为研发同时预防和控制猪圆环病毒2d基因型(PCV2d)和猪伪狂犬病病毒(PRV)的疫苗,将PCV2dORF2基因克隆到含有绿色荧光蛋白(EGFP)基因的PRV转移质粒pG中BamHⅠ位点,获得重组质粒pG-PCV2d-EGFP。运用转染试剂ZLip2000将其与PRV变异株3基因...为研发同时预防和控制猪圆环病毒2d基因型(PCV2d)和猪伪狂犬病病毒(PRV)的疫苗,将PCV2dORF2基因克隆到含有绿色荧光蛋白(EGFP)基因的PRV转移质粒pG中BamHⅠ位点,获得重组质粒pG-PCV2d-EGFP。运用转染试剂ZLip2000将其与PRV变异株3基因缺失毒株gE^(-)/g^(-)/TK^(-)PRV NY DNA转入ST细胞中,经绿色荧光蚀斑纯化,得到表达EGFP的重组病毒rPRV-PCV2d-EGFP。采用CRISPR/Cas9基因双敲除质粒敲除重组病毒中EGFP基因,经蚀斑纯化,拯救出不表达EGFP的重组病毒rPRV-PCV2d。重组病毒rPRV-PCV2d与亲本株gE^(-)/g^(-)/TK^(-)PRV NY具有相近的遗传稳定性,且能够表达PCV2d衣壳(Cap)蛋白。在6周龄小鼠免疫试验中,与商品化PCV2灭活疫苗相比,rPRV-PCV2d刺激小鼠机体诱导了更高的PCV2特异性抗体,且用PCV2d强毒株攻毒后,rPRV-PCV2d显著降低了小鼠心脏、肝脏、脾脏等组织中PCV2d载量。此外,rPRV-PCV2d在小鼠体内激发PRV特异性免疫应答,并能阻止PRV强毒对小鼠的侵袭。表明rPRV-PCV2d具有良好的免疫原性。展开更多
Mutations and rearrangements of mitochondrial genes cause plant cytoplasmic male sterility. It is a significant way to utilize hybrid vigor to enhance crop yield. Ogu cytoplasmic male sterility(CMS) is a natural cytop...Mutations and rearrangements of mitochondrial genes cause plant cytoplasmic male sterility. It is a significant way to utilize hybrid vigor to enhance crop yield. Ogu cytoplasmic male sterility(CMS) is a natural cytoplasmic male sterility type discovered in radishes, being successfully transferred to rapeseed and cruciferous vegetables. However, current studies lack depth in exploring the molecular mechanisms of its male sterility. This study confirmed that orf138 is the causal gene for Ogu CMS through the genetic transformation in Arabidopsis. Transcriptome analysis of aborted anthers in different stages suggested that differentially expressed genes(DEGs) are mainly enriched in pathways such as glycerophospholipid metabolism and arginine and proline metabolism. It reveals that key genes involved in lipid metabolism pathways are significantly down-regulated in the sterile line(OguA), including BnaGPAT1, localized within the tapetum mitochondrial and endoplasmic reticulum. This could lead to changes in the metabolism of substances like acylglycerols within the tapetum, causing disruptions in lipid metabolism. This is consistent with morphological and subcellular structural changes in the tapetum and microspore cells, as observed in the transmission electron microscopy. This abnormal lipid metabolism may trigger specific reactive oxygen species(ROS) accumulation in an oxidative stress response, ultimately leading to an aborted microspore. Our study based on transcriptome has deepened our understanding of the molecular mechanisms in Ogu CMS.展开更多
文摘为研发同时预防和控制猪圆环病毒2d基因型(PCV2d)和猪伪狂犬病病毒(PRV)的疫苗,将PCV2dORF2基因克隆到含有绿色荧光蛋白(EGFP)基因的PRV转移质粒pG中BamHⅠ位点,获得重组质粒pG-PCV2d-EGFP。运用转染试剂ZLip2000将其与PRV变异株3基因缺失毒株gE^(-)/g^(-)/TK^(-)PRV NY DNA转入ST细胞中,经绿色荧光蚀斑纯化,得到表达EGFP的重组病毒rPRV-PCV2d-EGFP。采用CRISPR/Cas9基因双敲除质粒敲除重组病毒中EGFP基因,经蚀斑纯化,拯救出不表达EGFP的重组病毒rPRV-PCV2d。重组病毒rPRV-PCV2d与亲本株gE^(-)/g^(-)/TK^(-)PRV NY具有相近的遗传稳定性,且能够表达PCV2d衣壳(Cap)蛋白。在6周龄小鼠免疫试验中,与商品化PCV2灭活疫苗相比,rPRV-PCV2d刺激小鼠机体诱导了更高的PCV2特异性抗体,且用PCV2d强毒株攻毒后,rPRV-PCV2d显著降低了小鼠心脏、肝脏、脾脏等组织中PCV2d载量。此外,rPRV-PCV2d在小鼠体内激发PRV特异性免疫应答,并能阻止PRV强毒对小鼠的侵袭。表明rPRV-PCV2d具有良好的免疫原性。
基金supported by the National Natural Science Foundation of China (31930032)。
文摘Mutations and rearrangements of mitochondrial genes cause plant cytoplasmic male sterility. It is a significant way to utilize hybrid vigor to enhance crop yield. Ogu cytoplasmic male sterility(CMS) is a natural cytoplasmic male sterility type discovered in radishes, being successfully transferred to rapeseed and cruciferous vegetables. However, current studies lack depth in exploring the molecular mechanisms of its male sterility. This study confirmed that orf138 is the causal gene for Ogu CMS through the genetic transformation in Arabidopsis. Transcriptome analysis of aborted anthers in different stages suggested that differentially expressed genes(DEGs) are mainly enriched in pathways such as glycerophospholipid metabolism and arginine and proline metabolism. It reveals that key genes involved in lipid metabolism pathways are significantly down-regulated in the sterile line(OguA), including BnaGPAT1, localized within the tapetum mitochondrial and endoplasmic reticulum. This could lead to changes in the metabolism of substances like acylglycerols within the tapetum, causing disruptions in lipid metabolism. This is consistent with morphological and subcellular structural changes in the tapetum and microspore cells, as observed in the transmission electron microscopy. This abnormal lipid metabolism may trigger specific reactive oxygen species(ROS) accumulation in an oxidative stress response, ultimately leading to an aborted microspore. Our study based on transcriptome has deepened our understanding of the molecular mechanisms in Ogu CMS.
