠(dextran sulphate sodium,DSS)诱导的结肠炎小鼠中结肠炎症和肠道菌群的影响。方法采用CRISPR/Cas9技术构建SEC1^(-/-)C57BL/6小鼠,通过连续口服3%葡聚糖硫酸钠5天构建实验性结肠炎模型,设置SEC1^(-/-)结肠炎组(14只)、野生型结肠炎组(15只)和野生型对照组(6只)共3组。通过疾病活动度评分和体质量变化评估结肠炎严重程度,收集小鼠升结肠内粪便样本,采用高通量16s-rDNA测序技术检测粪便菌群组分,通过生物信息分析比较肠道菌群组分的差异。组间比较采用方差分析、独立样本t检验或秩和检验。结果饮用DSS第7天,与野生型结肠炎组比较,SEC1^(-/-)结肠炎小鼠体质量显著下降(77.9%±4.7%vs 87.0%±6.2%,P<0.05),而疾病活动指数显著升高(7.3±2.8 vs 9.4±2.6,P<0.05)。野生型结肠炎小鼠Simpson指数显著低于对照组小鼠(0.83 vs 0.91,P<0.05),但两组小鼠肠道菌群OTU个数、Shannon和chao1指数比较,差异均无统计学意义(P均>0.05)。与野生型结肠炎组比较,SEC1^(-/-)结肠炎组肠道菌群的OTU个数显著增多(305 vs 194,P<0.05),Shannon和chao1指数均显著增加(4.71 vs 4.09;332 vs 207;P均<0.05),乳酸杆菌属(28.95 vs 2.23)、梭菌属(0.13 vs 0.01)和糖单胞菌属(1.82 vs 0.09)的丰度均显著增多(P均<0.05)。结论SEC1基因敲除使葡聚糖硫酸钠诱导的结肠炎小鼠模型严重程度加重,并影响结肠炎小鼠的肠道菌群组成。展开更多
目的探讨IQSEC1是否通过病毒蛋白PB1调控甲型流感病毒的增殖。方法首先克隆甲型流感病毒[A/Shanghai/02/2013(H7N9)]的8个基因;其次,通过免疫共沉淀检测IQ模体Sec7结构域蛋白1(IQSEC1)与聚合酶PB1(PB1)存在相互作用;此外,通过过表达或...目的探讨IQSEC1是否通过病毒蛋白PB1调控甲型流感病毒的增殖。方法首先克隆甲型流感病毒[A/Shanghai/02/2013(H7N9)]的8个基因;其次,通过免疫共沉淀检测IQ模体Sec7结构域蛋白1(IQSEC1)与聚合酶PB1(PB1)存在相互作用;此外,通过过表达或者敲低IQSEC1的方法检测IQSEC1对PB1核定位的影响;最后,过表达或者敲低IQSEC1后检测Influenza A virus[A/Shanghai/02/2013(H7N9)]。结果病毒感染条件下,外源IQSEC1和PB1存在相互作用。当过表达IQSEC1时,细胞中IQSEC1的表达量上升,相应的PB1在细胞核中的定位减少;当用敲低IQSEC1时,细胞中IQSEC1的表达量下降,相应的PB1在细胞核中的定位上升。过表达IQSEC1后,甲型流感病毒的增殖水平下降(P<0.05)。敲低IQSEC1后,甲型流感病毒的增殖水平上升(P<0.05)。结论IQSEC1通过减少甲型流感病毒蛋白PB1的核定位抑制甲型流感病毒的增殖。展开更多
目的:探讨脂多糖(lipopolysaccharide,LPS)模拟的炎症环境是否通过下调转运及高尔基体组织蛋白1(transport and Golgi organization protein 1,TANGO1)表达干扰含Sec1家族结构域蛋白1(Sec1 family domain-containing 1,SCFD1)功能,进而...目的:探讨脂多糖(lipopolysaccharide,LPS)模拟的炎症环境是否通过下调转运及高尔基体组织蛋白1(transport and Golgi organization protein 1,TANGO1)表达干扰含Sec1家族结构域蛋白1(Sec1 family domain-containing 1,SCFD1)功能,进而导致血管内皮细胞功能障碍和炎症恶化。方法:以人脐静脉内皮细胞为细胞模型,设置二甲基亚砜(dimethyl sulfoxide,DMSO)组(培养液中加入0.1%DMSO);LPS组培养液中加入(1 mg/L的LPS);OE-NC组(细胞转染过表达质粒空载体);LPS+TANGO1过表达组(细胞转染过表达质粒后,再加入1 mg/L的LPS处理),每组重复样本数为3。采用内质网与高尔基体荧光探针进行共标记,观察荧光强度变化;Actin-Tracker Red检测TANGO1过表达对细胞骨架重塑的影响;Western blot检测TANGO1、SCFD1、肌醇需求酶1和剪接型X盒结合蛋白1的表达;流式细胞仪检测细胞的凋亡情况;通过检测活性氧、一氧化氮、超氧化物歧化酶和丙二醛水平以评估内皮细胞氧化应激水平。利用免疫共沉淀实验和敲减SCFD1同时过表达TANGO1的共转染实验,验证TANGO1与SCFD1的相互作用关系。结果:与DMSO组相比,LPS组显著促进细胞氧化应激水平(P<0.01),并增加细胞凋亡率(P<0.01);同时,LPS显著增强内质网荧光强度(P<0.01),降低高尔基体荧光强度(P<0.01),诱导细胞骨架重塑异常(伪足收缩),并下调TANGO1和SCFD1蛋白表达(P<0.01)。在LPS刺激下,过表达TANGO1可有效拮抗上述LPS诱导的细胞损伤(P<0.01)。免疫共沉淀实验证实TANGO1与SCFD1存在直接相互作用,过表达SCFD1对TANGO1的表达水平无显著影响,敲减SCFD1后,TANGO1缓解内质网应激的功能受阻。结论:LPS可通过下调TANGO1/SCFD1表达,破坏内质网-高尔基体稳态,导致血管内皮细胞氧化应激与凋亡,而过表达TANGO1可维持内质网-高尔基体稳态,降低细胞凋亡且发挥保护作用。展开更多
基金supported by the National Natural Science Foundation of China (32170330, 32170331 and 31970195)Hainan Yazhou Bay Seed Lab (B23YQ1510)the Fundamental Research Funds for the Central Universities (lzujbky-2022-ey06)。
文摘Pollen tube polar growth is a key physiological activity for angiosperms to complete double fertilization, which is highly dependent on the transport of polar substances mediated by secretory vesicles.The exocyst and Sec1/Munc18(SM) proteins are involved in the regulation of the tethering and fusion of vesicles and plasma membranes, but the molecular mechanism by which they regulate pollen tube polar growth is still unclear. In this study, we found that loss of function of SEC1A, a member of the SM protein family in Arabidopsis thaliana, resulted in reducing pollen tube growth and a significant increase in pollen tube width. SEC1A was diffusely distributed in the pollen tube cytoplasm, and was more concentrated at the tip of the pollen tube. Through coimmunoprecipitation-mass spectrometry screening,protein interaction analysis and in vivo microscopy,we found that SEC1A interacted with the exocyst subunit SEC6, and they mutually affected the distribution and secretion rate at the tip of the pollen tube. Meanwhile, the functional loss of SEC1A and SEC6 significantly affected the distribution of the SNARE(soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex member SYP125 at the tip of the pollen tube, and led to the disorder of pollen tube cell wall components. Genetic analysis revealed that the pollen tube-related phenotype of the sec1a sec6 double mutant was significantly enhanced compared with their respective single mutants. Therefore, we speculated that SEC1A and SEC6 cooperatively regulate the fusion of secretory vesicles and plasma membranes in pollen tubes, thereby affecting the length and the width of pollen tubes.
文摘目的探讨一种小鼠α-(1,2)-岩藻糖基转移酶SEC1基因敲除(SEC1^(-/-))对葡聚糖硫酸钠(dextran sulphate sodium,DSS)诱导的结肠炎小鼠中结肠炎症和肠道菌群的影响。方法采用CRISPR/Cas9技术构建SEC1^(-/-)C57BL/6小鼠,通过连续口服3%葡聚糖硫酸钠5天构建实验性结肠炎模型,设置SEC1^(-/-)结肠炎组(14只)、野生型结肠炎组(15只)和野生型对照组(6只)共3组。通过疾病活动度评分和体质量变化评估结肠炎严重程度,收集小鼠升结肠内粪便样本,采用高通量16s-rDNA测序技术检测粪便菌群组分,通过生物信息分析比较肠道菌群组分的差异。组间比较采用方差分析、独立样本t检验或秩和检验。结果饮用DSS第7天,与野生型结肠炎组比较,SEC1^(-/-)结肠炎小鼠体质量显著下降(77.9%±4.7%vs 87.0%±6.2%,P<0.05),而疾病活动指数显著升高(7.3±2.8 vs 9.4±2.6,P<0.05)。野生型结肠炎小鼠Simpson指数显著低于对照组小鼠(0.83 vs 0.91,P<0.05),但两组小鼠肠道菌群OTU个数、Shannon和chao1指数比较,差异均无统计学意义(P均>0.05)。与野生型结肠炎组比较,SEC1^(-/-)结肠炎组肠道菌群的OTU个数显著增多(305 vs 194,P<0.05),Shannon和chao1指数均显著增加(4.71 vs 4.09;332 vs 207;P均<0.05),乳酸杆菌属(28.95 vs 2.23)、梭菌属(0.13 vs 0.01)和糖单胞菌属(1.82 vs 0.09)的丰度均显著增多(P均<0.05)。结论SEC1基因敲除使葡聚糖硫酸钠诱导的结肠炎小鼠模型严重程度加重,并影响结肠炎小鼠的肠道菌群组成。
文摘目的探讨IQSEC1是否通过病毒蛋白PB1调控甲型流感病毒的增殖。方法首先克隆甲型流感病毒[A/Shanghai/02/2013(H7N9)]的8个基因;其次,通过免疫共沉淀检测IQ模体Sec7结构域蛋白1(IQSEC1)与聚合酶PB1(PB1)存在相互作用;此外,通过过表达或者敲低IQSEC1的方法检测IQSEC1对PB1核定位的影响;最后,过表达或者敲低IQSEC1后检测Influenza A virus[A/Shanghai/02/2013(H7N9)]。结果病毒感染条件下,外源IQSEC1和PB1存在相互作用。当过表达IQSEC1时,细胞中IQSEC1的表达量上升,相应的PB1在细胞核中的定位减少;当用敲低IQSEC1时,细胞中IQSEC1的表达量下降,相应的PB1在细胞核中的定位上升。过表达IQSEC1后,甲型流感病毒的增殖水平下降(P<0.05)。敲低IQSEC1后,甲型流感病毒的增殖水平上升(P<0.05)。结论IQSEC1通过减少甲型流感病毒蛋白PB1的核定位抑制甲型流感病毒的增殖。
文摘目的:探讨脂多糖(lipopolysaccharide,LPS)模拟的炎症环境是否通过下调转运及高尔基体组织蛋白1(transport and Golgi organization protein 1,TANGO1)表达干扰含Sec1家族结构域蛋白1(Sec1 family domain-containing 1,SCFD1)功能,进而导致血管内皮细胞功能障碍和炎症恶化。方法:以人脐静脉内皮细胞为细胞模型,设置二甲基亚砜(dimethyl sulfoxide,DMSO)组(培养液中加入0.1%DMSO);LPS组培养液中加入(1 mg/L的LPS);OE-NC组(细胞转染过表达质粒空载体);LPS+TANGO1过表达组(细胞转染过表达质粒后,再加入1 mg/L的LPS处理),每组重复样本数为3。采用内质网与高尔基体荧光探针进行共标记,观察荧光强度变化;Actin-Tracker Red检测TANGO1过表达对细胞骨架重塑的影响;Western blot检测TANGO1、SCFD1、肌醇需求酶1和剪接型X盒结合蛋白1的表达;流式细胞仪检测细胞的凋亡情况;通过检测活性氧、一氧化氮、超氧化物歧化酶和丙二醛水平以评估内皮细胞氧化应激水平。利用免疫共沉淀实验和敲减SCFD1同时过表达TANGO1的共转染实验,验证TANGO1与SCFD1的相互作用关系。结果:与DMSO组相比,LPS组显著促进细胞氧化应激水平(P<0.01),并增加细胞凋亡率(P<0.01);同时,LPS显著增强内质网荧光强度(P<0.01),降低高尔基体荧光强度(P<0.01),诱导细胞骨架重塑异常(伪足收缩),并下调TANGO1和SCFD1蛋白表达(P<0.01)。在LPS刺激下,过表达TANGO1可有效拮抗上述LPS诱导的细胞损伤(P<0.01)。免疫共沉淀实验证实TANGO1与SCFD1存在直接相互作用,过表达SCFD1对TANGO1的表达水平无显著影响,敲减SCFD1后,TANGO1缓解内质网应激的功能受阻。结论:LPS可通过下调TANGO1/SCFD1表达,破坏内质网-高尔基体稳态,导致血管内皮细胞氧化应激与凋亡,而过表达TANGO1可维持内质网-高尔基体稳态,降低细胞凋亡且发挥保护作用。
