大豆PM1蛋白表達(dá)可提高酵母突變株ΔTPS2的耐熱性
發(fā)布時(shí)間：2018-08-17 13:30
【摘要】：高溫脅迫是影響植物生長發(fā)育的重要因素之一,主要損傷蛋白質(zhì)和膜系統(tǒng),導(dǎo)致植物生長不良、農(nóng)作物減產(chǎn)、甚至死亡。LEA(Late embryogenesis abundant)蛋白的表達(dá)與植物抗逆保護(hù)作用有著密切聯(lián)系。根據(jù)LEA蛋白的序列的保守性和一些特殊的基元序列可將其分為6組,大豆PM1蛋白屬于LEA4蛋白。已有研究結(jié)果證明PM1蛋白具有結(jié)合金屬離子Cu2+和Fe3+;減少羥基自由基的產(chǎn)生;在凍融脅迫下,PM1蛋白對(duì)LDH酶活具有保護(hù)作用,顯示出PM1具有多功能保護(hù)作用。然而,PM1蛋白是否使植物具有抗高溫脅迫的能力,還不清楚。本文從15種酵母突變株中篩選、并鑒定了對(duì)高溫敏感的酵母突變株ΔTPS2;對(duì)氧化脅迫、Cu2+和Cd2+敏感的酵母突變株ΔYAP1;NaCl敏感的酵母突變株ΔHOG1和ΔCNB1;Cu2+和Cd2+敏感的酵母突變株ΔSOD1。其中高溫敏感突變株中的TPS2基因缺失導(dǎo)致6-磷酸海藻糖磷酸酯酶(TPP)活性喪失,導(dǎo)致海藻糖合成受阻,進(jìn)而細(xì)胞生長抑制、甚至死亡。將空載體pYES2-CT和帶有PM1基因的pYES2-PM1兩種載體轉(zhuǎn)染酵母突變株ΔTPS2中。ΔTPS2/pYES2-PM1重組酵母在受到熱脅迫(42℃,2h)后,在固體和液體培養(yǎng)基中的生長速度明顯快于對(duì)照ΔTPS2/pYES2-CT,表明PM1蛋白的表達(dá)可提高突變株ΔTPS2的耐高溫脅迫能力。之后,我們測(cè)量了野生型酵母BY4742/pYES2-CT、突變株ΔTPS2/pYES2-CT和重組酵母ΔTPS2/pYES2-PM1在熱脅迫前后體內(nèi)的海藻糖含量,三種酵母在熱脅迫前海藻糖含量分別為4.0 mg/g(濕重)、0.55 mg/g(濕重)和0.78mg/g(濕重),熱脅迫后分別為22.6 mg/g(濕重)、5.4 mg/g(濕重)、7.3 mg/g(濕重),表明在在ΔTPS2酵母突變株內(nèi)PM1蛋白可起到提高細(xì)胞耐高溫能力。為研究PM1蛋白對(duì)保護(hù)蛋白和膜系統(tǒng)的能力保護(hù)功能,提取了酵母可溶性總蛋白,與PM1混合后進(jìn)行熱處理(42℃,2h),測(cè)量A340并計(jì)算聚集度,結(jié)果表明BSA(Albumin from bovine serum)蛋白不能抑制酵母可溶性總蛋白的熱聚集;低濃度HSP(Heat Shock Protein)蛋白使酵母可溶性總蛋白聚集度下降43%,高濃度HSP沒有保護(hù)作用反而將酵母可溶性總蛋白的聚集度升高至105%;低濃度PM1蛋白(0.5mg/ml)不能抑制酵母可溶性總蛋白的熱聚集;高濃度PM1蛋白(2mg/ml)使酵母可溶性總蛋白聚集度下降32%。海藻糖(50mg/ml)與可溶性總蛋白混合后,可使后者的熱聚集度下降10%;海藻糖與低濃度PM1蛋白(0.5mg/ml)混合,可使可溶性總蛋白聚集度下降22%,表明PM1蛋白與海藻糖具有協(xié)同作用。我們進(jìn)一步研究PM1蛋白對(duì)線粒體膜(電位)的保護(hù)。用染料JC-1對(duì)熱脅迫前后的野生型BY4742/pYES2-CT、突變株ΔTPS2/pYES2-CT和ΔTPS2/pYES2-PM1酵母進(jìn)行染色。結(jié)果表明,熱脅迫前三種酵母膜損傷率分別為13.7%、12.7%和10.8%;熱脅迫后,對(duì)照ΔTPS2/pYES2-CT的線粒體膜損傷率為77.0%,野生型BY4742/pYES2-CT的膜損傷率為32.8%,ΔTPS2/pYES2-PM1重組酵母的膜損傷率(44.1%)。表明PM1蛋白表達(dá)可保護(hù)酵母細(xì)胞體內(nèi)的線粒體膜。綜上所述,PM1蛋白可通過保護(hù)酵母蛋白質(zhì),防止蛋白的聚集,與海藻糖協(xié)同作用,可保護(hù)酵母線粒體膜及膜系統(tǒng),這是PM1蛋白保護(hù)經(jīng)熱脅迫酵母細(xì)胞的分子機(jī)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.
【學(xué)位授予單位】：深圳大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q946
本文編號(hào)：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.
【學(xué)位授予單位】：深圳大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q946
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