大豆乙醛酸途径关键酶应答甲醛胁迫的模式及在甲醛代谢中的作用研究

发布时间：2018-10-19 07:10

【摘要】：甲醛作为室内主要污染物之一,因为毒性大,持续挥发的特性而危害健康。环境中的甲醛存在形式多种多样:如以液体的形式存在于水体中;以固体化合物的形式存在于服装、家具、土壤基质中;以气体的形式存在于大气以及室内空气中。近年来,针对甲醛的污染治理,国内外很多研究者开发了多种技术。在众多室内甲醛污染修复技术中,利用植物清除环境中甲醛的污染,具有简单、经济、环保的特点。最近的研究表明用植物根系和土壤基质构成的生物反应器可有效去除室内环境中污染的气体甲醛。本实验室谭浩博士论文研究阐明黑大豆SB吸收甲醛的代谢机理,发现乙醛酸途径在SB甲醛代谢过程中发挥重要作用。本论文利用乙醛酸途径关键酶ICL(柠檬酸裂解酶)和MS(苹果酸合成酶)的激活剂和抑制剂改变SB根系中ICL和MS基因的表达水平和酶活性,考察ICL和MS在SB根系代谢甲醛中的作用,同时利用响应面优化SB根系吸收甲醛的最佳组合条件,然后用SB根系构建悬浮式生物反应器,分析反应器在去除室内甲醛污染中的表现,取得如下结果。用RT-PCR分析不同浓度甲醛胁迫处理的SB大豆根系中ICL、MS基因的表达谱,结果表明ICL及MS基因在低浓度甲醛(2mM)处理4、12、24h,高浓度(4、6mM)甲醛处理2h表达上调,qRT-PCR分析结果说明ICL基因在2mM甲醛处理0.5h、2h、4h、12h、24h后,表达量分别达到对照的6.2、1.97、6.4、2倍,在4、6mM甲醛处理2h后增加5.3、1.9倍,MS基因在2mM甲醛处理0.5h、4h、12h、24h后,表达量分别为对照的9.8、1.7、1.2、3.4倍,在4mM处理2h后表达量是对照的1.6倍,这些数据证实ICL、MS的表达被甲醛胁迫诱导。用ICL、MS表达的抑制剂(葡萄糖、甘露糖、蔗糖)和激活剂(甲醇、MgCl2、IAA)分别和甲醛共处理SB根系,分析结果说明添加10mM葡萄糖、5mM甘露糖、10mM蔗糖后ICL和MS的表达量和酶活均显著下降。添加4mM甲醇、1mM MgCl2处理时ICL和MS基因的表达量显著上升,ICL达到对照的16.5倍、7倍,MS分别为对照的6.4倍、5.6倍,ICL和MS酶活性也大幅度提高。13C-NMR分析结果说明添加甘露糖或葡萄糖与H13CHO同处理SB根系,导致通过乙醛酸循环产生的代谢产物Malate、Cit、Glucose的生成减少。添加甲醇与H13CHO同处理SB根系,Cit、Glucose产量显著增加,这些结果证实乙醛酸循环在SB根系甲醛代谢中的重要作用。采用Central Composite Design设计数学模型,应用响应面优化法获得SB植株根系对液体甲醛吸收的最佳条件,拟合获得各试验因子对响应值影响的回归二次方程为:Y=23.14-17.35A-6.84B+3.78C+3.55AB-0.90AC-3.86BC+11.63A2+6.00B2+1.56C2,模型预测甲醛浓度、处理液体积和大豆鲜重分别为0.099mM、0.273L和38.74g的组合条件下,SB植株对甲醛的吸收效率最高,预测值为73.74%,而通过实验验证的真实值是71.40%,结果说明模型的拟合性很好。分析SB根系吸收甲醛通过茎叶的转移对甲醛去除的贡献,结果显示在1h、2h、4h根中残留有少量(～0.5%)的游离甲醛,处理24至48 h后根中检测不到残留甲醛。茎叶中的游离甲醛在4h时达到最高水平(1～3%)。在处理1～12h期间转移到茎叶周围空气中的甲醛占比未0.13%,这些结果表明通过根系吸收的甲醛可被转移至茎叶然后进入空气中,但甲醛的转移对于整个实验体系中甲醛去除的贡献很小(少于～4%)。分析SB植株蒸腾速率的变化,结果说明在2h蒸腾速率最大,同时甲醛在空气中的浓度也达到了最高水平。当处理时间持续12h时,蒸腾速率大幅度下降,同时甲醛在空气中蒸发量也快速下降,这些结果说明SB大豆植株中甲醛转移到空气的过程依赖于蒸腾拉力,同时甲醛胁迫显著抑制SB大豆植株的蒸腾作用。用SB根系构建悬浮式生物反应器,分别考察SB植株鲜重、处理液体积、环境中甲醛初始浓度对反应器去除甲醛效率的影响,结果说明SB植株鲜重为284g时反应器对甲醛的清除效率最好(保持在60%～70%);用4L的培养液处理时对甲醛清除效率最高(60%～70%),固定这两个条件后把反应器置于含有不同初始浓度甲醛、TVOC的环境中检测其清除效率,结果说明该悬浮式处理系统对于初始浓度为3mg/m3甲醛污染净化效果较好达到60%,而对于含低浓度(1.2 mg/m3)和高浓度(7.4 mg/m3)甲醛污染环境的净化效果较差低于50%。对于环境中较高浓度(7 mg/m3、9.6 mg/m3)的TVOC净化效果不理想,但是对于较低浓度(5.6 mg/m3)的TVOC该反应器的清除效率较高。
[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.
【学位授予单位】：昆明理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：X173;X51

【参考文献】