大豆PM1蛋白表达可提高酵母突变株ΔTPS2的耐热性
发布时间：2018-08-17 13:30
【摘要】：高温胁迫是影响植物生长发育的重要因素之一,主要损伤蛋白质和膜系统,导致植物生长不良、农作物减产、甚至死亡。LEA(Late embryogenesis abundant)蛋白的表达与植物抗逆保护作用有着密切联系。根据LEA蛋白的序列的保守性和一些特殊的基元序列可将其分为6组,大豆PM1蛋白属于LEA4蛋白。已有研究结果证明PM1蛋白具有结合金属离子Cu2+和Fe3+;减少羟基自由基的产生;在冻融胁迫下,PM1蛋白对LDH酶活具有保护作用,显示出PM1具有多功能保护作用。然而,PM1蛋白是否使植物具有抗高温胁迫的能力,还不清楚。本文从15种酵母突变株中筛选、并鉴定了对高温敏感的酵母突变株ΔTPS2;对氧化胁迫、Cu2+和Cd2+敏感的酵母突变株ΔYAP1;NaCl敏感的酵母突变株ΔHOG1和ΔCNB1;Cu2+和Cd2+敏感的酵母突变株ΔSOD1。其中高温敏感突变株中的TPS2基因缺失导致6-磷酸海藻糖磷酸酯酶(TPP)活性丧失,导致海藻糖合成受阻,进而细胞生长抑制、甚至死亡。将空载体pYES2-CT和带有PM1基因的pYES2-PM1两种载体转染酵母突变株ΔTPS2中。ΔTPS2/pYES2-PM1重组酵母在受到热胁迫(42℃,2h)后,在固体和液体培养基中的生长速度明显快于对照ΔTPS2/pYES2-CT,表明PM1蛋白的表达可提高突变株ΔTPS2的耐高温胁迫能力。之后,我们测量了野生型酵母BY4742/pYES2-CT、突变株ΔTPS2/pYES2-CT和重组酵母ΔTPS2/pYES2-PM1在热胁迫前后体内的海藻糖含量,三种酵母在热胁迫前海藻糖含量分别为4.0 mg/g(湿重)、0.55 mg/g(湿重)和0.78mg/g(湿重),热胁迫后分别为22.6 mg/g(湿重)、5.4 mg/g(湿重)、7.3 mg/g(湿重),表明在在ΔTPS2酵母突变株内PM1蛋白可起到提高细胞耐高温能力。为研究PM1蛋白对保护蛋白和膜系统的能力保护功能,提取了酵母可溶性总蛋白,与PM1混合后进行热处理(42℃,2h),测量A340并计算聚集度,结果表明BSA(Albumin from bovine serum)蛋白不能抑制酵母可溶性总蛋白的热聚集;低浓度HSP(Heat Shock Protein)蛋白使酵母可溶性总蛋白聚集度下降43%,高浓度HSP没有保护作用反而将酵母可溶性总蛋白的聚集度升高至105%;低浓度PM1蛋白(0.5mg/ml)不能抑制酵母可溶性总蛋白的热聚集;高浓度PM1蛋白(2mg/ml)使酵母可溶性总蛋白聚集度下降32%。海藻糖(50mg/ml)与可溶性总蛋白混合后,可使后者的热聚集度下降10%;海藻糖与低浓度PM1蛋白(0.5mg/ml)混合,可使可溶性总蛋白聚集度下降22%,表明PM1蛋白与海藻糖具有协同作用。我们进一步研究PM1蛋白对线粒体膜(电位)的保护。用染料JC-1对热胁迫前后的野生型BY4742/pYES2-CT、突变株ΔTPS2/pYES2-CT和ΔTPS2/pYES2-PM1酵母进行染色。结果表明,热胁迫前三种酵母膜损伤率分别为13.7%、12.7%和10.8%;热胁迫后,对照ΔTPS2/pYES2-CT的线粒体膜损伤率为77.0%,野生型BY4742/pYES2-CT的膜损伤率为32.8%,ΔTPS2/pYES2-PM1重组酵母的膜损伤率(44.1%)。表明PM1蛋白表达可保护酵母细胞体内的线粒体膜。综上所述,PM1蛋白可通过保护酵母蛋白质,防止蛋白的聚集,与海藻糖协同作用,可保护酵母线粒体膜及膜系统,这是PM1蛋白保护经热胁迫酵母细胞的分子机理。
[Abstract]:High temperature stress is one of the important factors affecting plant growth and development. It mainly damages the protein and membrane system, resulting in poor plant growth, crop yield reduction, and even death. The expression of LEA (Late embryogenesis abundant nt) protein is closely related to plant stress resistance and protection. According to the conservation of LEA protein sequence and some special bases. Soybean PM1 proteins belong to LEA4 proteins. Previous studies have shown that PM1 proteins bind to metal ions Cu2+ and Fe3+; reduce the production of hydroxyl radicals; and protect LDH enzyme activity under freeze-thaw stress, indicating that PM1 proteins have multifunctional protective effects. However, whether PM1 proteins make plants have multifunctional protective effects. The ability to resist high temperature stress is still unclear.Fifteen yeast mutants were screened and identified as heat-sensitive mutants TPS2, Cu2+ and Cd2+ sensitive mutants YAP1, NaCl-sensitive mutants HOG1 and CNB1, Cu2+ and Cd2+ sensitive mutants SOD1. The deletion of TPS2 gene in the strain resulted in the loss of the activity of trehalose 6-phosphate phosphatase (TPP), resulting in the inhibition of trehalose biosynthesis and cell growth, even death. The growth rate of the mutant strain TPS2/pYES2-CT was faster than that of the control strain TPS2/pYES2-CT in both solid and liquid media, indicating that the expression of PM1 protein could improve the tolerance of the mutant strain TPS2 to high temperature stress. After that, we measured the trehalose content of wild-type yeast BY4742/pYES2-CT, mutant TPS2/pYES2-CT and recombinant yeast TPS2/pYES2-PM1 before and after heat stress. The trehalose content of the three yeasts were 4.0 mg/g (wet weight), 0.55 mg/g (wet weight) and 0.78 mg/g (wet weight) before heat stress, 22.6 mg/g (wet weight), 5.4 mg/g (wet weight) and 7.3 mg/g (wet weight) after heat stress, respectively. This indicated that PM1 protein could improve the cell heat tolerance in the mutant of TPS2. The results showed that BSA (Albumin from bovine serum) protein could not inhibit the thermal aggregation of yeast soluble total protein, and low concentration of HSP (Heat Shock Protein) could make yeast soluble total egg. The albumin aggregation decreased by 43%, but the total soluble protein aggregation of yeast was increased to 105% by high concentration of HSP without protection; low concentration of PM1 protein (0.5mg/ml) could not inhibit the thermal aggregation of total soluble protein of yeast; high concentration of PM1 protein (2mg/ml) could reduce the aggregation of total soluble protein of yeast by 32%. Trehalose (50mg/ml) and total soluble protein of yeast decreased by 32%. When mixed, the thermal aggregation of the latter decreased by 10%; when trehalose was mixed with low concentration PM1 protein (0.5mg/ml), the total soluble protein aggregation decreased by 22%, indicating that PM1 protein and trehalose had a synergistic effect. We further studied the protection of PM1 protein on mitochondrial membrane (potential). The wild type BY4742/pYE before and after heat stress was treated with JC-1 dye. The results showed that the membrane damage rates of the three yeasts before heat stress were 13.7%, 12.7% and 10.8%, respectively; after heat stress, the mitochondrial membrane damage rate of the control TPS2/pYES2-CT was 77.0%, and that of the wild type BY4742/pYES2-CT was 32.8%, and that of the recombinant yeast TPS2/pYES2-PM1 was 10.8%. In summary, PM1 proteins can protect yeast mitochondrial membrane and membrane system by protecting yeast protein, preventing protein aggregation and cooperating with trehalose, which is the molecular mechanism of PM1 proteins protecting heat-stressed yeast cells.
【学位授予单位】：深圳大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q946
本文编号：2187786
[Abstract]:High temperature stress is one of the important factors affecting plant growth and development. It mainly damages the protein and membrane system, resulting in poor plant growth, crop yield reduction, and even death. The expression of LEA (Late embryogenesis abundant nt) protein is closely related to plant stress resistance and protection. According to the conservation of LEA protein sequence and some special bases. Soybean PM1 proteins belong to LEA4 proteins. Previous studies have shown that PM1 proteins bind to metal ions Cu2+ and Fe3+; reduce the production of hydroxyl radicals; and protect LDH enzyme activity under freeze-thaw stress, indicating that PM1 proteins have multifunctional protective effects. However, whether PM1 proteins make plants have multifunctional protective effects. The ability to resist high temperature stress is still unclear.Fifteen yeast mutants were screened and identified as heat-sensitive mutants TPS2, Cu2+ and Cd2+ sensitive mutants YAP1, NaCl-sensitive mutants HOG1 and CNB1, Cu2+ and Cd2+ sensitive mutants SOD1. The deletion of TPS2 gene in the strain resulted in the loss of the activity of trehalose 6-phosphate phosphatase (TPP), resulting in the inhibition of trehalose biosynthesis and cell growth, even death. The growth rate of the mutant strain TPS2/pYES2-CT was faster than that of the control strain TPS2/pYES2-CT in both solid and liquid media, indicating that the expression of PM1 protein could improve the tolerance of the mutant strain TPS2 to high temperature stress. After that, we measured the trehalose content of wild-type yeast BY4742/pYES2-CT, mutant TPS2/pYES2-CT and recombinant yeast TPS2/pYES2-PM1 before and after heat stress. The trehalose content of the three yeasts were 4.0 mg/g (wet weight), 0.55 mg/g (wet weight) and 0.78 mg/g (wet weight) before heat stress, 22.6 mg/g (wet weight), 5.4 mg/g (wet weight) and 7.3 mg/g (wet weight) after heat stress, respectively. This indicated that PM1 protein could improve the cell heat tolerance in the mutant of TPS2. The results showed that BSA (Albumin from bovine serum) protein could not inhibit the thermal aggregation of yeast soluble total protein, and low concentration of HSP (Heat Shock Protein) could make yeast soluble total egg. The albumin aggregation decreased by 43%, but the total soluble protein aggregation of yeast was increased to 105% by high concentration of HSP without protection; low concentration of PM1 protein (0.5mg/ml) could not inhibit the thermal aggregation of total soluble protein of yeast; high concentration of PM1 protein (2mg/ml) could reduce the aggregation of total soluble protein of yeast by 32%. Trehalose (50mg/ml) and total soluble protein of yeast decreased by 32%. When mixed, the thermal aggregation of the latter decreased by 10%; when trehalose was mixed with low concentration PM1 protein (0.5mg/ml), the total soluble protein aggregation decreased by 22%, indicating that PM1 protein and trehalose had a synergistic effect. We further studied the protection of PM1 protein on mitochondrial membrane (potential). The wild type BY4742/pYE before and after heat stress was treated with JC-1 dye. The results showed that the membrane damage rates of the three yeasts before heat stress were 13.7%, 12.7% and 10.8%, respectively; after heat stress, the mitochondrial membrane damage rate of the control TPS2/pYES2-CT was 77.0%, and that of the wild type BY4742/pYES2-CT was 32.8%, and that of the recombinant yeast TPS2/pYES2-PM1 was 10.8%. In summary, PM1 proteins can protect yeast mitochondrial membrane and membrane system by protecting yeast protein, preventing protein aggregation and cooperating with trehalose, which is the molecular mechanism of PM1 proteins protecting heat-stressed yeast cells.
【学位授予单位】：深圳大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q946
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