黑大豆根系吸收和代谢甲醛的机理与应用研究

发布时间：2018-06-21 00:26

  本文选题：黑大豆 + 根系甲醛吸收　； 参考：《昆明理工大学》2017年博士论文

【摘要】：甲醛(HCHO)被广泛用于化工合成、工业制造、医药合成等工业领域,在化学农药及其中间体合成领域甲醛也有着举足轻重的作用,因此在现代化工和化学农药生产过程中不可避免地要产生大量含液体甲醛的生产废水。另一方面随着生活质量的不断提高装修已成为现代人生活的一部分,甲醛是很多装修材料中广泛使用的胶黏剂,胶黏剂中未交联的游离甲醛持续地释放造成严重的室内空气甲醛污染。很多研究观察到不同植物品种具有不同的甲醛吸收能力。由于甲醛污染的危害很广泛,因此有很多研究致力于开发治理甲醛污染的技术和方法,在众多治理方法中,基于微生物和植物吸收甲醛能力的治理法是一种行之有效且经济实惠的方法,大多数的研究认为空气中甲醛的吸收通过由叶片完成,因此有些研究已经阐明甲醛在植物叶片中的代谢机理。最近的研究表明当含有污染甲醛的空气或废水流经植物根系和土壤基质构成的生物反应器时,空气或废水中的甲醛可通过土壤吸收进入植物根系,在根系细胞内代谢酶的作用下被降解。植株根系白天和晚上都能有效去除甲醛,晚上根去除甲醛的量可达到地上部分的10倍,但是至今没有研究系统考察植物根系去除甲醛的代谢机理。本研究通过采用种子颗粒较大的栽培种丹波黑大豆(RB)和豆粒较小的黑大豆栽培种(SB)根系为材料,分析比较RB和SB根系吸收甲醛的动力学模型,同时通过13C-NMR分析结合生理生化方法系统考察两种黑大豆根系甲醛代谢机理,为科学利用植物根系修复甲醛污染提供理论据。用2-6mM甲醛处理RB和SB植株根系,测定0.5-48h期间的甲醛吸收,结果说明RB和SB根系都可有效吸收处理液中的甲醛,在48 h处理时期内两种黑大豆根系吸收甲醛的动力学相似,都呈现先慢后快的模式,对甲醛的吸收与时间的关系符合幂函数模型。13C-NMR分析结果表明在H13CHO胁迫早期(0-4h),根吸收的H13CHO首先通过依赖于谷胱甘肽依赖型甲醛脱氢酶(FALDH)的氧化途径被氧化为甲酸,甲酸可能通过乙醛酸合酶(GXS)的作用缩合为乙醛酸;在随后的代谢中,有部分乙醛酸可能进入乙醛酸循环产生柠檬酸(Cit)和异柠檬酸(Icit);有部分乙醛酸通过转氨酶(GAT)的作用被转化为甘氨酸(Gly)或通过未知途径代谢为天冬酰胺(Asn)和谷氨酰胺(Gln)。此外,还有部分乙醛酸被氧化为草酸(OA)。在H13CHO处理的后期(24-48h),Asn和Gln、OA通过三羧酸(TCA)循环被转化为Cit和Icit;Cit和Icit进入乙醛酸循环产生苹果酸(Mal);从乙醛酸循环中输出的Mal进入糖异生途径被转化为[U-13C]Glucose。由此可见乙醛酸循环可能是根系甲醛代谢的中心,在甲醛处理晚期由于乙醛酸途径在大豆根中充分发挥作用使根对甲醛吸收能力迅速增大,因而这一时期根吸收的甲醛对甲醛去除的贡献显著增加。仔细比较RB和SB根系甲醛代谢机理的差异,结果发现RB根系中甲醛代谢产生OA和Gly途径的作用强于SB。此外,13C-NMR分析证实根系吸收的甲醛可能并未转移到叶片中进行代谢转化。在2mM甲醛胁迫下RB叶片中积累的脯氨酸(Pro)显著高于SB,因此甲醛胁迫SB植株叶片萎蔫比RB严重。但是SB根中积累的Pro显著大于RB,氧化胁迫指标低于RB,因此随后的大部分实验材料用SB根系进行。环孢素A(CSA)是一种特异性线粒体渗透性转运孔抑制剂。通过CSA预处理分析RB和SB根系各甲醛代谢途径的亚细胞定位。对比用CSA预处理和未预处理根系在2mM H13CHO处理4h和24h代谢谱的差异。分析4h甲醛代谢谱,结果说明CSA完全抑制甲醛代谢产生Cit途径的作用,对Icit的生成有部分抑制作用,由此推测Cit和Icit产生途径的作用定位在线粒体或乙醛酸循环体内。OA、Gly2、Gln、Asn和丝氨酸(Ser3)的产量没有被抑制反而增加,说明产生这些有机酸的代谢途径均在根的细胞质中发挥作用,CSA预处理使产生Cit和Ici的13C代谢流进入这些有机酸的代谢途径。在24h甲醛代谢谱中,葡萄糖的生成也受到显著影响,这是乙醛酸循环途径的作用被抑制的另一结果。13C-NMR分析证实CSA预处理也影响叶片叶绿体中卡尔文循环的作用,使叶片葡萄糖含量显著降低。考察甲醛吸收曲线的结果表明CSA预处理对RB和SB根系甲醛吸收仅有轻微的抑制作用,这是由于CSA仅抑制乙醛酸循途径的作用而不影响OA、Gly2、Gln、Asn和Ser3产生途径的作用所致,这两个途径的作用是独立的。利用甲醛代谢产物对代谢途径的反馈抑制作用和影响乙醛酸循环途径关键酶基因表达的化合物深入考察到各代谢途径在黑大豆根系甲醛吸收中的作用,结果说明乙醛酸(GX)、Gly、Ser和OA预处理显著抑制SB根系甲醛吸收,证实GX、Gly、Ser和OA产生途径在SB根系甲醛吸收过程中发挥作用。Cit的预处理也有相似的作用效果,证实乙醛酸途径在SB根系甲醛吸收过程中的作用;在其他植物中证明能抑制乙醛酸途径关键酶基因异柠檬酸裂解酶(ICL)和苹果酸合酶(MS)表达的抑制剂葡萄糖(Glucose)、蔗糖(Sucrose)和甘露糖(Mannose)的预处理也抑制SB根系甲醛吸收,实验结果表明10 mM Glucose、5 mM Sucrose和10 mM Mannose产生的抑制作用最强。此外,分析抗氧化剂抗坏血酸(ASA)和还原性谷胱甘肽(GSH)预处理的结果表明,2mM ASA预处理作用效果最好,能使SB根系甲醛吸收增加6倍,4mM ASA预处理作用效果最好,能使SB根系甲醛吸收增加5倍,说明ASA和GSH预处理缓解甲醛的氧化胁迫,使SB根系甲醛吸收效率大幅度提高。甲醇(CH_3OH)能够刺激植物生长以及增加植物生物量在高等植物和农作物中已经被证实。在固体培养基中加入合适浓度的甲醇促进SB生根作用明显。甲醇叶面喷施能够促进SB茎伸长。用13C-NMR分析结果说明13CH_3OH在SB根内代谢非常缓慢,其代谢产物只有Ser2和Cit(2,4),处理24h后根系吸收的13CH_30H大部分仍以游离态存在。通过Central Composite Design设计了数学模型,应用响应面优化法获得甲醇刺激SB植株根系甲醛吸收的最佳条件,即甲醇预处理浓度0.73mM,甲醛处理浓度1.3mM和甲醛处理时间22h,在此条件下SB植物对甲醛的吸收的模型预测值是85.86%,而通过实验验证的真实值是84.72%,说明模型的拟合性很好。用0.73mM甲醇预处理SB植株根系12h,再用1.3mM H13CHO处理22h,13C-NMR分析说明甲醇预处理促进甲酸(FA)、Gly2、Ser3、Cit、Icit、Asn、Gln以及葡萄糖的生成,在甲醇预处理阶段这些代谢产物的产量并没有增加,这排除了由甲醇代谢作用增加这些代谢产物的可能性,因此推测甲醇预处理可能通过影响SB根中甲醛代谢相关酶基因的表达来改变其甲醛代谢机理,促进甲醛的吸收。为了在分子水平上更好理解甲醇的应用促进SB植株生长的机理,本研究应用cDNA芯片鉴定叶面喷施5%甲醇后SB叶片中的甲醇应答基因。结果鉴定得到731个可能的甲醇响应基因,其中上调基因和下调基因的数目分别为522和209。
[Abstract]:Formaldehyde (HCHO) is widely used in chemical synthesis, industrial manufacture, pharmaceutical synthesis and other industrial fields. It plays an important role in the field of chemical pesticide and its intermediate synthesis. Therefore, it is inevitable to produce a large number of liquid formaldehyde production wastewater in the process of modern chemical and chemical pesticide production. On the other hand, with the quality of life. The continuous improvement of the quantity has become a part of modern people's life. Formaldehyde is a widely used adhesive in many decoration materials. The unlinked free formaldehyde in the adhesive is continuously released to cause serious indoor air formaldehyde pollution. Many studies have observed that different plant varieties have different formaldehyde absorption capacity. Many studies have been devoted to the development of techniques and methods for the development of formaldehyde pollution. In many treatment methods, the treatment method based on the ability of microorganisms and plants to absorb formaldehyde is an effective and economical method. Most of the studies believe that the absorption of formaldehyde in the air is accomplished by the leaves, so some of them are done. Studies have elucidated the metabolic mechanism of formaldehyde in plant leaves. Recent studies have shown that when air or wastewater containing polluted formaldehyde flows through plant roots and soil substrates, the formaldehyde in air or wastewater can be absorbed into the plant roots through soil and degraded under the role of metabolic enzymes in the root cells of the root system. The root system can effectively remove formaldehyde during the day and night, and the amount of formaldehyde removal in the root can reach 10 times that of the upper part of the ground in the evening. However, there has been no systematic study on the metabolic mechanism of the plant root system to remove formaldehyde. This study was based on the roots of black soybean (RB) with large seed grains and the root of black soybean (SB). The kinetic model of formaldehyde absorption by RB and SB roots was analyzed and compared. At the same time, the formaldehyde metabolism mechanism of two kinds of black soybean roots was investigated by 13C-NMR analysis combined with physiological and biochemical methods. The scientific argument was provided for the scientific use of plant roots to repair formaldehyde pollution. RB and SB plant roots were treated with 2-6mM formaldehyde, and the formaldehyde absorption during 0.5-48h was measured. The results showed that both RB and SB roots could effectively absorb formaldehyde in the solution. During the 48 h treatment period, the kinetics of the absorption of formaldehyde in the two kinds of black soybean roots was similar, and all of them showed a slow and slow mode. The relationship between the absorption of formaldehyde and the time was in accordance with the.13C-NMR analysis of the power function model (0-4h) and the H1 of the root absorption in the early stage of H13CHO stress. 3CHO is oxidized to formic acid by the oxidation pathway that relies on the glutathione dependent formalin dehydrogenase (FALDH), and formic acid may be condensed into glyoxylic acid through the action of glyoxylate synthase (GXS); in subsequent metabolism, some glyoxylic acid may enter the glyoxylic acid cycle to produce citric acid (Cit) and ISO citrate (Icit); there are some alalic acids. The effect of transaminase (GAT) is converted to glycine (Gly) or metabolized to asparagine (Asn) and glutamine (Gln) through unknown pathways. In addition, some glyoxylic acid is oxidized to oxalic acid (OA). At the later stage of H13CHO treatment (24-48h), Asn and Gln, and OA are converted into Cit and aldehydes through the three carboxylic acid (TCA) cycle. Raw malic acid (Mal); the Mal output from the glyoxylic acid cycle into the sugar isogenesis pathway is converted to [U-13C]Glucose., thus it can be seen that the acetaldehyde acid cycle may be the center of formaldehyde metabolism in the root system. In the late formaldehyde treatment, the root to formaldehyde absorpability was rapidly increased because of the alalic acid pathway in the root of the soybean root. The contribution of the absorbed formaldehyde to the formaldehyde removal was significantly increased. The difference between the formaldehyde metabolism mechanism of RB and SB roots was carefully compared. The results showed that the metabolism of formaldehyde and Gly pathway in the root system of RB was stronger than that of SB.. The 13C-NMR analysis confirmed that the formaldehyde absorbed by the root system may not be transferred to the leaf slices for metabolic transformation. RB under the stress of 2mM formaldehyde. The accumulation of proline (Pro) in the leaves was significantly higher than that of SB, so the leaf wilting of SB plants was more severe than that of RB, but the accumulated Pro in SB roots was significantly greater than that of RB, and the oxidative stress index was lower than RB. Therefore, most of the experimental materials followed the SB root system. Cyclosporin A (CSA) was a specific mitochondrial permeable transport pore inhibitor. Analysis of the subcellular localization of formaldehyde metabolic pathways in RB and SB roots by preconditioning. Compared the metabolic profiles of 4H and 24h by CSA pretreatment and untreated roots in 2mM H13CHO treatment, the metabolic spectrum of 4H formaldehyde was analyzed. The results showed that CSA completely inhibited the role of the Cit pathway in the formation of formaldehyde metabolism, partly inhibiting the production of Icit, and thus speculated Cit. The role of the Icit production pathway is located in the mitochondria or glyoxylate cycle.OA, the production of Gly2, Gln, Asn and serine (Ser3) has not been suppressed but increased, indicating that the metabolic pathways that produce these organic acids play a role in the cytoplasm of the root, and CSA preconditioning makes Cit and Ici 13C metabolites into the metabolic pathways of these organic acids. Diameter. In the 24h formaldehyde metabolism spectrum, the formation of glucose was also significantly affected, which was another result of the inhibition of the effect of glyoxylic acid cycle pathway..13C-NMR analysis confirmed that CSA preconditioning also affected the effect of Calvin cycle in leaves chloroplasts and reduced the glucose content of leaves significantly. It has only a slight inhibitory effect on the absorption of formaldehyde in RB and SB roots, which is due to the effect of CSA only on the action of glyoxylate pathways without affecting the pathway of OA, Gly2, Gln, Asn and Ser3. The effects of these two pathways are independent. The feedback inhibition effect of formaldehyde metabolites on the metabolite path and the effect of glyoxylic acid cycle pathway The role of key enzyme gene expression compounds to investigate the role of various metabolic pathways in the absorption of formaldehyde in the root system of black soybean, the results showed that the pretreatment of glyoxylic acid (GX), Gly, Ser and OA significantly inhibited the absorption of formaldehyde in the root system of SB, and confirmed that the GX, Gly, Ser and OA production pathway played a similar role in the process of.Cit on the absorption of formaldehyde in SB roots. The effect of glyoxylate pathway on the absorption of formaldehyde in SB roots was confirmed. In other plants, it was proved that the inhibitor of ISO lysate lyase (ICL) and malate synthase (MS) expression of glyoxylic acid pathway inhibitor glucose (Glucose), the pretreatment of sucrose (Sucrose) and mannose (Mannose) also inhibited the absorption of formaldehyde in the root system of SB. The experimental results showed that the inhibitory effects of 10 mM Glucose, 5 mM Sucrose and 10 mM Mannose were the strongest. Furthermore, the results of the pretreatment of antioxidant ascorbic acid (ASA) and reduced glutathione (GSH) showed that the pre treatment of 2mM ASA was best, and the absorption of formaldehyde in the SB root system could be increased by 6 times, and the effect of 4mM pretreatment was the best. The absorption of formaldehyde in roots increased by 5 times, indicating that ASA and GSH pretreatment alleviated the oxidative stress of formaldehyde and increased the efficiency of formaldehyde absorption in SB roots greatly. Methanol (CH_3OH) could stimulate plant growth and increase plant biomass in higher plants and crops. The proper concentration of methanol was added to SB rooting in solid medium. The effect is obvious. The foliar spraying of methanol can promote the elongation of SB stem. The result of 13C-NMR analysis shows that the metabolism of 13CH_3OH in SB root is very slow, its metabolites are only Ser2 and Cit (2,4), and the majority of the root absorption of the root system still exists in the free state. The mathematical model is designed by Central Composite Design, and the response surface is optimized. The optimum conditions for the absorption of formaldehyde in the roots of SB plants were obtained by methanol, that is, methanol preconditioning concentration 0.73mM, formaldehyde treatment concentration 1.3mM and formaldehyde treatment time 22h. Under these conditions, the model prediction value of SB plant to formaldehyde absorption is 85.86%, and the true value of the experiment verified by the experiment is 84.72%, indicating that the model is very good. 0.73mM A is used. SB plant root 12h was pretreated with alcohol, then 22h was treated with 1.3mM H13CHO, and 13C-NMR analysis showed that methanol pretreatment promoted formic acid (FA), Gly2, Ser3, Cit, Icit, Asn, and glucose production. The production of these metabolites did not increase in the phase of methanol pretreatment, which excluded the possibility of increasing these metabolites by methanol metabolism. Therefore, it is presumed that methanol pretreatment may change the mechanism of formaldehyde metabolism and promote the absorption of formaldehyde by affecting the expression of formaldehyde metabolism related enzyme gene in SB roots. In order to better understand the mechanism of the application of methanol to promote the growth of SB plants at the molecular level, this study used cDNA chip to determine the methanol in the leaves of SB leaves after the foliar spraying of 5% methanol. The results showed that 731 possible methanol responsive genes were identified, of which the number of up regulated genes and down regulated genes was 522 and 209. respectively.
【学位授予单位】：昆明理工大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：X51;X173
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本文编号：2046320