本文選題：黑大豆 + 根系甲醛吸收　； 參考：《昆明理工大學(xué)》2017年博士論文

【摘要】：甲醛(HCHO)被廣泛用于化工合成、工業(yè)制造、醫(yī)藥合成等工業(yè)領(lǐng)域,在化學(xué)農(nóng)藥及其中間體合成領(lǐng)域甲醛也有著舉足輕重的作用,因此在現(xiàn)代化工和化學(xué)農(nóng)藥生產(chǎn)過(guò)程中不可避免地要產(chǎn)生大量含液體甲醛的生產(chǎn)廢水。另一方面隨著生活質(zhì)量的不斷提高裝修已成為現(xiàn)代人生活的一部分,甲醛是很多裝修材料中廣泛使用的膠黏劑,膠黏劑中未交聯(lián)的游離甲醛持續(xù)地釋放造成嚴(yán)重的室內(nèi)空氣甲醛污染。很多研究觀察到不同植物品種具有不同的甲醛吸收能力。由于甲醛污染的危害很廣泛,因此有很多研究致力于開(kāi)發(fā)治理甲醛污染的技術(shù)和方法,在眾多治理方法中,基于微生物和植物吸收甲醛能力的治理法是一種行之有效且經(jīng)濟(jì)實(shí)惠的方法,大多數(shù)的研究認(rèn)為空氣中甲醛的吸收通過(guò)由葉片完成,因此有些研究已經(jīng)闡明甲醛在植物葉片中的代謝機(jī)理。最近的研究表明當(dāng)含有污染甲醛的空氣或廢水流經(jīng)植物根系和土壤基質(zhì)構(gòu)成的生物反應(yīng)器時(shí),空氣或廢水中的甲醛可通過(guò)土壤吸收進(jìn)入植物根系,在根系細(xì)胞內(nèi)代謝酶的作用下被降解。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達(dá)到地上部分的10倍,但是至今沒(méi)有研究系統(tǒng)考察植物根系去除甲醛的代謝機(jī)理。本研究通過(guò)采用種子顆粒較大的栽培種丹波黑大豆(RB)和豆粒較小的黑大豆栽培種(SB)根系為材料,分析比較RB和SB根系吸收甲醛的動(dòng)力學(xué)模型,同時(shí)通過(guò)13C-NMR分析結(jié)合生理生化方法系統(tǒng)考察兩種黑大豆根系甲醛代謝機(jī)理,為科學(xué)利用植物根系修復(fù)甲醛污染提供理論據(jù)。用2-6mM甲醛處理RB和SB植株根系,測(cè)定0.5-48h期間的甲醛吸收,結(jié)果說(shuō)明RB和SB根系都可有效吸收處理液中的甲醛,在48 h處理時(shí)期內(nèi)兩種黑大豆根系吸收甲醛的動(dòng)力學(xué)相似,都呈現(xiàn)先慢后快的模式,對(duì)甲醛的吸收與時(shí)間的關(guān)系符合冪函數(shù)模型。13C-NMR分析結(jié)果表明在H13CHO脅迫早期(0-4h),根吸收的H13CHO首先通過(guò)依賴于谷胱甘肽依賴型甲醛脫氫酶(FALDH)的氧化途徑被氧化為甲酸,甲酸可能通過(guò)乙醛酸合酶(GXS)的作用縮合為乙醛酸;在隨后的代謝中,有部分乙醛酸可能進(jìn)入乙醛酸循環(huán)產(chǎn)生檸檬酸(Cit)和異檸檬酸(Icit);有部分乙醛酸通過(guò)轉(zhuǎn)氨酶(GAT)的作用被轉(zhuǎn)化為甘氨酸(Gly)或通過(guò)未知途徑代謝為天冬酰胺(Asn)和谷氨酰胺(Gln)。此外,還有部分乙醛酸被氧化為草酸(OA)。在H13CHO處理的后期(24-48h),Asn和Gln、OA通過(guò)三羧酸(TCA)循環(huán)被轉(zhuǎn)化為Cit和Icit;Cit和Icit進(jìn)入乙醛酸循環(huán)產(chǎn)生蘋(píng)果酸(Mal);從乙醛酸循環(huán)中輸出的Mal進(jìn)入糖異生途徑被轉(zhuǎn)化為[U-13C]Glucose。由此可見(jiàn)乙醛酸循環(huán)可能是根系甲醛代謝的中心,在甲醛處理晚期由于乙醛酸途徑在大豆根中充分發(fā)揮作用使根對(duì)甲醛吸收能力迅速增大,因而這一時(shí)期根吸收的甲醛對(duì)甲醛去除的貢獻(xiàn)顯著增加。仔細(xì)比較RB和SB根系甲醛代謝機(jī)理的差異,結(jié)果發(fā)現(xiàn)RB根系中甲醛代謝產(chǎn)生OA和Gly途徑的作用強(qiáng)于SB。此外,13C-NMR分析證實(shí)根系吸收的甲醛可能并未轉(zhuǎn)移到葉片中進(jìn)行代謝轉(zhuǎn)化。在2mM甲醛脅迫下RB葉片中積累的脯氨酸(Pro)顯著高于SB,因此甲醛脅迫SB植株葉片萎蔫比RB嚴(yán)重。但是SB根中積累的Pro顯著大于RB,氧化脅迫指標(biāo)低于RB,因此隨后的大部分實(shí)驗(yàn)材料用SB根系進(jìn)行。環(huán)孢素A(CSA)是一種特異性線粒體滲透性轉(zhuǎn)運(yùn)孔抑制劑。通過(guò)CSA預(yù)處理分析RB和SB根系各甲醛代謝途徑的亞細(xì)胞定位。對(duì)比用CSA預(yù)處理和未預(yù)處理根系在2mM H13CHO處理4h和24h代謝譜的差異。分析4h甲醛代謝譜,結(jié)果說(shuō)明CSA完全抑制甲醛代謝產(chǎn)生Cit途徑的作用,對(duì)Icit的生成有部分抑制作用,由此推測(cè)Cit和Icit產(chǎn)生途徑的作用定位在線粒體或乙醛酸循環(huán)體內(nèi)。OA、Gly2、Gln、Asn和絲氨酸(Ser3)的產(chǎn)量沒(méi)有被抑制反而增加,說(shuō)明產(chǎn)生這些有機(jī)酸的代謝途徑均在根的細(xì)胞質(zhì)中發(fā)揮作用,CSA預(yù)處理使產(chǎn)生Cit和Ici的13C代謝流進(jìn)入這些有機(jī)酸的代謝途徑。在24h甲醛代謝譜中,葡萄糖的生成也受到顯著影響,這是乙醛酸循環(huán)途徑的作用被抑制的另一結(jié)果。13C-NMR分析證實(shí)CSA預(yù)處理也影響葉片葉綠體中卡爾文循環(huán)的作用,使葉片葡萄糖含量顯著降低�？疾旒兹┪涨�的結(jié)果表明CSA預(yù)處理對(duì)RB和SB根系甲醛吸收僅有輕微的抑制作用,這是由于CSA僅抑制乙醛酸循途徑的作用而不影響OA、Gly2、Gln、Asn和Ser3產(chǎn)生途徑的作用所致,這兩個(gè)途徑的作用是獨(dú)立的。利用甲醛代謝產(chǎn)物對(duì)代謝途徑的反饋抑制作用和影響乙醛酸循環(huán)途徑關(guān)鍵酶基因表達(dá)的化合物深入考察到各代謝途徑在黑大豆根系甲醛吸收中的作用,結(jié)果說(shuō)明乙醛酸(GX)、Gly、Ser和OA預(yù)處理顯著抑制SB根系甲醛吸收,證實(shí)GX、Gly、Ser和OA產(chǎn)生途徑在SB根系甲醛吸收過(guò)程中發(fā)揮作用。Cit的預(yù)處理也有相似的作用效果,證實(shí)乙醛酸途徑在SB根系甲醛吸收過(guò)程中的作用;在其他植物中證明能抑制乙醛酸途徑關(guān)鍵酶基因異檸檬酸裂解酶(ICL)和蘋(píng)果酸合酶(MS)表達(dá)的抑制劑葡萄糖(Glucose)、蔗糖(Sucrose)和甘露糖(Mannose)的預(yù)處理也抑制SB根系甲醛吸收,實(shí)驗(yàn)結(jié)果表明10 mM Glucose、5 mM Sucrose和10 mM Mannose產(chǎn)生的抑制作用最強(qiáng)。此外,分析抗氧化劑抗壞血酸(ASA)和還原性谷胱甘肽(GSH)預(yù)處理的結(jié)果表明,2mM ASA預(yù)處理作用效果最好,能使SB根系甲醛吸收增加6倍,4mM ASA預(yù)處理作用效果最好,能使SB根系甲醛吸收增加5倍,說(shuō)明ASA和GSH預(yù)處理緩解甲醛的氧化脅迫,使SB根系甲醛吸收效率大幅度提高。甲醇(CH_3OH)能夠刺激植物生長(zhǎng)以及增加植物生物量在高等植物和農(nóng)作物中已經(jīng)被證實(shí)。在固體培養(yǎng)基中加入合適濃度的甲醇促進(jìn)SB生根作用明顯。甲醇葉面噴施能夠促進(jìn)SB莖伸長(zhǎng)。用13C-NMR分析結(jié)果說(shuō)明13CH_3OH在SB根內(nèi)代謝非常緩慢,其代謝產(chǎn)物只有Ser2和Cit(2,4),處理24h后根系吸收的13CH_30H大部分仍以游離態(tài)存在。通過(guò)Central Composite Design設(shè)計(jì)了數(shù)學(xué)模型,應(yīng)用響應(yīng)面優(yōu)化法獲得甲醇刺激SB植株根系甲醛吸收的最佳條件,即甲醇預(yù)處理濃度0.73mM,甲醛處理濃度1.3mM和甲醛處理時(shí)間22h,在此條件下SB植物對(duì)甲醛的吸收的模型預(yù)測(cè)值是85.86%,而通過(guò)實(shí)驗(yàn)驗(yàn)證的真實(shí)值是84.72%,說(shuō)明模型的擬合性很好。用0.73mM甲醇預(yù)處理SB植株根系12h,再用1.3mM H13CHO處理22h,13C-NMR分析說(shuō)明甲醇預(yù)處理促進(jìn)甲酸(FA)、Gly2、Ser3、Cit、Icit、Asn、Gln以及葡萄糖的生成,在甲醇預(yù)處理階段這些代謝產(chǎn)物的產(chǎn)量并沒(méi)有增加,這排除了由甲醇代謝作用增加這些代謝產(chǎn)物的可能性,因此推測(cè)甲醇預(yù)處理可能通過(guò)影響SB根中甲醛代謝相關(guān)酶基因的表達(dá)來(lái)改變其甲醛代謝機(jī)理,促進(jìn)甲醛的吸收。為了在分子水平上更好理解甲醇的應(yīng)用促進(jìn)SB植株生長(zhǎng)的機(jī)理,本研究應(yīng)用cDNA芯片鑒定葉面噴施5%甲醇后SB葉片中的甲醇應(yīng)答基因。結(jié)果鑒定得到731個(gè)可能的甲醇響應(yīng)基因,其中上調(diào)基因和下調(diào)基因的數(shù)目分別為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.
【學(xué)位授予單位】：昆明理工大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：X51;X173
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本文編號(hào)：2046320