为初步鉴定并挖掘出猪戊型肝炎病毒(Hepatitis E virus,HEV)ORF3蛋白影响HepG2细胞核黄素代谢信号通路的lncRNA-mRNA调控网络,本试验通过构建腺病毒过表达载体,制备高滴度过表达腺病毒,介导猪HEV-ORF3在HepG2细胞中实现过表达。Western ...为初步鉴定并挖掘出猪戊型肝炎病毒(Hepatitis E virus,HEV)ORF3蛋白影响HepG2细胞核黄素代谢信号通路的lncRNA-mRNA调控网络,本试验通过构建腺病毒过表达载体,制备高滴度过表达腺病毒,介导猪HEV-ORF3在HepG2细胞中实现过表达。Western blotting检测ORF3蛋白过表达成功后,运用lncRNA高通量组学测序,筛选出差异表达的lncRNA并进行靶向差异基因预测,对lncRNA靶向差异基因进行GO功能和KEGG通路富集分析,初步鉴定出与核黄素代谢信号通路相关的lncRNA-mRNA调控网络。Western blotting结果显示,在约为12 ku处出现目的条带,说明成功实现腺病毒介导猪HEV-ORF3在HepG2细胞中过表达。lncRNA高通量组学测序结果显示,共发现102个显著差异表达lncRNAs表达量上调,80个显著差异表达lncRNAs表达量下调。GO功能和KEGG通路富集分析显示,初步挖掘出lncRNA(MSTRG.13995.2)和lncRNA(MSTRG.1960.1)可能是与核黄素代谢信号通路相关的显著差异表达lncRNA,分别通过顺式调控其靶向基因APC-5和FLAD1来影响核黄素代谢信号通路。本试验初步鉴定出lncRNA(MSTRG.13995.2)-APC5和lncRNA(MSTRG.1960.1)-FLAD1可能是影响HepG2细胞核黄素代谢信号通路的lncRNA-mRNA调控网络。展开更多
The nitrogen source requirements for riboflavin production by ccpA mutant Bacillus subtilis 24A1/pMX45 were optimized using linear regression. The optimal medium components considered included 8% glucose as carbon sou...The nitrogen source requirements for riboflavin production by ccpA mutant Bacillus subtilis 24A1/pMX45 were optimized using linear regression. The optimal medium components considered included 8% glucose as carbon source, 2% yeast powder, 0.05% MgSO4 ·7H2O, and four types of nitrogen sources ： 0.1% yeast extract, 2% soybean powder, 1% corn plasm, and 0.2% （ NH4 ） 2 HPO4 in shake flask tests. Predictive ellipsoid was applied to determining the response values under the optimal levels for riboflavin production and glucose consumption. The optimal concentrations of the four types of nitrogen sources can remedy ammonium assimilative defection of ccpA mutant. Under the optimal conditions, the riboflavin yield increases to more than 5.0 g/L and 8%, glucose can be consumed completely after 60 h.展开更多
3,4-Dihydroxy-2-butanone 4-phosphate （DHBP） and GTP are the precursors for riboflavin biosynthesis. In this research, improving the precursor supply for riboflavin production was attempted by overexpressing ribB and...3,4-Dihydroxy-2-butanone 4-phosphate （DHBP） and GTP are the precursors for riboflavin biosynthesis. In this research, improving the precursor supply for riboflavin production was attempted by overexpressing ribB and engineering purine pathway in a riboflavin-producing Escherichia colt strain. Initially, ribB gene was overexpressed to increase the flux from ribulose 5-phosphate （Ru-5-P） to DHBP. Then ndk and grnk genes were overexpressed to enhance GTP supply. Subsequently, a R419L mutation was introduced into purA to reduce the flux from IMP to AMP. Finally, co-overexpression of mutant purF and prs genes further increased riboflavin production. The final strain RF18S produced 387.6 mg riboflavin · L-1 with a yield of 44.8 mg riboflavin per gram glucose in shake-flask fermentations. The final titer and yield were 72.2% and 55.6% higher than those of RF01S, respectively. It was concluded that simultaneously engineering the DHBP synthase and GTP biosynthetic pathway by rational metabolic engineering can efficiently boost riboflavin production in E. coll.展开更多
文摘基于血清代谢组学探讨乌药水提物(Linderae Radix water extract,LRWE)防治腹泻型肠易激综合征(diarrhea-predominant irritable bowel syndrome,IBS-D)的作用机制。选取2周龄雄性SD大鼠18只,随机分为对照组(control)、IBS-D组、LRWE干预组。除对照组外,其余大鼠接受“番泻叶灌胃联合束缚应激”刺激,构建IBS-D大鼠模型。LRWE干预组大鼠灌胃LRWE(5.4 g·kg^(-1)),control组、IBS-D组灌胃等体积蒸馏水,共计14 d;采用结肠扩张实验结合腹壁撤退反射(abdominal withdrawal reflex,AWR)评分评价大鼠内脏敏感性;检测大鼠粪便含水率(fecal water content,FWC)评价大鼠腹泻程度;采用苏木素-伊红(hematoxylin-eosin,HE)、过碘酸席夫(periodic acid-schiff,PAS)染色分别观察结肠组织形态学变化、杯状细胞形态及数量;应用超高效液相色谱串联质谱(ultra-high performance liquid chromatography tandem mass spectrometry,UPLC-MS/MS)进行大鼠血清代谢组学分析,筛选潜在生物标志物及其相关代谢通路。结果显示,LRWE可降低IBS-D大鼠AWR评分,改善内脏敏感性;降低IBS-D大鼠FWC,改善腹泻症状。HE染色和PAS染色显示,LRWE能改善IBS-D大鼠肠道轻度炎症,增加结肠上皮中成熟分泌态杯状细胞数量。血清代谢组学分析共筛选出25种LRWE干预IBS-D大鼠的潜在生物标志物,涉及代谢通路包括核黄素、色氨酸、甘氨酸、丝氨酸和苏氨酸代谢、乙醛酸和二羧酸代谢、半胱氨酸和蛋氨酸代谢等,其中核黄素及色氨酸代谢通路调控作用最明显。LRWE改善IBS-D大鼠内脏高敏性、腹泻等症状,其作用机制可能与促进IBS-D大鼠能量代谢及氨基酸代谢,增强肠道屏障功能、改善肠道免疫功能有关。
文摘The nitrogen source requirements for riboflavin production by ccpA mutant Bacillus subtilis 24A1/pMX45 were optimized using linear regression. The optimal medium components considered included 8% glucose as carbon source, 2% yeast powder, 0.05% MgSO4 ·7H2O, and four types of nitrogen sources ： 0.1% yeast extract, 2% soybean powder, 1% corn plasm, and 0.2% （ NH4 ） 2 HPO4 in shake flask tests. Predictive ellipsoid was applied to determining the response values under the optimal levels for riboflavin production and glucose consumption. The optimal concentrations of the four types of nitrogen sources can remedy ammonium assimilative defection of ccpA mutant. Under the optimal conditions, the riboflavin yield increases to more than 5.0 g/L and 8%, glucose can be consumed completely after 60 h.
基金supported by National High-tech R&D Program of China [2012AA02A702, 2012AA022103]
文摘3,4-Dihydroxy-2-butanone 4-phosphate （DHBP） and GTP are the precursors for riboflavin biosynthesis. In this research, improving the precursor supply for riboflavin production was attempted by overexpressing ribB and engineering purine pathway in a riboflavin-producing Escherichia colt strain. Initially, ribB gene was overexpressed to increase the flux from ribulose 5-phosphate （Ru-5-P） to DHBP. Then ndk and grnk genes were overexpressed to enhance GTP supply. Subsequently, a R419L mutation was introduced into purA to reduce the flux from IMP to AMP. Finally, co-overexpression of mutant purF and prs genes further increased riboflavin production. The final strain RF18S produced 387.6 mg riboflavin · L-1 with a yield of 44.8 mg riboflavin per gram glucose in shake-flask fermentations. The final titer and yield were 72.2% and 55.6% higher than those of RF01S, respectively. It was concluded that simultaneously engineering the DHBP synthase and GTP biosynthetic pathway by rational metabolic engineering can efficiently boost riboflavin production in E. coll.
