代谢组学技术研究氯磺隆和镉对玉米幼苗和菠菜代谢的影响

发布时间：2018-06-11 17:54

本文选题：氯磺隆 + Cd　；参考：《山西大学》2016年博士论文

【摘要】：磺酰脲类除草剂是目前田间使用量较大的一类除草剂,常被用于田间阔叶杂草的防除。氯磺隆作为磺酰脲类除草剂的代表品种,在土壤中残效期较长,容易对后茬敏感作物产生药害。土壤长期污水和工业废水混合灌溉、降尘污染、以及农药和化肥的长期使用等,都会导致土壤镉的积累。在农田土壤体系中残留除草剂氯磺隆和重金属同时存在,对作物的影响及其作用机制研究引起研究者广泛关注,因此研究两种污染物同时存在,对玉米幼苗和菠菜生长过程、抗氧化作用机制、光合作用机制、以及植株代谢产物和代谢途径的影响和变化等,对食品安全评价、污染物治理以及环境污染与健康的关系至关重要。根据ISO11269-2(2013)的方法,将土壤分别用不同含量的氯磺隆单一处理、固定镉含量(5.0mg/kg)单一处理、以及固定镉含量(5.0mg/kg)改变氯磺隆含量的复合处理后,采用盆栽试验的方法,对玉米幼苗和菠菜分别进行发芽和早期生长试验。通过观察生长过程、对植株相关抗氧化物酶的活性测定,评价氯磺隆和镉对两种作物生长及抗氧化作用机制的影响；采用叶绿素荧光技术评价氯磺隆和镉对两种作物光合作用的影响；从代谢组学的角度出发,采用核磁技术、气质联用和液质联用技术分析玉米幼苗和菠菜受到氯磺隆和镉单一及复合胁迫后,代谢物及代谢途径的变化。多种代谢组学技术、叶绿素荧光、以及对植株生理生化指标的研究结果显示,氯磺隆、镉及其复合污染对玉米幼苗和菠菜多种代谢途径产生了影响。1、氯磺隆单一污染结果表明,土壤残留氯磺隆,会诱导玉米幼苗和菠菜活性氧产生、使植株自身抗氧化系统和膜质过氧化自我修复机制被激活；光合作用系统能量的利用和传递受限,阻碍植株三羧酸循环的正常进行、导致部分氨基酸代谢异常、能量代谢和细胞壁的合成受到影响。首先,土壤残留氯磺隆,诱导玉米幼苗和菠菜活性氧的产生,使其自我保护机制开启,以阻碍氧化应激反应对植株的伤害,包括抗氧化系统的激活(SOD、CAT和GST酶活性显著升高,P0.05)和膜质过氧化的修复(一定浓度的氯磺隆诱导丙二醛含量显著升高,P0.05)。其次,氯磺隆会影响菠菜叶片的荧光产量及PSⅡ中QA、QB和质体醌之间的电子传递(OJIP曲线荧光强度下降,P0.05),而对玉米幼苗则没有影响。第三,氯磺隆影响玉米幼苗和菠菜的光合反应中心,使得能量的利用和传递受到阻碍(ABS/RC显著升高,P0.05),植株的防御系统也被激发,将过多的能量以热耗散的形式释放(DI0/RC也显著升高,P0.05),但对还原QA和电子传递没有影响(TRo/RC和ET0/RC没有显著变化,P0.05)。第四,随着氯磺隆含量的不断增大,指示反应PSⅡ生理活性状态的PIABS呈先上升后下降的趋势,表明低含量氯磺隆诱导植株防御系统启动,而高含量则导致作物光合作用系统受到不可逆转的损害。第五,植株生理生化指标的改变,在代谢上表现为：阻碍了三羧酸循环的正常进行(柠檬酸和苹果酸含量显著升高,P0.05),造成了能量代谢从有氧呼吸到无氧呼吸的转移(丙酮酸和乳酸含量的显著升高,P0.05),植物体内糖类物质(蔗糖、葡萄糖、果糖等)的大量积累也反应植物能量代谢及细胞壁(半乳糖醛酸、鼠李糖)合成受到影响。第六,土壤中残留氯磺隆还导致植株部分氨基酸代谢异常(支链氨基酸(Branch chain amino acids, BCAAs)、芳香族氨基酸(Aromatic amino acid, AAAs)、丝氨酸(Ser)、谷氨酸(Glu)等,与抗逆相关物质(谷氨酰胺、脯氨酸、腐胺、肌醇和GABA)的显著升高。2、镉单一污染结果指出,土壤镉含量为5.0 mg/kg时,对玉米幼苗和菠菜的生长、光合作用过程没有显著影响；但造成了玉米幼苗和菠菜代谢途径三羧酸循环的异常、其能量代谢、氨基酸和糖代谢在玉米幼苗和菠菜中的表现各异。首先,与空白对照组比,土壤镉含量(5.0 mg/kg)没有造成植株体内活性氧的积累,植株抗氧化的自我保护系统没有被激活(SOD、POD、CAT和GST酶活性没有显著变化),丙二醛含量也没有显著积累；其次,镉对玉米幼苗和菠菜的光合反应中心、能量反应及耗散的相关参数变化也没有显著影响。但是,对代谢产物和代谢途径却存在明显的影响。第一,镉造成了玉米幼苗和菠菜三羧酸循环的异常(玉米幼苗苹果酸和柠檬酸含量显著降低；菠菜苹果酸和柠檬酸含量显著升高,P0.05)；第二,能量代谢在玉米幼苗和菠菜中的表现各异,镉诱导菠菜中能量代谢从有氧到无氧呼吸的转移(乳酸含量显著升高,P0.05),而玉米幼苗能量代谢却没有发生转移(乳酸含量显著(P0.05)下降,丙酮酸含量没有显著变化,P0.05)；与抗逆相关物质的含量变化也不同,镉诱导玉米幼苗中腐胺和肌醇含量积累,脯氨酸没有显著变化,而菠菜则是脯氨酸和肌醇含量积累,腐胺含量下降。第三,镉还诱导玉米幼苗和菠菜几种氨基酸代谢异常(天冬氨酸、谷氨酸、丝氨酸、丙氨酸等)和糖类物质的积累。3、与氯磺隆和镉的单一污染相比,复合污染结果显示,植株叶片叶绿素含量显著升高；高含量氯磺隆复合处理,菠菜叶片的荧光产量及PSⅡ中QA、QB和质体醌之间的电子传递受抑、对菠菜光合作用的影响二者存在协同作用,而对玉米幼苗没有协同作用。对某些特定代谢物的联合作用机制因植株种类而不同。首先,与单一污染相比,复合污染均导致玉米幼苗和菠菜植株叶片叶绿素含量显著升高(P0.05),有利于光合作用的正常进行。同时,还能刺激植物体内自我保护机制,即抗氧化物酶活性更高(POD和CAT活性显著升高,GST酶活性显著降低,P0.05),有利于植物体内活性氧的清除。其次,不同含量的氯磺隆和镉复合污染对玉米幼苗和菠菜荧光产量及PSⅡ中QA、QB和质体醌之间电子传递影响表现不同。具体表现为,低含量氯磺隆(0.001mg/kg)和Cd复合对玉米幼苗和菠菜植株光合作用均没有影响(与各自对照相比,ABS/RC、DI0/RC和PIABS没有显著变化,P0.05)；而高含量氯磺隆和镉复合污染,对玉米幼苗没有协同作用(与单一污染相比较,ABS/RC、DI0/RC和PIABS没有显著变化,P0.05)；但菠菜叶片的荧光产量及PSⅡ中QA、QB和质体醌之间的电子传递则受到抑制,且随着氯磺隆浓度越高,抑制作用越强；特别是对ABS/RC和DI0/RC值的影响较氯磺隆单一污染更为明显(P0.05),表明镉加重了氯磺隆对菠菜光合系统反应中心能量的影响,二者存在协同作用。同时,复合处理组PIABS显著下降也表明二者对菠菜光合作用具有协同作用。第三,从代谢物含量的变化可以看出,氯磺隆和镉复合污染对玉米幼苗某些特定代谢物如异亮氨酸、缬氨酸、丝氨酸、乳酸等含量具有拮抗作用,其余代谢物如丙酮酸、苹果酸、柠檬酸和蛋氨酸在低含量复合处理组表现为拮抗作用,而高含量则为协同作用；氯磺隆和镉复合污染对菠菜中异亮氨酸、谷氨酸、乳酸和柠檬酸等具有协同作用,其余代谢物缬氨酸、苹果酸、γ-氨基丁酸在低含量复合处理组表现为协同作用,高含量则为拮抗作用。综上所述,统计检验结合定量分析结果表明,土壤残留氯磺隆会诱导玉米幼苗和菠菜活性氧产生,植物自身防御系统开启(抗氧化系统的激活和膜质过氧的修复)；光合作用系统能量的利用和传递受限,植物代谢途径三羧酸循环、部分氨基酸代谢、能量代谢及细胞壁合成受阻。土壤镉(5mg/kg)虽然对玉米幼苗和菠菜的生长和光合作用过程没有明显影响,但对玉米幼苗和菠菜代谢物及代谢途径产生了明显影响。如玉米幼苗和菠菜三羧酸循环、氨基酸、糖和能量代谢发生异常。该研究结果揭示,氯磺隆与镉复合污染存在交互作用,氯磺隆和镉胁迫下,玉米幼苗和菠菜代谢中最大的不同表现在莽草酸途径,推断该途径中多种主要代谢物变化可作为单子叶作物受胁迫的标记。
[Abstract]:Sulfonylurea herbicides are one of the most widely used herbicides used in the field at present. They are often used for the prevention of broad leaved weeds in the field. Chlorsulfononide is a representative variety of sulfonylurea herbicides. It has a long residual effect in the soil and is easy to produce a drug damage to the later crop sensitive crops. The mixed irrigation of soil and industrial waste water, dust pollution, and agriculture are used in the field. The long-term use of drugs and chemical fertilizers will lead to the accumulation of cadmium in soil. The residual herbicide chlorsulfonide and heavy metals in the soil system in the farmland are simultaneous, the influence of the crop and the mechanism of its action have aroused widespread concern. Therefore, the study of two kinds of pollutants at the same time, the growth process of corn seedlings and spinach, antioxidant effect The mechanism, the photosynthesis mechanism, the effects and changes of the plant metabolites and metabolic pathways, and the relationship between the food safety assessment, the pollution control and the environmental pollution and health are essential. According to the method of ISO11269-2 (2013), the soil is treated with a single treatment with different content of chlorsulfonon, and the fixed cadmium content (5.0mg/kg) is treated by a single treatment, After a combined treatment of the content of chlorsulfonyl chloride with fixed cadmium content (5.0mg/kg), a pot experiment was used to test the germination and early growth of the maize seedlings and spinach. By observing the growth process, the activity of the related antioxidant enzymes was measured, and the growth of two crops and the antioxidants were evaluated by chlorononolonon and cadmium. The effects of the chlorophyll fluorescence technique were used to evaluate the effects of chlorsulfonyl and cadmium on the photosynthesis of two crops. From the perspective of metabolomics, the changes in metabolites and metabolic pathways of maize seedlings and spinach were analyzed by NMR technique, GC-MS and liquid chromatography-mass spectrometry. The results of metabonomics, chlorophyll fluorescence, and physiological and biochemical indexes of plant showed that chlorsulfonon, cadmium and its compound pollution had an effect on various metabolic pathways of maize seedlings and spinach. The single pollution result of chlorsulfonon showed that the residual chlorsulfonont in soil could induce the production of active oxygen of maize seedlings and spinach, and the plant itself could be induced by.1. The antioxidant system and the self repair mechanism of membranous peroxidation are activated; the utilization and transfer of energy in the photosynthesis system is limited, which hinders the normal progress of the three carboxylic acid cycle, resulting in abnormal metabolism of some amino acids, energy metabolism and the synthesis of cell walls. The production of oxygen, which opens the mechanism of self protection, prevents oxidative stress response to plants, including the activation of antioxidant systems (SOD, CAT and GST enzyme activity significantly increased, P0.05) and membranous peroxide repair (a certain concentration of chlorsulfonon induced malondialdehyde content increases significantly, P0.05). Secondly, chlorsulfonon will affect the fluorescence of spinach leaves Light yield and electron transfer between QA, QB and plastid quinone in PS II (OJIP curve fluorescence intensity decreased, P0.05), but no effect on maize seedlings. Third, chlorononolonon affects the photosynthetic reaction center of maize seedlings and spinach, which prevents the use and transmission of energy (ABS/RC significantly increased, P0.05), and the plant defense system is also stimulated. The excess energy was released in the form of heat dissipation (DI0/RC also increased significantly, P0.05), but had no effect on the reduction of QA and electron transfer (TRo/RC and ET0/RC did not change significantly, P0.05). Fourth, with the increase of the content of chlorsulfonyl methyl, the PIABS showing the physiological active state of PS II was rising and then decreasing, indicating the low content of chlorinated sulphur. The growth of plant defense system was induced, while high content resulted in irreversible damage to the photosynthesis system of crops. Fifth, the changes in physiological and biochemical indicators of plants were metabolized as hindering the normal cycle of the three carboxylic acid (citric acid and malic acid content increased significantly, P0.05), resulting in energy metabolism from aerobic respiration to A significant increase in the content of pyruvic acid and lactic acid, P0.05), the accumulation of carbohydrates (sucrose, glucose, fructose, etc.) in the plant also responds to the energy metabolism of plants and the synthesis of cell wall (galactoacid, Li Tang). Sixth, residual chlorosulonide in soil is also responsible for the abnormal metabolism of some amino acids. Chain amino acids (Branch chain amino acids, BCAAs), aromatic amino acids (Aromatic amino acid, AAAs), serine (Ser), glutamic acid (Glu), etc., with a significant increase in resistance related substances (glutamine, proline, proline, inositol and GABA). The cadmium single pollution results indicated that the soil cadmium content was 5, the growth of maize seedlings and spinach. Long, photosynthesis process was not significantly affected, but the metabolism of three carboxylic acid cycles in maize seedlings and spinach was abnormal, and its energy metabolism, amino acid and sugar metabolism in maize seedlings and spinach were different. First, the content of soil cadmium (5 mg/kg) did not cause the accumulation of active oxygen in the plant compared with the blank control group. The antioxidant self-protection system was not activated (SOD, POD, CAT and GST activity did not change significantly), and MDA content was not significantly accumulated; secondly, there was no significant influence on the photosynthetic reaction center of maize seedlings and spinach, energy response and dissipation related parameters. However, the metabolic products and metabolic pathways were present. First, cadmium resulted in abnormal cycling of three carboxylic acids in maize seedlings and spinach (the content of malic acid and citric acid in maize seedlings decreased significantly; the content of malic acid and citric acid in spinach increased significantly, P0.05); second, energy metabolism in maize seedlings and spinach were different. Cadmium induced energy metabolism in spinach from aerobic to no The transfer of oxygen respiration (the content of lactic acid increased significantly, P0.05), but the energy metabolism of maize seedlings did not change (P0.05), the content of pyruvic acid was not significantly changed, P0.05), and the content of anti inverse related substances was also different. The content of putrescine and inositol in the seedlings of cadmium induced jade rice seedlings was not significantly changed. The contents of proline and inositol were accumulated and the content of putrescine decreased. Third, cadmium also induced the accumulation of several amino acids in maize seedlings and spinach (aspartic acid, glutamic acid, serine, alanine, etc.) and the accumulation of carbohydrates. Compared with the single pollution of chlorosinolide and cadmium, the compound pollution results showed that the chlorophyll content of plant leaves was contained in.3. The amount of the high content chlorsulfonon combined treatment, the fluorescence yield of spinach leaves and the electron transfer between QA, QB and plastid quinone in PS II were inhibited. The synergistic effects on the photosynthesis of spinach were synergistic, but there was no synergistic effect on the maize seedlings. The joint action mechanism of some specific metabolites was different from the plant species. First, compared with the single pollution, the compound pollution resulted in a significant increase in the chlorophyll content of the leaves of the maize seedlings and spinach plants (P0.05), which was beneficial to the normal photosynthesis. At the same time, it could also stimulate the mechanism of self protection in the plant, that is, the activity of antioxidant enzymes is higher (the activity of POD and CAT increased significantly, the activity of GST decreased significantly, P0.05). The effects of chlorsulfonylon and cadmium on the fluorescence yield of maize seedlings and spinach and the electron transfer between QA, QB and plastid quinones in PS II were different. The specific performance was that low content of chlorononolonon (0.001mg/kg) and Cd had no effect on the photosynthesis of maize seedlings and spinach plants. Compared with the control, ABS/RC, DI0/RC and PIABS did not significantly change, P0.05); while the high content of chlorononolonon and cadmium pollution had no synergistic effect on maize seedlings (compared with single pollution, ABS/RC, DI0/RC and PIABS had no significant changes, P0.05), but the fluorescence yield of spinach leaves and the electron transfer between QA, QB and plastid quinones in PS II. It was inhibited, and with the higher concentration of chlorsulfonon, the stronger the inhibitory effect; especially the effect on ABS/RC and DI0/RC value was more obvious than that of chlorsulfonon single pollution (P0.05), indicating that cadmium aggravated the effect of chlorsulfonon on the central energy of the photosynthetic system reaction of spinach, the two had synergistic effect. At the same time, the PIABS of the compound treatment group decreased significantly. The two groups have a synergistic effect on the photosynthesis of spinach. Third, from the change of metabolite content, it can be seen that the compound pollution of chlorsulfonyl and cadmium has antagonistic effects on some specific metabolites such as isoleucine, valine, serine and lactic acid in maize seedlings, while the remaining metabolites such as pyruvic acid, malic acid, citric acid and methionine are low The compound treatment group showed antagonism, while the high content was synergistic, and the compound pollution of chlorsulfonon and cadmium had synergistic effect on isoleucine, glutamic acid, lactic acid and citric acid in spinach. The other metabolites, valine, malic acid and gamma aminobutyric acid, were synergistic in the low content compound treatment group and the high content was antagonistic. To sum up, the statistical test combined with quantitative analysis showed that the residual chlorsulfonon in soil could induce the production of active oxygen of maize seedlings and spinach, the plant self defense system opened (the activation of antioxidant system and the repair of membrane oxygen); the utilization and transmission of the photosynthesis system energy was limited, the plant metabolism pathway three carboxylic acid cycle, part The metabolism of amino acids, energy metabolism and cell wall synthesis were blocked. Although soil cadmium (5mg/kg) had no obvious influence on the growth and photosynthesis process of maize seedlings and spinach, it had a significant influence on the metabolites and metabolic pathways of maize seedlings and spinach, such as the cycle of three carboxylic acids, amino acids, sugar and energy metabolism in maize seedlings and spinach. The results revealed that the compound pollution of chlorsulfonon and cadmium was interactive. Under the stress of chlorsulfonyl and cadmium, the biggest difference in the metabolism of maize seedlings and spinach was shown in the way of shikimic acid. It was concluded that many major metabolites in this pathway could be used as a marker for the stress of mono cotyledon crops.
【学位授予单位】：山西大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：X592

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