盐胁迫与赤霉素（GA_3）处理下水稻幼苗的蛋白质组学分析
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摘要
盐胁迫常造成植物的生长受抑制,而植物逆境响应过程大多与内源激素的信号转导相关,研究激素调控植物对环境适应的机制对提高植物的耐盐性具有重要的意义。目前,外源水杨酸(SA)、脱落酸(ABA)、茉莉酸甲酯(Me-Ja)对盐胁迫下粳稻日本晴(Oryza sativa L. Nipponbare)种子萌发生长的影响尚未明确,虽然有研究认为赤霉酸(GA3)可以缓解盐胁迫对水稻生长的抑制作用,但对该过程内在机理的研究还有待深入展开。
本文研究了盐胁迫对日本晴萌发生长的影响;外源SA、ABA、Me-Ja和GA3对日本晴盐胁迫的作用;从蛋白质组学的角度研究了盐胁迫与GA3对水稻幼苗生长的影响,并从中找出与提高水稻幼苗耐盐性相关的蛋白。将水稻幼苗分别以H2O,5 g/L NaCl,5 g/L NaCl + 100μmol/L GA3,100μmol/L GA3培养48 h后提取其芽部的蛋白质,利用双向电泳(2-DE)技术分离分析出相关的差异表达蛋白质斑点,将蛋白质斑点酶切后,采用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)进行分析鉴定。同时,本文对基于Pro-Q Diamond荧光染色的蛋白质磷酸化修饰研究方法以及荧光差异凝胶电泳(2-D DIGE)分离技术在本课题中的应用进行了初步探索。通过上述研究,获得主要结果如下:
1.盐胁迫能够显著抑制日本晴种子的萌发;外源水杨酸、脱落酸和茉莉酸甲酯对日本晴的5 g/L NaCl胁迫无显著缓解作用,在高浓度时使盐胁迫对种子萌发的抑制效应更加明显;外源GA3可以显著的缓解5 g/L NaCl的抑制作用。
2.经双向电泳分离分析,得到12个受盐胁迫调控和1个受GA3调控的差异表达蛋白质斑点,其中11个蛋白质斑点分别被鉴定为谷氨酰-tRNA还原酶(Glutamyl-tRNA reductase,GluTR)、烯醇酶(Enolase)、Salt stress-induced protein (Salt protein)、Os09g0249700、Hypothetical protein OsJ_014066、Putative chaperonin 21 precursor、Os04g0659300、Hypothetical protein OsJ_025258、RuBisCO activase small isoform precursor、Isoflavone reductase-like蛋白和葡萄糖磷酸变位酶(phosphoglucomutase)。而isoflavone reductase-like蛋白与葡萄糖磷酸变位酶可能在外源GA3提高水稻幼苗耐盐性的途径中发挥了一定的作用。
3.利用Pro-Q Diamond荧光染料,在H2O,5 g/L NaCl,5 g/L NaCl + 100μmol/L GA3,100μmol/L GA3培养的四组材料的2-DE凝胶图谱中分别平均检测到602、566、649和697个磷酸化蛋白;在2-D DIGE分离试验所得的凝胶图谱中,平均有2 703个蛋白质斑点被检出。
Salt-stress always leads to growth inhibition of plants. For plant stress response process is mostly associated with endogenous hormone-related signal transduction, Study on the mechanism how hormones adapt plant to the environment is very important to improve the salt tolerance of plants. Hitherto, the effect of exogenous salicylic acid (SA), Abscisic Acid (ABA), methyl jasmonate (Me-JA) on salt-stressed rice (Oryza sativa L. Nipponbare) seed germination is uncertain. Though studies have shown that exogenous gibberellic acid (GA3) can alleviate salt stress on rice growth inhibition, the internal mechanism of this process yet need to be well studied.
In this paper, we studied the inhibition effect of salt stress on seed germination, and the effect of exogenous SA, ABA, Me-JA and GA3 on the inhibition of salt stress. We also traced the effect of salt-stress and GA3 on rice seedlings from the perspective of proteomics, and then found out proteins which relate to the mechanism GA3 promote salt tolerance of rice. Rice shoots of 5-day-old seedlings grown on H2O, 5 g/L NaCl, 5 g/L NaCl + 100μmol/L GA3, and 100μmol/L GA3 for 48 h were used for protein extraction. The differential expressed proteins revealed by two-dimensional polyacrylamide gel electrophoresis (2-DE) were digested and then analyzed and identified by Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS). This paper also preliminary studied on the application of protein phosphorylation research technology based on the Pro-Q Diamond fluorescent staining as well as fluorescence two dimensional difference gel electrophoresis (2-D DIGE) technology on the proteomics analysis affected by salt and GA3 in rice.
The main results of this experiment are as follows:
1. Salt stress can significantly inhibit seed germination Nipponbare; Exogenous Salicylic Acid, Abscisic Acid and Methyl Jasmonate can not relieve the inhibition of 5 g/L NaCl stress. While high concentrations of these hormones lead to more significant of the inhibition effect induced by salt; Exogenous GA3 can significantly relieve the inhibitory effect of 5 g/L NaCl.
2. After separated and analyzed by 2-DE, twelve salt regulated and 1 GA3 regulated protein were obtained. Eleven of these proteins were identified as Glutamyl-tRNA reductase (GluTR), Enolase, Salt stress-induced protein (Salt protein), Os09g0249700, Hypothetical protein OsJ_014066, Putative chaperonin 21 precursor, Os04g0659300, Hypothetical protein OsJ_025258, RuBisCO activase small isoform precursor, Isoflavone reductase-like protein and phosphoglucomutase. Isoflavone reductase-like protein and phosphoglucomutase may play important role in the mechanism, through which exogenous GA3 relieved the salt induced inhibition of rice seedlings.
3. By using Pro-Q Diamond fluorescent dye, an average of 602, 566, 649 and 697 phosphorylated proteins were detected from 2-DE gels from the four treatment groups (H2O, 5 g/L NaCl, 5 g/L NaCl + 100μmol/L GA3 and 100μmol/L GA3); In 2-D DIGE images, an average of 2 703 protein spots were detected.
本文研究了盐胁迫对日本晴萌发生长的影响;外源SA、ABA、Me-Ja和GA3对日本晴盐胁迫的作用;从蛋白质组学的角度研究了盐胁迫与GA3对水稻幼苗生长的影响,并从中找出与提高水稻幼苗耐盐性相关的蛋白。将水稻幼苗分别以H2O,5 g/L NaCl,5 g/L NaCl + 100μmol/L GA3,100μmol/L GA3培养48 h后提取其芽部的蛋白质,利用双向电泳(2-DE)技术分离分析出相关的差异表达蛋白质斑点,将蛋白质斑点酶切后,采用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)进行分析鉴定。同时,本文对基于Pro-Q Diamond荧光染色的蛋白质磷酸化修饰研究方法以及荧光差异凝胶电泳(2-D DIGE)分离技术在本课题中的应用进行了初步探索。通过上述研究,获得主要结果如下:
1.盐胁迫能够显著抑制日本晴种子的萌发;外源水杨酸、脱落酸和茉莉酸甲酯对日本晴的5 g/L NaCl胁迫无显著缓解作用,在高浓度时使盐胁迫对种子萌发的抑制效应更加明显;外源GA3可以显著的缓解5 g/L NaCl的抑制作用。
2.经双向电泳分离分析,得到12个受盐胁迫调控和1个受GA3调控的差异表达蛋白质斑点,其中11个蛋白质斑点分别被鉴定为谷氨酰-tRNA还原酶(Glutamyl-tRNA reductase,GluTR)、烯醇酶(Enolase)、Salt stress-induced protein (Salt protein)、Os09g0249700、Hypothetical protein OsJ_014066、Putative chaperonin 21 precursor、Os04g0659300、Hypothetical protein OsJ_025258、RuBisCO activase small isoform precursor、Isoflavone reductase-like蛋白和葡萄糖磷酸变位酶(phosphoglucomutase)。而isoflavone reductase-like蛋白与葡萄糖磷酸变位酶可能在外源GA3提高水稻幼苗耐盐性的途径中发挥了一定的作用。
3.利用Pro-Q Diamond荧光染料,在H2O,5 g/L NaCl,5 g/L NaCl + 100μmol/L GA3,100μmol/L GA3培养的四组材料的2-DE凝胶图谱中分别平均检测到602、566、649和697个磷酸化蛋白;在2-D DIGE分离试验所得的凝胶图谱中,平均有2 703个蛋白质斑点被检出。
Salt-stress always leads to growth inhibition of plants. For plant stress response process is mostly associated with endogenous hormone-related signal transduction, Study on the mechanism how hormones adapt plant to the environment is very important to improve the salt tolerance of plants. Hitherto, the effect of exogenous salicylic acid (SA), Abscisic Acid (ABA), methyl jasmonate (Me-JA) on salt-stressed rice (Oryza sativa L. Nipponbare) seed germination is uncertain. Though studies have shown that exogenous gibberellic acid (GA3) can alleviate salt stress on rice growth inhibition, the internal mechanism of this process yet need to be well studied.
In this paper, we studied the inhibition effect of salt stress on seed germination, and the effect of exogenous SA, ABA, Me-JA and GA3 on the inhibition of salt stress. We also traced the effect of salt-stress and GA3 on rice seedlings from the perspective of proteomics, and then found out proteins which relate to the mechanism GA3 promote salt tolerance of rice. Rice shoots of 5-day-old seedlings grown on H2O, 5 g/L NaCl, 5 g/L NaCl + 100μmol/L GA3, and 100μmol/L GA3 for 48 h were used for protein extraction. The differential expressed proteins revealed by two-dimensional polyacrylamide gel electrophoresis (2-DE) were digested and then analyzed and identified by Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS). This paper also preliminary studied on the application of protein phosphorylation research technology based on the Pro-Q Diamond fluorescent staining as well as fluorescence two dimensional difference gel electrophoresis (2-D DIGE) technology on the proteomics analysis affected by salt and GA3 in rice.
The main results of this experiment are as follows:
1. Salt stress can significantly inhibit seed germination Nipponbare; Exogenous Salicylic Acid, Abscisic Acid and Methyl Jasmonate can not relieve the inhibition of 5 g/L NaCl stress. While high concentrations of these hormones lead to more significant of the inhibition effect induced by salt; Exogenous GA3 can significantly relieve the inhibitory effect of 5 g/L NaCl.
2. After separated and analyzed by 2-DE, twelve salt regulated and 1 GA3 regulated protein were obtained. Eleven of these proteins were identified as Glutamyl-tRNA reductase (GluTR), Enolase, Salt stress-induced protein (Salt protein), Os09g0249700, Hypothetical protein OsJ_014066, Putative chaperonin 21 precursor, Os04g0659300, Hypothetical protein OsJ_025258, RuBisCO activase small isoform precursor, Isoflavone reductase-like protein and phosphoglucomutase. Isoflavone reductase-like protein and phosphoglucomutase may play important role in the mechanism, through which exogenous GA3 relieved the salt induced inhibition of rice seedlings.
3. By using Pro-Q Diamond fluorescent dye, an average of 602, 566, 649 and 697 phosphorylated proteins were detected from 2-DE gels from the four treatment groups (H2O, 5 g/L NaCl, 5 g/L NaCl + 100μmol/L GA3 and 100μmol/L GA3); In 2-D DIGE images, an average of 2 703 protein spots were detected.
引文
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