【目的】通过分析在逆境胁迫过程中绿僵菌菌株MAX-2的Hog1基因的转录表达,为揭示该菌株耐逆特性的分子机理提供理论依据。【方法】将具有不同耐逆能力的3个绿僵菌菌株MAX-2、BUM2495和BUM723的分生孢子分别用盐处理(0.4、0.8 mol/L Na ...【目的】通过分析在逆境胁迫过程中绿僵菌菌株MAX-2的Hog1基因的转录表达,为揭示该菌株耐逆特性的分子机理提供理论依据。【方法】将具有不同耐逆能力的3个绿僵菌菌株MAX-2、BUM2495和BUM723的分生孢子分别用盐处理(0.4、0.8 mol/L Na Cl)和紫外线照射(5、15 min),测定孢子萌发率,并提取RNA,通过半定量RT-PCR方法分析其Hog1基因的转录表达差异。【结果】从盐和紫外线逆境条件下菌株的孢子萌发率可以看出:这3个菌株的耐逆性能为MAX-2>BUM2495>BUM723,菌株间的差异都达到了极显著水平(P<0.01)。盐处理和紫外线辐射都能诱导绿僵菌Hog1基因的表达上调,不同菌株表达差异较大,MAX-2表达水平最高,BUM723表达水平最低。在较低程度的胁迫条件下(0.4 mol/L Na Cl和紫外线照射5 min),MAX-2的表达水平略高于BUM2495,而在较高程度的胁迫条件下(0.8 mol/L Na Cl和紫外线照射15 min)MAX-2的表达水平明显高于BUM2495,表明Hog1基因在较高程度压力条件下的高效表达与MAX-2的耐逆能力密切相关。【结论】绿僵菌菌株MAX-2较高的耐逆能力与其Hog1基因的高水平转录表达相关。展开更多
Arsenic is a highly toxic and carcinogenic metalloid widely dispersed in the environment, contaminating water and soil and accumulating in crops. Paradoxically, arsenic is also part of modern therapy and employed in t...Arsenic is a highly toxic and carcinogenic metalloid widely dispersed in the environment, contaminating water and soil and accumulating in crops. Paradoxically, arsenic is also part of modern therapy and employed in treating numerous ailments and diseases. Hence, inventing strategies to tune cellular arsenic uptake based on purpose is striking. Here, we describe an approach in which the arsenite uptake can be increased using a MAPK inhibitor. Employing microfluidic flow chambers in combination with optical tweezers and fluorescent microscopy, we elevated the influx of arsenite into the yeast Saccharomyces cerevisiae cells following short-term treatment with a Hog1 kinase inhibitor. The increase in arsenite uptake was followed on arsenite triggered redistribution of a reporter protein, Hsp104-GFP, which was imaged over time. The effect was even more pronounced when the yeast mother and daughter cells were analyzed disjointedly, an opportunity provided owing to single-cell analysis. Our data firstly provide a strategy to increase arsenite uptake and secondly show that arsenite triggered aggregates, previously shown to be sites of damaged proteins, are distributed asymmetrically and less accumulated in daughter cells. Inventing approaches to tune arsenite uptake has a great value for its use in environmental as well as medical applications.展开更多
Autophagy is crucial for maintaining cellular homeostasis and is linked to various dis-eases.In Saccharomyces cerevisiae,the Polymyxin B Sensitivity 2(Pbs2)protein is a member of the mitogen-activated protein kinase(M...Autophagy is crucial for maintaining cellular homeostasis and is linked to various dis-eases.In Saccharomyces cerevisiae,the Polymyxin B Sensitivity 2(Pbs2)protein is a member of the mitogen-activated protein kinase(MAPK)family and plays a role in mitophagy.To explore the potential role of Pbs2 in macroautophagy,we engineered wild-type and PBS2-deficient cells using plasmid construction and yeast transforma-tion techniques,followed by a series of autophagy assays.First,after nitrogen star-vation,the levels of autophagic activity were evaluated with the classical GFP-Atg8 cleavage assay and the Pho8Δ60 activity assay at different time points.Deleting PBS2 significantly decreased both GFP-Atg8 protein cleavage and Pho8Δ60 activity,indicat-ing that Pbs2 is essential for macroautophagy.Furthermore,the influence of Pbs2 on macroautophagy was shown to be independent of Hog1,a well-known downstream factor of Pbs2.Second,the Atg8 lipidation assay demonstrated that Atg8 lipidation levels increased upon PBS2 deletion,suggesting that Pbs2 acts after Atg8 lipidation.Third,the proteinase K protection assay indicated that the loss of PBS2 led to a higher proportion of closed autophagosomes,implying that Pbs2 impacts the later stages of macroautophagy following autophagosome closure.In conclusion,Pbs2 regulates the late stages of macroautophagy induced by nitrogen starvation.展开更多
文摘【目的】通过分析在逆境胁迫过程中绿僵菌菌株MAX-2的Hog1基因的转录表达,为揭示该菌株耐逆特性的分子机理提供理论依据。【方法】将具有不同耐逆能力的3个绿僵菌菌株MAX-2、BUM2495和BUM723的分生孢子分别用盐处理(0.4、0.8 mol/L Na Cl)和紫外线照射(5、15 min),测定孢子萌发率,并提取RNA,通过半定量RT-PCR方法分析其Hog1基因的转录表达差异。【结果】从盐和紫外线逆境条件下菌株的孢子萌发率可以看出:这3个菌株的耐逆性能为MAX-2>BUM2495>BUM723,菌株间的差异都达到了极显著水平(P<0.01)。盐处理和紫外线辐射都能诱导绿僵菌Hog1基因的表达上调,不同菌株表达差异较大,MAX-2表达水平最高,BUM723表达水平最低。在较低程度的胁迫条件下(0.4 mol/L Na Cl和紫外线照射5 min),MAX-2的表达水平略高于BUM2495,而在较高程度的胁迫条件下(0.8 mol/L Na Cl和紫外线照射15 min)MAX-2的表达水平明显高于BUM2495,表明Hog1基因在较高程度压力条件下的高效表达与MAX-2的耐逆能力密切相关。【结论】绿僵菌菌株MAX-2较高的耐逆能力与其Hog1基因的高水平转录表达相关。
文摘Arsenic is a highly toxic and carcinogenic metalloid widely dispersed in the environment, contaminating water and soil and accumulating in crops. Paradoxically, arsenic is also part of modern therapy and employed in treating numerous ailments and diseases. Hence, inventing strategies to tune cellular arsenic uptake based on purpose is striking. Here, we describe an approach in which the arsenite uptake can be increased using a MAPK inhibitor. Employing microfluidic flow chambers in combination with optical tweezers and fluorescent microscopy, we elevated the influx of arsenite into the yeast Saccharomyces cerevisiae cells following short-term treatment with a Hog1 kinase inhibitor. The increase in arsenite uptake was followed on arsenite triggered redistribution of a reporter protein, Hsp104-GFP, which was imaged over time. The effect was even more pronounced when the yeast mother and daughter cells were analyzed disjointedly, an opportunity provided owing to single-cell analysis. Our data firstly provide a strategy to increase arsenite uptake and secondly show that arsenite triggered aggregates, previously shown to be sites of damaged proteins, are distributed asymmetrically and less accumulated in daughter cells. Inventing approaches to tune arsenite uptake has a great value for its use in environmental as well as medical applications.
基金National Natural Science Foundation of China,Grant/Award Number:31970044 and 32370805。
文摘Autophagy is crucial for maintaining cellular homeostasis and is linked to various dis-eases.In Saccharomyces cerevisiae,the Polymyxin B Sensitivity 2(Pbs2)protein is a member of the mitogen-activated protein kinase(MAPK)family and plays a role in mitophagy.To explore the potential role of Pbs2 in macroautophagy,we engineered wild-type and PBS2-deficient cells using plasmid construction and yeast transforma-tion techniques,followed by a series of autophagy assays.First,after nitrogen star-vation,the levels of autophagic activity were evaluated with the classical GFP-Atg8 cleavage assay and the Pho8Δ60 activity assay at different time points.Deleting PBS2 significantly decreased both GFP-Atg8 protein cleavage and Pho8Δ60 activity,indicat-ing that Pbs2 is essential for macroautophagy.Furthermore,the influence of Pbs2 on macroautophagy was shown to be independent of Hog1,a well-known downstream factor of Pbs2.Second,the Atg8 lipidation assay demonstrated that Atg8 lipidation levels increased upon PBS2 deletion,suggesting that Pbs2 acts after Atg8 lipidation.Third,the proteinase K protection assay indicated that the loss of PBS2 led to a higher proportion of closed autophagosomes,implying that Pbs2 impacts the later stages of macroautophagy following autophagosome closure.In conclusion,Pbs2 regulates the late stages of macroautophagy induced by nitrogen starvation.
