真姬菇低温胁迫下菌丝体酶活变化及差异蛋白质组学研究
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摘要
真姬菇(Hypsizigus marmoreus)是一种具有良好的食用和药用价值的珍稀食用菌,在内销和外贸上有很大的潜力。真姬菇需要在低温刺激下才能出菇,温度是影响其生长和品质的重要因素。本研究应用蛋白质组学方法和技术,通过比较最适温度和低温胁迫条件下菌丝体蛋白质组的变化,寻找与低温胁迫紧密相关的差异蛋白,并探讨其功能;同时对两种条件下的菌丝体保护酶的活性加以测定,以期对食用菌低温诱导出菇的适应性机制有所了解,为今后克隆低温诱导相关基因奠定基础。
本研究以最适温(25℃)为对照,低温14℃为处理,通过双向电泳技术比较了处理不同时间对照和处理之间的蛋白质组差异。比较分析2-DE图谱后发现26个上调或下调的差异表达的蛋白质,经过MALDI-TOF-MS/MS初步鉴定和数据库检索,有14个差异表达的蛋白质得到鉴定。
在14个鉴定到的差异蛋白中,肌动蛋白、微管蛋白是细胞骨架的重要成分;蛋白酶体和乙酰乳酸合酶参与蛋白质的合成与分解;ATP合成酶、转醛醇酶、甲酸脱氢酶、1,5-二磷酸羧化酶参与细胞内的物质与能量代谢;鸟苷酸结合蛋白、14-3-3蛋白参与信号转导调控;新生多肽相关复合物可能参与细胞分化、发育的调控。说明真姬菇在低温胁迫下的调控可能是一个比较复杂的多蛋白和酶参与的过程。
通过酶活测定发现,低温胁迫下CAT、POD、SOD的活性都在最初的9个小时内上升较快,且均高于对照,之后开始逐渐下降,MDA含量也在12h内保持上升,之后略有下降,说明低温胁迫下真姬菇细胞膜受到一定伤害,但真姬菇对一定范围内的低温有很强的适应性。
Hypsizigus marmoreus is a kind of edible fungi which is valuable in edible and medical. It plays an important role in domestic market and foreign trade. It is need to be stimulated in low temperature while it begins fruiting. Temperature is the main factor that effects the growth and quality. In this research, we used proteomic methods and techniques, analyzed the variation of proteome in mycelium with suitable and low temperature. In order to search the different proteins related closely to cold stress and to investigat the function. Meanwhile we analyzed the activities of protective enzymes under two different conditions to know the adaptive mechanism of edible fungi in cold stress, as the basis of clone the genes inducted by low temperature in the future.
This study use the optimize tempertature (25℃) as control, 14℃as treatment. By using two-dimensional electrophoresis (2DE) and mass Spectrometry (MS) techniques, we analyzed the proteins between CK and treatment in different treating time. Twenty-six protein spots were found changed under cold stress conditions. Changed protein spots were identified first by MALDI-TOF-MS/MS, there were fourteen proteins were successfully identified.
In the fourteen differental proteins, actin proteins and tublin proteins are the important components of cytoskeleton; Proteasome and acetolactate synthase are involved in the synthesis and decomposition of protein; ATP synthase, transaldolase, formate dehydrogenase, and RuBisCO are concerned with the metabolize of energy and substance; guanine nucleotide binding protein and 14-3-3protein participate in control of signal transcription; nascent polypeptide-associated complex may be related to the differentiation and development of cell. It shows that there may be kinds of proteins and enzymes joined in the control of Hypsizygus marmoreus in low temperature.
We also studied the activity of protective enzymes. The activity of CAT、POD、SOD raised rapidly in the pre-9 hours of treatment. And were higher than those in control. After that they dropped gradually. MDA increased in the early 12 hour, later slightly declined. It accounted for the membrane of hypsizigus marmoreus were injured. But the hypsizigus marmoreus possess strong adaptability in a range of low temperature.
本研究以最适温(25℃)为对照,低温14℃为处理,通过双向电泳技术比较了处理不同时间对照和处理之间的蛋白质组差异。比较分析2-DE图谱后发现26个上调或下调的差异表达的蛋白质,经过MALDI-TOF-MS/MS初步鉴定和数据库检索,有14个差异表达的蛋白质得到鉴定。
在14个鉴定到的差异蛋白中,肌动蛋白、微管蛋白是细胞骨架的重要成分;蛋白酶体和乙酰乳酸合酶参与蛋白质的合成与分解;ATP合成酶、转醛醇酶、甲酸脱氢酶、1,5-二磷酸羧化酶参与细胞内的物质与能量代谢;鸟苷酸结合蛋白、14-3-3蛋白参与信号转导调控;新生多肽相关复合物可能参与细胞分化、发育的调控。说明真姬菇在低温胁迫下的调控可能是一个比较复杂的多蛋白和酶参与的过程。
通过酶活测定发现,低温胁迫下CAT、POD、SOD的活性都在最初的9个小时内上升较快,且均高于对照,之后开始逐渐下降,MDA含量也在12h内保持上升,之后略有下降,说明低温胁迫下真姬菇细胞膜受到一定伤害,但真姬菇对一定范围内的低温有很强的适应性。
Hypsizigus marmoreus is a kind of edible fungi which is valuable in edible and medical. It plays an important role in domestic market and foreign trade. It is need to be stimulated in low temperature while it begins fruiting. Temperature is the main factor that effects the growth and quality. In this research, we used proteomic methods and techniques, analyzed the variation of proteome in mycelium with suitable and low temperature. In order to search the different proteins related closely to cold stress and to investigat the function. Meanwhile we analyzed the activities of protective enzymes under two different conditions to know the adaptive mechanism of edible fungi in cold stress, as the basis of clone the genes inducted by low temperature in the future.
This study use the optimize tempertature (25℃) as control, 14℃as treatment. By using two-dimensional electrophoresis (2DE) and mass Spectrometry (MS) techniques, we analyzed the proteins between CK and treatment in different treating time. Twenty-six protein spots were found changed under cold stress conditions. Changed protein spots were identified first by MALDI-TOF-MS/MS, there were fourteen proteins were successfully identified.
In the fourteen differental proteins, actin proteins and tublin proteins are the important components of cytoskeleton; Proteasome and acetolactate synthase are involved in the synthesis and decomposition of protein; ATP synthase, transaldolase, formate dehydrogenase, and RuBisCO are concerned with the metabolize of energy and substance; guanine nucleotide binding protein and 14-3-3protein participate in control of signal transcription; nascent polypeptide-associated complex may be related to the differentiation and development of cell. It shows that there may be kinds of proteins and enzymes joined in the control of Hypsizygus marmoreus in low temperature.
We also studied the activity of protective enzymes. The activity of CAT、POD、SOD raised rapidly in the pre-9 hours of treatment. And were higher than those in control. After that they dropped gradually. MDA increased in the early 12 hour, later slightly declined. It accounted for the membrane of hypsizigus marmoreus were injured. But the hypsizigus marmoreus possess strong adaptability in a range of low temperature.
引文
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