狗牙根干旱相关蛋白及代谢产物研究
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摘要
干旱是限制植物生长的主要非生物因素之一,能够引起植物细胞在各个水平上发生改变。目前,与狗牙根抗旱相关的研究多集中在形态与生理方面,对干旱相关蛋白及代谢产物的研究甚少。本论文以抗旱性不同的狗牙根基因型为试验材料,借助蛋白质组、免疫印迹分析、代谢组等技术,开展了以下三个方面的工作:一、狗牙根干旱相关蛋白的研究;二、狗牙根干旱相关蛋白的免疫印迹分析;三、狗牙根干旱相关代谢产物的研究。主要研究内容与结果如下:
1、以狗牙根基因型‘Tifway’与‘C299’为试验材料,在生长箱内进行了为期15 d的干旱处理。干旱胁迫降低了狗牙根2个基因型的叶片相对含水量(RWC)与光化学效率(Fv/Fm),提高了相对电导率(EL)的水平,但上述指标在‘Tifway’中的变化幅度比在‘C299’中要小。在试验处理第10 d时,分别选取正常生长与干旱处理的2个基因型叶片,进行了全蛋白的提取与双向电泳分离(2-DE)。凝胶图像分析结果显示,干旱胁迫总共引起了54个蛋白表达量的显著性改变,其中有32个蛋白的表达量上调,有22个蛋白的表达量下调。质谱(MS)分析结果显示,这些干旱响应蛋白的功能涉及到代谢、能量、细胞生长/分化、蛋白合成以及逆境防御等多个过程。通过对抗旱性不同的‘Tifway’与‘C299’进行差异蛋白比较,发现狗牙根的抗旱性可能与光合作用相关蛋白(Chl a-b、ATPSα、PRK、Rubisco)表达量的维持,抗氧化蛋白酶(SOD、APX、DHAR、MDHAR、Prx)表达量的升高有关。
2、以狗牙根基因型‘Midrion’、‘Tifgreen’、‘Tifway’、‘Champion’、‘Pariot’、‘Celebration’为试验材料,在生长箱内进行了为期15 d的干旱处理。通过测定目测质量、叶片RWC、Fv/Fm、净光合速率(Pn)以及EL的变化差异,综合评价了狗牙根6个基因型的抗旱性。在干旱处理第0 d与10 d时,选取较抗旱的‘Midrion’、‘Tifway’与干旱敏感的‘Pariot’、‘Celebration’为试验材料,用免疫印迹的方法验证了干旱相关蛋白Rubisco LSU、SOD、DHAR以及MDHAR的表达。与干旱敏感的基因型相比,抗旱基因型的Rubisco LSU下降幅度较小,而SOD、DHAR以及MDHAR的上升幅度较大。
3、以狗牙根基因型‘Tifway’与‘C299’为试验材料,比较了干旱胁迫下2个基因型的叶片RWC、叶绿素含量、Fv/Fm以及EL的变化差异,所有测定指标均表明‘Tifway’较‘C299’更抗旱。在试验处理第5、10、15 d时,分别选取2个基因型的干旱处理叶片与相应的正常生长的对照叶片,以甲醇为溶剂进行代谢产物的提取。在狗牙根2个基因型的叶片或根系中,GC-MS分析总共鉴定出44种代谢产物:15种有机酸、14种氨基酸、10种糖与糖醇、3种脂肪酸以及2种含氮化合物。通过对抗旱性不同的‘Tifway’与‘C299’进行差异代谢产物比较,发现狗牙根的抗旱性可能与某些代谢产物的高水平表达或者含量维持有关,这些代谢产物包括:3种有机酸(阿魏酸、甘油酸、衣康酸),3种氨基酸(丙氨酸、脯氨酸、缬氨酸),5种糖与糖醇(半乳糖、蔗糖、蜜二糖、山梨醇、甘露醇)、2种不饱和脂肪酸(α-亚麻酸、亚油酸)以及2种含氮化合物(乙醇胺、腐胺)。
Drought is one of the major abiotic factors limiting plant growth, which induces various changes at different cellular levels. Although morphological and physiological mechanisms of drought tolerance have been examined extensively in bermudagrass, limited information is available on the drought-responsive proteins or metabolites. By technologies of proteomics, western blotting and metabolomics, three experiments were carried out with different bermudagrass genotypes. The first was study of drought-responsive proteins in bermudagrass leaves. The second was responses of protein expression to drought stress in bermudagrass leaves. The third was study of drought-responsive metabolites in leaf and root of bermudagrass. The main contents and results were as follows:
1. Plants of bermudagrass‘Tifway’and‘C299’were exposed to drought stress for 15 d by withholding irrigation in a growth chamber. Leaf electrolyte leakage increased and photochemical efficiency and relative water content declined under drought stress, but the extent of changes in each of the physiological parameters for‘Tifway’was less pronounced than those for‘C299’. Total proteins of leaves were extracted from well-watered and drought-stressed plants at 10 d of treatment, and separated by two-dimensional gel electrophoresis. According to the quantitative analysis of protein gels, a total of 54 protein spots in the two genotypes showed reproducible and significant changes in the abundance, with 32 increasing and 22 decreasing. All drought-responsive proteins were subjected to mass spectrometry analysis, which were mainly involved in metabolism, energy, cell growth/division, protein synthesis, and stress defense. Functional analysis of differential drought-responsive proteins between the two genotypes suggested that the superior drought tolerance in‘Tifway’could be mainly associated with less severe decline in the abundance level of proteins involved in photosynthesis (Chl a-b, ATPSα, PRK and Rubisco) and greater increase in the abundance level of antioxidant defense proteins (SOD, APX, DHAR, MDHAR and Prx).
2. Plants of bermudagrass‘Midrion’,‘Tifgreen’,‘Tifway’,‘Champion’,‘Pariot’and‘Celebration’were exposed to drought stress for 15 d by withholding irrigation in a growth chamber. Evaluation of drought tolerance in different genotypes of bermudagrass was based on turf quality, leaf relative water content, photochemical efficiency, net photosynthetic rate and electrolyte leakage. Expression of Rubisco LSU, SOD, DHAR and MDHAR under well-watered and drought-stressed conditions was detected by western blotting analysis. Drought-tolerant‘Midrion’and‘Tifway’had lower degradation of Rubisco LSU and greater increase of SOD, DHAR and MDHAR compared to drought-sensitive‘Pariot’and‘Celebration’.
3. Physiological responses to drought stress were evaluated by measuring leaf relative water content, chlorophyll content, photochemical efficiency and cell membrane leakage, and all parameters indicated that‘Tifway’exhibited better drought tolerance than‘C299’. Methanol was used for metabolites extraction from well-watered and drought-stressed plants at 5, 10 and 15 d of treatment. The gas chromatography-mass spectrometry analysis identified 44 drought-responsive metabolites in leaves or roots of two genotypes, including 15 organic acids, 14 amino acids, 10 sugar and sugar alcohols, 3 fatty acids and 2 N-compounds. The differential metabolic expression pattern between two genotypes differing in drought tolerance demonstrated that the superior drought tolerance in‘Tifway’could be related to the accumulation or maintenance of a series of metabolites, including 3 organic acids (ferulate, glycerate and itaconate), 3 amino acids (alanine, proline and valine), 5 sugar and sugar alcohols (galactose, sucrose, melibiose, gluticol and mannitol), 2 unsaturated fatty acids (α-linolenate and linoleate), and 2 N-compounds (ethanolamine and putrescine).
1、以狗牙根基因型‘Tifway’与‘C299’为试验材料,在生长箱内进行了为期15 d的干旱处理。干旱胁迫降低了狗牙根2个基因型的叶片相对含水量(RWC)与光化学效率(Fv/Fm),提高了相对电导率(EL)的水平,但上述指标在‘Tifway’中的变化幅度比在‘C299’中要小。在试验处理第10 d时,分别选取正常生长与干旱处理的2个基因型叶片,进行了全蛋白的提取与双向电泳分离(2-DE)。凝胶图像分析结果显示,干旱胁迫总共引起了54个蛋白表达量的显著性改变,其中有32个蛋白的表达量上调,有22个蛋白的表达量下调。质谱(MS)分析结果显示,这些干旱响应蛋白的功能涉及到代谢、能量、细胞生长/分化、蛋白合成以及逆境防御等多个过程。通过对抗旱性不同的‘Tifway’与‘C299’进行差异蛋白比较,发现狗牙根的抗旱性可能与光合作用相关蛋白(Chl a-b、ATPSα、PRK、Rubisco)表达量的维持,抗氧化蛋白酶(SOD、APX、DHAR、MDHAR、Prx)表达量的升高有关。
2、以狗牙根基因型‘Midrion’、‘Tifgreen’、‘Tifway’、‘Champion’、‘Pariot’、‘Celebration’为试验材料,在生长箱内进行了为期15 d的干旱处理。通过测定目测质量、叶片RWC、Fv/Fm、净光合速率(Pn)以及EL的变化差异,综合评价了狗牙根6个基因型的抗旱性。在干旱处理第0 d与10 d时,选取较抗旱的‘Midrion’、‘Tifway’与干旱敏感的‘Pariot’、‘Celebration’为试验材料,用免疫印迹的方法验证了干旱相关蛋白Rubisco LSU、SOD、DHAR以及MDHAR的表达。与干旱敏感的基因型相比,抗旱基因型的Rubisco LSU下降幅度较小,而SOD、DHAR以及MDHAR的上升幅度较大。
3、以狗牙根基因型‘Tifway’与‘C299’为试验材料,比较了干旱胁迫下2个基因型的叶片RWC、叶绿素含量、Fv/Fm以及EL的变化差异,所有测定指标均表明‘Tifway’较‘C299’更抗旱。在试验处理第5、10、15 d时,分别选取2个基因型的干旱处理叶片与相应的正常生长的对照叶片,以甲醇为溶剂进行代谢产物的提取。在狗牙根2个基因型的叶片或根系中,GC-MS分析总共鉴定出44种代谢产物:15种有机酸、14种氨基酸、10种糖与糖醇、3种脂肪酸以及2种含氮化合物。通过对抗旱性不同的‘Tifway’与‘C299’进行差异代谢产物比较,发现狗牙根的抗旱性可能与某些代谢产物的高水平表达或者含量维持有关,这些代谢产物包括:3种有机酸(阿魏酸、甘油酸、衣康酸),3种氨基酸(丙氨酸、脯氨酸、缬氨酸),5种糖与糖醇(半乳糖、蔗糖、蜜二糖、山梨醇、甘露醇)、2种不饱和脂肪酸(α-亚麻酸、亚油酸)以及2种含氮化合物(乙醇胺、腐胺)。
Drought is one of the major abiotic factors limiting plant growth, which induces various changes at different cellular levels. Although morphological and physiological mechanisms of drought tolerance have been examined extensively in bermudagrass, limited information is available on the drought-responsive proteins or metabolites. By technologies of proteomics, western blotting and metabolomics, three experiments were carried out with different bermudagrass genotypes. The first was study of drought-responsive proteins in bermudagrass leaves. The second was responses of protein expression to drought stress in bermudagrass leaves. The third was study of drought-responsive metabolites in leaf and root of bermudagrass. The main contents and results were as follows:
1. Plants of bermudagrass‘Tifway’and‘C299’were exposed to drought stress for 15 d by withholding irrigation in a growth chamber. Leaf electrolyte leakage increased and photochemical efficiency and relative water content declined under drought stress, but the extent of changes in each of the physiological parameters for‘Tifway’was less pronounced than those for‘C299’. Total proteins of leaves were extracted from well-watered and drought-stressed plants at 10 d of treatment, and separated by two-dimensional gel electrophoresis. According to the quantitative analysis of protein gels, a total of 54 protein spots in the two genotypes showed reproducible and significant changes in the abundance, with 32 increasing and 22 decreasing. All drought-responsive proteins were subjected to mass spectrometry analysis, which were mainly involved in metabolism, energy, cell growth/division, protein synthesis, and stress defense. Functional analysis of differential drought-responsive proteins between the two genotypes suggested that the superior drought tolerance in‘Tifway’could be mainly associated with less severe decline in the abundance level of proteins involved in photosynthesis (Chl a-b, ATPSα, PRK and Rubisco) and greater increase in the abundance level of antioxidant defense proteins (SOD, APX, DHAR, MDHAR and Prx).
2. Plants of bermudagrass‘Midrion’,‘Tifgreen’,‘Tifway’,‘Champion’,‘Pariot’and‘Celebration’were exposed to drought stress for 15 d by withholding irrigation in a growth chamber. Evaluation of drought tolerance in different genotypes of bermudagrass was based on turf quality, leaf relative water content, photochemical efficiency, net photosynthetic rate and electrolyte leakage. Expression of Rubisco LSU, SOD, DHAR and MDHAR under well-watered and drought-stressed conditions was detected by western blotting analysis. Drought-tolerant‘Midrion’and‘Tifway’had lower degradation of Rubisco LSU and greater increase of SOD, DHAR and MDHAR compared to drought-sensitive‘Pariot’and‘Celebration’.
3. Physiological responses to drought stress were evaluated by measuring leaf relative water content, chlorophyll content, photochemical efficiency and cell membrane leakage, and all parameters indicated that‘Tifway’exhibited better drought tolerance than‘C299’. Methanol was used for metabolites extraction from well-watered and drought-stressed plants at 5, 10 and 15 d of treatment. The gas chromatography-mass spectrometry analysis identified 44 drought-responsive metabolites in leaves or roots of two genotypes, including 15 organic acids, 14 amino acids, 10 sugar and sugar alcohols, 3 fatty acids and 2 N-compounds. The differential metabolic expression pattern between two genotypes differing in drought tolerance demonstrated that the superior drought tolerance in‘Tifway’could be related to the accumulation or maintenance of a series of metabolites, including 3 organic acids (ferulate, glycerate and itaconate), 3 amino acids (alanine, proline and valine), 5 sugar and sugar alcohols (galactose, sucrose, melibiose, gluticol and mannitol), 2 unsaturated fatty acids (α-linolenate and linoleate), and 2 N-compounds (ethanolamine and putrescine).
引文
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