植物硝酸盐转运蛋白(Nitrate transporter,NRT)可有效转运NO-3,提升氮素利用效率。为了解析草地早熟禾(Poa pratensis)适应不同浓度及不同形态氮素、干旱胁迫机理,本研究以草地早熟禾为试材,对NRT1/PTR FAMILY 8.3基因进行克隆、生物信...植物硝酸盐转运蛋白(Nitrate transporter,NRT)可有效转运NO-3,提升氮素利用效率。为了解析草地早熟禾(Poa pratensis)适应不同浓度及不同形态氮素、干旱胁迫机理,本研究以草地早熟禾为试材,对NRT1/PTR FAMILY 8.3基因进行克隆、生物信息学分析,并分析了不同浓度及不同形态氮素以及干旱下该基因表达情况。研究结果:草地早熟禾NRT1/PTR FAMILY 8.3基因包含典型的主要协同转运蛋白超家族(Major facilitator superfamily,MFS)结构域,与二穗短柄草(Brachypodium distachyon)高度同源;荧光定量PCR分析表明,该基因根部、叶部的表达量显著高于茎部;氮浓度为7.5 mM时,不同形态氮素诱导下,NaNO3处理组有利于该基因表达;无氮、高氮组(0,15 mM NaNO3)基因的表达量显著高于适氮组(7.5 mM NaNO3);干旱胁迫可促进其表达。研究结果为探究NRT1/PTR FAMILY 8.3在不同氮素环境中的调节机制,培育氮素利用率高的优质草种提供理论基础。展开更多
Small heat shock proteins (sHSPs) are diverse and mainly function as molecular chaperones to protect organisms and cells from various stresses. In this study, hsp 18.3, one Tribolium castaneum species-specific shsp、h...Small heat shock proteins (sHSPs) are diverse and mainly function as molecular chaperones to protect organisms and cells from various stresses. In this study, hsp 18.3, one Tribolium castaneum species-specific shsp、has been identified. Quantitative real-time polymerase chain reaction illustrated that Tchsp 18.3 is expressed in all developmental stages, and is highly expressed at early pupal and late adult stages, while it is highly expressed in ovary and fat body at the adult period. Moreover, it was up-regulated 4532 ± 396-fold in response to enhanced heat stress but not to cold stress;meanwhile the lifespan of adults in ds-Tchsp 18.3 group reduced by 15.8% from control group under starvation. Laval RNA interference (RNAi) of Tchsp 18.3 caused 86.1%±4.5% arrested pupal eclosion and revealed that Tchsp 18.3 played an important role in insect development. In addition, parental RNAi of Tchsp 18.3 reduced the oviposition amount by 94.7%. These results suggest that Tchsp 18.3 is not only essential for the resistance to heat and starvation stress, but also is critical for normal development and reproduction in T. castaneum.展开更多
The cytochrome P450 (CYP) superfamily plays a key role in the oxidative metabolism of a wide range of exogenous chemicals. CYP2C8 is the principal enzyme responsible for the metabolism of the anti-cancer drug paclitax...The cytochrome P450 (CYP) superfamily plays a key role in the oxidative metabolism of a wide range of exogenous chemicals. CYP2C8 is the principal enzyme responsible for the metabolism of the anti-cancer drug paclitaxel in the human liver, and carries out the oxidative metabolism of at least 5% of clinical drugs. Polymorphisms in CYP2C8 have been closely implicated in individualized medication. CYP2C8.3, a common polymorph of CYP2C8 with dual amino acid substitutions R139K and K399R, is found primarily in Caucasians. In this study, CYP2C8.3 and its wild type (WT) CYP2C8 were expressed in E. coli, and their purified proteins were characterized by UV-visible spectroscopy, mass spectrometry, and circular dichroism. Their thermal stability, substrate binding ability, and metabolic activity against paclitaxel were investigated. The electron transfer kinetics during paclitaxel metabolism by WT CYP2C8 or CYP2C8.3 was studied by stopped-flow kinetics. The results revealed that mutations in CYP2C8.3 did not greatly influence the heme active site or protein thermal stability and paclitaxel binding ability, but the metabolic activity against paclitaxel was significantly depressed to just 11% of that of WT CYP2C8. Electron transfer from CYP reductase to CYP2C8.3 was found to be significantly slower than that to WT CYP2C8 during catalysis, and this might be the main reason for the depressed metabolic activity. Since the polymorph CYP2C8.3 is defective in catalyzing substrates of CYP2C8 in vitro, it might be expected to have important clinical and pathophysiological consequences in homozygous individuals, and this study provides valuable information in this aspect.展开更多
文摘植物硝酸盐转运蛋白(Nitrate transporter,NRT)可有效转运NO-3,提升氮素利用效率。为了解析草地早熟禾(Poa pratensis)适应不同浓度及不同形态氮素、干旱胁迫机理,本研究以草地早熟禾为试材,对NRT1/PTR FAMILY 8.3基因进行克隆、生物信息学分析,并分析了不同浓度及不同形态氮素以及干旱下该基因表达情况。研究结果:草地早熟禾NRT1/PTR FAMILY 8.3基因包含典型的主要协同转运蛋白超家族(Major facilitator superfamily,MFS)结构域,与二穗短柄草(Brachypodium distachyon)高度同源;荧光定量PCR分析表明,该基因根部、叶部的表达量显著高于茎部;氮浓度为7.5 mM时,不同形态氮素诱导下,NaNO3处理组有利于该基因表达;无氮、高氮组(0,15 mM NaNO3)基因的表达量显著高于适氮组(7.5 mM NaNO3);干旱胁迫可促进其表达。研究结果为探究NRT1/PTR FAMILY 8.3在不同氮素环境中的调节机制,培育氮素利用率高的优质草种提供理论基础。
基金the National Natural Science Foundation of China (No. 31572326 & 31172146)the Natural Science Foundation of Jiangsu Province, China (No. BK2011785)the PAPD of Jiangsu Higher Education Institutions.
文摘Small heat shock proteins (sHSPs) are diverse and mainly function as molecular chaperones to protect organisms and cells from various stresses. In this study, hsp 18.3, one Tribolium castaneum species-specific shsp、has been identified. Quantitative real-time polymerase chain reaction illustrated that Tchsp 18.3 is expressed in all developmental stages, and is highly expressed at early pupal and late adult stages, while it is highly expressed in ovary and fat body at the adult period. Moreover, it was up-regulated 4532 ± 396-fold in response to enhanced heat stress but not to cold stress;meanwhile the lifespan of adults in ds-Tchsp 18.3 group reduced by 15.8% from control group under starvation. Laval RNA interference (RNAi) of Tchsp 18.3 caused 86.1%±4.5% arrested pupal eclosion and revealed that Tchsp 18.3 played an important role in insect development. In addition, parental RNAi of Tchsp 18.3 reduced the oviposition amount by 94.7%. These results suggest that Tchsp 18.3 is not only essential for the resistance to heat and starvation stress, but also is critical for normal development and reproduction in T. castaneum.
基金supported by the National Natural Science Foundation of China (20771029)Shanghai Pujiang Talent Project (08PJ14017)+1 种基金Shang-hai Leading Academic Discipline Project (B108)Beijing Synchrotron Radiation Facility (BSRF) in the Institute of High Energy Physics,Chinese Academy of Sciences
文摘The cytochrome P450 (CYP) superfamily plays a key role in the oxidative metabolism of a wide range of exogenous chemicals. CYP2C8 is the principal enzyme responsible for the metabolism of the anti-cancer drug paclitaxel in the human liver, and carries out the oxidative metabolism of at least 5% of clinical drugs. Polymorphisms in CYP2C8 have been closely implicated in individualized medication. CYP2C8.3, a common polymorph of CYP2C8 with dual amino acid substitutions R139K and K399R, is found primarily in Caucasians. In this study, CYP2C8.3 and its wild type (WT) CYP2C8 were expressed in E. coli, and their purified proteins were characterized by UV-visible spectroscopy, mass spectrometry, and circular dichroism. Their thermal stability, substrate binding ability, and metabolic activity against paclitaxel were investigated. The electron transfer kinetics during paclitaxel metabolism by WT CYP2C8 or CYP2C8.3 was studied by stopped-flow kinetics. The results revealed that mutations in CYP2C8.3 did not greatly influence the heme active site or protein thermal stability and paclitaxel binding ability, but the metabolic activity against paclitaxel was significantly depressed to just 11% of that of WT CYP2C8. Electron transfer from CYP reductase to CYP2C8.3 was found to be significantly slower than that to WT CYP2C8 during catalysis, and this might be the main reason for the depressed metabolic activity. Since the polymorph CYP2C8.3 is defective in catalyzing substrates of CYP2C8 in vitro, it might be expected to have important clinical and pathophysiological consequences in homozygous individuals, and this study provides valuable information in this aspect.
