大豆乙醛酸途徑關鍵酶應答甲醛脅迫的模式及在甲醛代謝中的作用研究
發(fā)布時間：2018-10-19 07:10
【摘要】：甲醛作為室內(nèi)主要污染物之一,因為毒性大,持續(xù)揮發(fā)的特性而危害健康。環(huán)境中的甲醛存在形式多種多樣:如以液體的形式存在于水體中;以固體化合物的形式存在于服裝、家具、土壤基質(zhì)中;以氣體的形式存在于大氣以及室內(nèi)空氣中。近年來,針對甲醛的污染治理,國內(nèi)外很多研究者開發(fā)了多種技術。在眾多室內(nèi)甲醛污染修復技術中,利用植物清除環(huán)境中甲醛的污染,具有簡單、經(jīng)濟、環(huán)保的特點。最近的研究表明用植物根系和土壤基質(zhì)構成的生物反應器可有效去除室內(nèi)環(huán)境中污染的氣體甲醛。本實驗室譚浩博士論文研究闡明黑大豆SB吸收甲醛的代謝機理,發(fā)現(xiàn)乙醛酸途徑在SB甲醛代謝過程中發(fā)揮重要作用。本論文利用乙醛酸途徑關鍵酶ICL(檸檬酸裂解酶)和MS(蘋果酸合成酶)的激活劑和抑制劑改變SB根系中ICL和MS基因的表達水平和酶活性,考察ICL和MS在SB根系代謝甲醛中的作用,同時利用響應面優(yōu)化SB根系吸收甲醛的最佳組合條件,然后用SB根系構建懸浮式生物反應器,分析反應器在去除室內(nèi)甲醛污染中的表現(xiàn),取得如下結果。用RT-PCR分析不同濃度甲醛脅迫處理的SB大豆根系中ICL、MS基因的表達譜,結果表明ICL及MS基因在低濃度甲醛(2mM)處理4、12、24h,高濃度(4、6mM)甲醛處理2h表達上調(diào),qRT-PCR分析結果說明ICL基因在2mM甲醛處理0.5h、2h、4h、12h、24h后,表達量分別達到對照的6.2、1.97、6.4、2倍,在4、6mM甲醛處理2h后增加5.3、1.9倍,MS基因在2mM甲醛處理0.5h、4h、12h、24h后,表達量分別為對照的9.8、1.7、1.2、3.4倍,在4mM處理2h后表達量是對照的1.6倍,這些數(shù)據(jù)證實ICL、MS的表達被甲醛脅迫誘導。用ICL、MS表達的抑制劑(葡萄糖、甘露糖、蔗糖)和激活劑(甲醇、MgCl2、IAA)分別和甲醛共處理SB根系,分析結果說明添加10mM葡萄糖、5mM甘露糖、10mM蔗糖后ICL和MS的表達量和酶活均顯著下降。添加4mM甲醇、1mM MgCl2處理時ICL和MS基因的表達量顯著上升,ICL達到對照的16.5倍、7倍,MS分別為對照的6.4倍、5.6倍,ICL和MS酶活性也大幅度提高。13C-NMR分析結果說明添加甘露糖或葡萄糖與H13CHO同處理SB根系,導致通過乙醛酸循環(huán)產(chǎn)生的代謝產(chǎn)物Malate、Cit、Glucose的生成減少。添加甲醇與H13CHO同處理SB根系,Cit、Glucose產(chǎn)量顯著增加,這些結果證實乙醛酸循環(huán)在SB根系甲醛代謝中的重要作用。采用Central Composite Design設計數(shù)學模型,應用響應面優(yōu)化法獲得SB植株根系對液體甲醛吸收的最佳條件,擬合獲得各試驗因子對響應值影響的回歸二次方程為:Y=23.14-17.35A-6.84B+3.78C+3.55AB-0.90AC-3.86BC+11.63A2+6.00B2+1.56C2,模型預測甲醛濃度、處理液體積和大豆鮮重分別為0.099mM、0.273L和38.74g的組合條件下,SB植株對甲醛的吸收效率最高,預測值為73.74%,而通過實驗驗證的真實值是71.40%,結果說明模型的擬合性很好。分析SB根系吸收甲醛通過莖葉的轉(zhuǎn)移對甲醛去除的貢獻,結果顯示在1h、2h、4h根中殘留有少量(～0.5%)的游離甲醛,處理24至48 h后根中檢測不到殘留甲醛。莖葉中的游離甲醛在4h時達到最高水平(1～3%)。在處理1～12h期間轉(zhuǎn)移到莖葉周圍空氣中的甲醛占比未0.13%,這些結果表明通過根系吸收的甲醛可被轉(zhuǎn)移至莖葉然后進入空氣中,但甲醛的轉(zhuǎn)移對于整個實驗體系中甲醛去除的貢獻很小(少于～4%)。分析SB植株蒸騰速率的變化,結果說明在2h蒸騰速率最大,同時甲醛在空氣中的濃度也達到了最高水平。當處理時間持續(xù)12h時,蒸騰速率大幅度下降,同時甲醛在空氣中蒸發(fā)量也快速下降,這些結果說明SB大豆植株中甲醛轉(zhuǎn)移到空氣的過程依賴于蒸騰拉力,同時甲醛脅迫顯著抑制SB大豆植株的蒸騰作用。用SB根系構建懸浮式生物反應器,分別考察SB植株鮮重、處理液體積、環(huán)境中甲醛初始濃度對反應器去除甲醛效率的影響,結果說明SB植株鮮重為284g時反應器對甲醛的清除效率最好(保持在60%～70%);用4L的培養(yǎng)液處理時對甲醛清除效率最高(60%～70%),固定這兩個條件后把反應器置于含有不同初始濃度甲醛、TVOC的環(huán)境中檢測其清除效率,結果說明該懸浮式處理系統(tǒng)對于初始濃度為3mg/m3甲醛污染凈化效果較好達到60%,而對于含低濃度(1.2 mg/m3)和高濃度(7.4 mg/m3)甲醛污染環(huán)境的凈化效果較差低于50%。對于環(huán)境中較高濃度(7 mg/m3、9.6 mg/m3)的TVOC凈化效果不理想,但是對于較低濃度(5.6 mg/m3)的TVOC該反應器的清除效率較高。
[Abstract]:Formaldehyde is one of the main pollutants in the room, which is harmful to health because of its high toxicity and persistent volatilization. There are various forms of formaldehyde in the environment: for example, in the form of a liquid in a body of water; in the form of a solid compound in clothing, furniture, soil matrix; in the form of a gas in the atmosphere and in the indoor air. In recent years, many researchers at home and abroad have developed a variety of technologies for the pollution treatment of formaldehyde. in a plurality of indoor formaldehyde pollution repair technologies, the pollution of formaldehyde in the environment of the plant removal is utilized, and the method has the characteristics of simplicity, economy and environmental protection. Recent studies have shown that bioreactors consisting of plant roots and soil matrices can effectively remove contaminated gaseous formaldehyde in indoor environments. In this study, the metabolic mechanism of the absorption of formaldehyde by black soybean SB was studied in this laboratory, and it was found that the metabolic pathway plays an important role in the metabolic process of SB formaldehyde. In this paper, the expression level and enzyme activity of ICL and MS gene in SB root system were changed by using the key enzyme ICL (citrate lyase) and MS (malic acid synthase) activator and inhibitor, and the effects of ICL and MS on the metabolism of formaldehyde in SB root system were investigated. At the same time, using the response surface to optimize the optimal combination condition of the absorption of formaldehyde by the SB root system, the suspension bioreactor was constructed with SB root system, and the performance of the reactor in removing formaldehyde in the room was analyzed, and the following results were obtained. The expression profiles of ICL and MS genes were analyzed by RT-PCR. The results showed that ICL and MS genes were upregulated at low concentrations of formaldehyde (2mM) for 4, 12, 24h, high concentration (4, 6mM) formaldehyde treatment. The results of qRT-PCR showed that ICL gene was treated with 2mM formaldehyde for 0. 5h, 2h. After 24h, 12h and 24h, the expression level reached 6. 2, 1. 97, 6. 4 and 2 times of the control respectively. After treatment with 4 and 6mM formaldehyde for 2 h, the expression level of MS gene increased by 5. 3, 1. 9 times, and the expression level of MS gene was 0.9. 8, 1. 7, 1. 2, 3. 4 times respectively after treatment with 2mM formaldehyde, and the expression level was 1. 6 times of the control after 2h after treatment with 4mM. These data confirm ICL. The expression of MS was induced by formaldehyde stress. Using ICL, MS expression inhibitor (glucose, mannose, sucrose) and activator (methanol, MgCl2, IAA) and formaldehyde co-processed SB root system, the results showed that the expression and enzyme activity of ICL and MS added with 10mM glucose, 5mM mannose and 10mM sucrose decreased significantly. By adding 4mM methanol and 1mM MgCl2, the expression level of ICL and MS increased significantly. ICL reached 16. 5 times and 7 times of control. The activity of ICL and MS was 6. 4 times, 5. 6 times, ICL and MS respectively. The results of 13C-NMR analysis showed that addition of mannose or glucose and H13CHO were treated with SB root system. resulting in a reduction in the production of metabolic products malate, cit, glucose produced by the granulation cycle. The addition of methanol and H13CHO in the treatment of SB roots, Cit and Glucose yield increased significantly, which confirmed the important role of yeast circulation in the metabolism of formaldehyde in SB root system. The optimal conditions for the absorption of liquid formaldehyde by the root system of SB plants were obtained by using the Central Composite Design, and the regression quadratic equation of the effect of each test factor on response values was obtained: Y = 23.14-17. 35A-6.84B + 3.78C + 3.55AB-0.90AC-3.78C + 11.63A2 + 6.00B2 + 1.56C2, the model predicted formaldehyde concentration, the treated liquid product and the fresh weight of soybean were 0.099mM, respectively. Under the combined condition of 0.273L and 384.74g, the absorption efficiency of SB plants to formaldehyde was the highest, the predicted value was 73. 74%, and the real value obtained through the experiment was 71. 40%, and the result indicates that the model has good fit. The contribution of the absorption of formaldehyde from the roots of SB to the removal of formaldehyde was analyzed. The results showed that a small amount (~ 0.5%) of free formaldehyde remained in the roots of 1h, 2h and 4h, and no residual formaldehyde was found in the root after treatment for 24 to 48 hours. Free formaldehyde in stem leaves reached the highest level (1-3%) at 4h. The percentage of formaldehyde transferred to the air around the stem leaves during the treatment of 1 to 12h was less than 0. 13%. These results showed that formaldehyde absorbed by the root system could be transferred to the stem and then into the air, but the transfer of formaldehyde was very small (less than -4%) for formaldehyde removal throughout the experimental system. The transpiration rate of SB plants was analyzed. The results showed that the transpiration rate was the highest at 2h, while the concentration of formaldehyde in air reached the highest level. When the treatment time lasted for 12h, the transpiration rate decreased greatly, while the evaporation rate of formaldehyde decreased rapidly in the air, which indicated that the process of formaldehyde transfer to air in SB soybean plants depended on transpiration pull force, while formaldehyde stress significantly inhibited the transpiration of SB soybean plants. The effect of the initial concentration of formaldehyde on the removal efficiency of formaldehyde in the reactor was studied by using SB root system, and the effect of the initial concentration of formaldehyde on the formaldehyde removal efficiency of the reactor was investigated. The results indicated that the removal efficiency of the reactor to formaldehyde was best (kept at 60% ~ 70%) when the fresh weight of SB was 284g. the removal efficiency of formaldehyde is highest (60-70%) when treating with 4L culture solution, and after the two conditions are fixed, the reactor is placed in an environment containing different initial concentrations of formaldehyde and TVOC to detect the removal efficiency, The results show that the suspended treatment system has a good purification effect of 60% for the initial concentration of 3mg/ m3 formaldehyde, but the purification effect of formaldehyde with low concentration (1,2 mg/ m3) and high concentration (7. 4 mg/ m3) is lower than 50%. TVOC purification was not ideal for higher concentrations (7 mg/ m3, 9. 6 mg/ m3) in the environment, but the removal efficiency of TVOC for lower concentrations (5. 6 mg/ m3) was higher.
【學位授予單位】：昆明理工大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：X173;X51
本文編號：2280491
[Abstract]:Formaldehyde is one of the main pollutants in the room, which is harmful to health because of its high toxicity and persistent volatilization. There are various forms of formaldehyde in the environment: for example, in the form of a liquid in a body of water; in the form of a solid compound in clothing, furniture, soil matrix; in the form of a gas in the atmosphere and in the indoor air. In recent years, many researchers at home and abroad have developed a variety of technologies for the pollution treatment of formaldehyde. in a plurality of indoor formaldehyde pollution repair technologies, the pollution of formaldehyde in the environment of the plant removal is utilized, and the method has the characteristics of simplicity, economy and environmental protection. Recent studies have shown that bioreactors consisting of plant roots and soil matrices can effectively remove contaminated gaseous formaldehyde in indoor environments. In this study, the metabolic mechanism of the absorption of formaldehyde by black soybean SB was studied in this laboratory, and it was found that the metabolic pathway plays an important role in the metabolic process of SB formaldehyde. In this paper, the expression level and enzyme activity of ICL and MS gene in SB root system were changed by using the key enzyme ICL (citrate lyase) and MS (malic acid synthase) activator and inhibitor, and the effects of ICL and MS on the metabolism of formaldehyde in SB root system were investigated. At the same time, using the response surface to optimize the optimal combination condition of the absorption of formaldehyde by the SB root system, the suspension bioreactor was constructed with SB root system, and the performance of the reactor in removing formaldehyde in the room was analyzed, and the following results were obtained. The expression profiles of ICL and MS genes were analyzed by RT-PCR. The results showed that ICL and MS genes were upregulated at low concentrations of formaldehyde (2mM) for 4, 12, 24h, high concentration (4, 6mM) formaldehyde treatment. The results of qRT-PCR showed that ICL gene was treated with 2mM formaldehyde for 0. 5h, 2h. After 24h, 12h and 24h, the expression level reached 6. 2, 1. 97, 6. 4 and 2 times of the control respectively. After treatment with 4 and 6mM formaldehyde for 2 h, the expression level of MS gene increased by 5. 3, 1. 9 times, and the expression level of MS gene was 0.9. 8, 1. 7, 1. 2, 3. 4 times respectively after treatment with 2mM formaldehyde, and the expression level was 1. 6 times of the control after 2h after treatment with 4mM. These data confirm ICL. The expression of MS was induced by formaldehyde stress. Using ICL, MS expression inhibitor (glucose, mannose, sucrose) and activator (methanol, MgCl2, IAA) and formaldehyde co-processed SB root system, the results showed that the expression and enzyme activity of ICL and MS added with 10mM glucose, 5mM mannose and 10mM sucrose decreased significantly. By adding 4mM methanol and 1mM MgCl2, the expression level of ICL and MS increased significantly. ICL reached 16. 5 times and 7 times of control. The activity of ICL and MS was 6. 4 times, 5. 6 times, ICL and MS respectively. The results of 13C-NMR analysis showed that addition of mannose or glucose and H13CHO were treated with SB root system. resulting in a reduction in the production of metabolic products malate, cit, glucose produced by the granulation cycle. The addition of methanol and H13CHO in the treatment of SB roots, Cit and Glucose yield increased significantly, which confirmed the important role of yeast circulation in the metabolism of formaldehyde in SB root system. The optimal conditions for the absorption of liquid formaldehyde by the root system of SB plants were obtained by using the Central Composite Design, and the regression quadratic equation of the effect of each test factor on response values was obtained: Y = 23.14-17. 35A-6.84B + 3.78C + 3.55AB-0.90AC-3.78C + 11.63A2 + 6.00B2 + 1.56C2, the model predicted formaldehyde concentration, the treated liquid product and the fresh weight of soybean were 0.099mM, respectively. Under the combined condition of 0.273L and 384.74g, the absorption efficiency of SB plants to formaldehyde was the highest, the predicted value was 73. 74%, and the real value obtained through the experiment was 71. 40%, and the result indicates that the model has good fit. The contribution of the absorption of formaldehyde from the roots of SB to the removal of formaldehyde was analyzed. The results showed that a small amount (~ 0.5%) of free formaldehyde remained in the roots of 1h, 2h and 4h, and no residual formaldehyde was found in the root after treatment for 24 to 48 hours. Free formaldehyde in stem leaves reached the highest level (1-3%) at 4h. The percentage of formaldehyde transferred to the air around the stem leaves during the treatment of 1 to 12h was less than 0. 13%. These results showed that formaldehyde absorbed by the root system could be transferred to the stem and then into the air, but the transfer of formaldehyde was very small (less than -4%) for formaldehyde removal throughout the experimental system. The transpiration rate of SB plants was analyzed. The results showed that the transpiration rate was the highest at 2h, while the concentration of formaldehyde in air reached the highest level. When the treatment time lasted for 12h, the transpiration rate decreased greatly, while the evaporation rate of formaldehyde decreased rapidly in the air, which indicated that the process of formaldehyde transfer to air in SB soybean plants depended on transpiration pull force, while formaldehyde stress significantly inhibited the transpiration of SB soybean plants. The effect of the initial concentration of formaldehyde on the removal efficiency of formaldehyde in the reactor was studied by using SB root system, and the effect of the initial concentration of formaldehyde on the formaldehyde removal efficiency of the reactor was investigated. The results indicated that the removal efficiency of the reactor to formaldehyde was best (kept at 60% ~ 70%) when the fresh weight of SB was 284g. the removal efficiency of formaldehyde is highest (60-70%) when treating with 4L culture solution, and after the two conditions are fixed, the reactor is placed in an environment containing different initial concentrations of formaldehyde and TVOC to detect the removal efficiency, The results show that the suspended treatment system has a good purification effect of 60% for the initial concentration of 3mg/ m3 formaldehyde, but the purification effect of formaldehyde with low concentration (1,2 mg/ m3) and high concentration (7. 4 mg/ m3) is lower than 50%. TVOC purification was not ideal for higher concentrations (7 mg/ m3, 9. 6 mg/ m3) in the environment, but the removal efficiency of TVOC for lower concentrations (5. 6 mg/ m3) was higher.
【學位授予單位】：昆明理工大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：X173;X51
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