盐胁迫下小麦诱变系M3代幼苗生长及比较蛋白质组学研究
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摘要
本研究以小麦(Triticum aestivum L.)GA0602经化学诱变获得的小麦诱变系M3代为材料,研究了盐胁迫条件下诱变前后小麦发芽及幼苗生长的变化,进而鉴定出具有优良抗盐性的诱变系；同时利用双向电泳和质谱技术,并结合生物信息学研究方法,从蛋白质水平研究了盐胁迫下诱变前后小麦蛋白质表达丰度的变化,解析优良诱变系的盐胁迫应答蛋白的功能,为揭示诱变系耐盐机制提供实验依据。本研究获得了以下实验结果：
通过高原602及化学诱变M3代种子在盐胁迫条件下的发芽和生理指标(SOD、POD、CAT、MDA、可溶性蛋白质)的变化,并利用模糊数学隶属函数分析进行综合评价,确定了400mmol·L-1NaCl为本实验小麦材料发芽能力的临界盐浓度,化学诱变后代CA08113K的耐盐能力优于其它诱变系。
通过3种方法(TCA/丙酮法、尿素法、酚法)制备小麦幼苗总蛋白样品,利用SDS-PAGE和2-DE图谱分析确定了酚法为适合本实验材料的蛋白制备方法；对优良诱变系(GAO8113K)和对照(GAO811CK)在400mmol·L-1NaCl盐胁迫下的差异蛋白质组进行研究,以蛋白表达丰度变化上调2倍、下调1.5倍为标准,共检测到23个差异蛋白点,对其进行MALDI-TOF/TOF质谱鉴定分析,共有21个蛋白点获得了阳性鉴定,主要包括二磷酸核酮糖羧化加氧酶、果糖二磷酸醛缩酶、ATP酶合成亚基、热休克蛋白、V型氢离子ATP酶p亚基、S-腺苷甲硫氨酸合酶2等,这些蛋白在光合作用、能量代谢、物质代谢、分子伴侣和离子转运等方面发挥着重要作用。
总之,化学诱变提高了高原小麦602的抗盐性,盐胁迫条件下诱变M3代种子发芽率和抗氧化酶活性增强,并在蛋白质水平上阐明了化学诱变导致小麦抗哉机制的变化,为深入研究化学诱变系的抗盐机制奠定了基础。
This research used the chemical mutagenic wheat GAO602(Triticum aestivum L.) as the experimental materials to study the effects on seed germination and seedling growth under the condition of the salt stresses,and then identified the one which have the excellent salt resistance. we measured changes in the proteome of the mutagenic wheat using two-dimensional gel electrophoresis(2-DE)via liquid chromatography-tandem mass spectrometry,illustrating the function of salt stress response proteins in the fine mutagenesis,This result provided the evidences to reveal the salt tolerance mechanisms.The experiment results showed as follows:
As the test duration prolonged, there was a gradual increase in the content of soluble protein and MDA of the four varieties under the400mmol·L-1NaCl content stress, so was the antioxidase activity (SOD, POD, CAT). Five physical signs were selected including soluble protein, MDA, SOD, POD and CAT to evaluate the capacity of salt-tolerance by empirical distribution function and fuzzy membership function comprehensively. As a result,the mutagenic wheat GAO8113K reveals a better capacity of salt-tolerance than others,and that400mmol·L-1NaCl concentration should be the critical salt concentration to illustrate the germination of wheat seeds.
We have established a suitable and effective protocol of protein extraction for two-dimensional gel electrophoresis(2-DE) analysis in the wheat leaf tissues by three extraction methods(Trichloroacetic acid/acetone method,Urea/thiourea method,Phenol extraction method).Then the results were compared in regard to protein extraction efficiency, sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)and2-DE gels.Only protein identifications with score greater than P<0.05were considered to be positive.Statistical analysis of the density of differential spots in the2-DE gels.Twenty-three protein spots were significantly altered by exposure to the salinity environment.These proteins including ribulose-1,5-bisphosphate carboxylase activase,fructose-bisphosphate aldolase,ATP synthase subunit beta,heat shock protein,S-adenosylmethionine synthase2,oxygen evolving enhancer protein and so on. These proteins were associated with a variety of functions,including energy and material metabolism, protein metabolism,photosynthesis.
Above all,the chemical mutagenesis improved the salt resistance of GAO602wheat variety and the seed germination and antioxidant enzyme activity of the M3generation wheat increased in the salt conditions,laying a foundation to the in-depth study about the mutagenic wheat salt tolerance mechanisms in the protein level.
通过高原602及化学诱变M3代种子在盐胁迫条件下的发芽和生理指标(SOD、POD、CAT、MDA、可溶性蛋白质)的变化,并利用模糊数学隶属函数分析进行综合评价,确定了400mmol·L-1NaCl为本实验小麦材料发芽能力的临界盐浓度,化学诱变后代CA08113K的耐盐能力优于其它诱变系。
通过3种方法(TCA/丙酮法、尿素法、酚法)制备小麦幼苗总蛋白样品,利用SDS-PAGE和2-DE图谱分析确定了酚法为适合本实验材料的蛋白制备方法；对优良诱变系(GAO8113K)和对照(GAO811CK)在400mmol·L-1NaCl盐胁迫下的差异蛋白质组进行研究,以蛋白表达丰度变化上调2倍、下调1.5倍为标准,共检测到23个差异蛋白点,对其进行MALDI-TOF/TOF质谱鉴定分析,共有21个蛋白点获得了阳性鉴定,主要包括二磷酸核酮糖羧化加氧酶、果糖二磷酸醛缩酶、ATP酶合成亚基、热休克蛋白、V型氢离子ATP酶p亚基、S-腺苷甲硫氨酸合酶2等,这些蛋白在光合作用、能量代谢、物质代谢、分子伴侣和离子转运等方面发挥着重要作用。
总之,化学诱变提高了高原小麦602的抗盐性,盐胁迫条件下诱变M3代种子发芽率和抗氧化酶活性增强,并在蛋白质水平上阐明了化学诱变导致小麦抗哉机制的变化,为深入研究化学诱变系的抗盐机制奠定了基础。
This research used the chemical mutagenic wheat GAO602(Triticum aestivum L.) as the experimental materials to study the effects on seed germination and seedling growth under the condition of the salt stresses,and then identified the one which have the excellent salt resistance. we measured changes in the proteome of the mutagenic wheat using two-dimensional gel electrophoresis(2-DE)via liquid chromatography-tandem mass spectrometry,illustrating the function of salt stress response proteins in the fine mutagenesis,This result provided the evidences to reveal the salt tolerance mechanisms.The experiment results showed as follows:
As the test duration prolonged, there was a gradual increase in the content of soluble protein and MDA of the four varieties under the400mmol·L-1NaCl content stress, so was the antioxidase activity (SOD, POD, CAT). Five physical signs were selected including soluble protein, MDA, SOD, POD and CAT to evaluate the capacity of salt-tolerance by empirical distribution function and fuzzy membership function comprehensively. As a result,the mutagenic wheat GAO8113K reveals a better capacity of salt-tolerance than others,and that400mmol·L-1NaCl concentration should be the critical salt concentration to illustrate the germination of wheat seeds.
We have established a suitable and effective protocol of protein extraction for two-dimensional gel electrophoresis(2-DE) analysis in the wheat leaf tissues by three extraction methods(Trichloroacetic acid/acetone method,Urea/thiourea method,Phenol extraction method).Then the results were compared in regard to protein extraction efficiency, sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)and2-DE gels.Only protein identifications with score greater than P<0.05were considered to be positive.Statistical analysis of the density of differential spots in the2-DE gels.Twenty-three protein spots were significantly altered by exposure to the salinity environment.These proteins including ribulose-1,5-bisphosphate carboxylase activase,fructose-bisphosphate aldolase,ATP synthase subunit beta,heat shock protein,S-adenosylmethionine synthase2,oxygen evolving enhancer protein and so on. These proteins were associated with a variety of functions,including energy and material metabolism, protein metabolism,photosynthesis.
Above all,the chemical mutagenesis improved the salt resistance of GAO602wheat variety and the seed germination and antioxidant enzyme activity of the M3generation wheat increased in the salt conditions,laying a foundation to the in-depth study about the mutagenic wheat salt tolerance mechanisms in the protein level.
引文
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