WO2021169154A1 - Method for obtaining 13c and 15n dual-labeled plant and use thereof in preparation of biomass charcoal - Google Patents
Method for obtaining 13c and 15n dual-labeled plant and use thereof in preparation of biomass charcoal Download PDFInfo
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- WO2021169154A1 WO2021169154A1 PCT/CN2020/103040 CN2020103040W WO2021169154A1 WO 2021169154 A1 WO2021169154 A1 WO 2021169154A1 CN 2020103040 W CN2020103040 W CN 2020103040W WO 2021169154 A1 WO2021169154 A1 WO 2021169154A1
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- C—CHEMISTRY; METALLURGY
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- C01B32/00—Carbon; Compounds thereof
- C01B32/05—Preparation or purification of carbon not covered by groups C01B32/15, C01B32/20, C01B32/25, C01B32/30
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
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- the invention belongs to the technical field of isotope labeling, and specifically relates to a method for obtaining 13 C and 15 N double-labeled plants and its application in the preparation of biomass charcoal.
- Stable isotope tracing technology can distinguish between exogenous materials and soil itself, thereby providing important technical means for the study of soil element cycle.
- Biomass charcoal is produced by the pyrolysis of biomass materials under anaerobic or anaerobic conditions. Due to its high stability, biomass charcoal has become a new type of carbon sequestration and emission reduction material. Research on the stability of biomass charcoal added to the soil and its influence on the element cycle of the soil itself has become a research hotspot, and obtaining isotope-labeled biomass charcoal is the basis for this research.
- Plants are one of the important raw materials for the preparation of biomass charcoal.
- the method of monoisotopic labeling of plants with 13 C or 15 N is relatively common.
- the methods for dual-labeling plants with 13 C and 15 N isotopes are still relatively limited.
- the existing method mainly uses 13 CO 2 gas to perform 13 C continuous labeling on plants, which requires the use of steel cylinders during the implementation process, which is inconvenient to transport and poses a certain safety risk.
- the method of drip irrigation and fertilization is to add 15 N solution or fertilizer to the soil and other substrates.
- the plant absorbs 15 N through roots and assimilates to achieve marking.
- the nutrient solution and fertilizer in the two methods need to be added repeatedly, and the time period is long.
- the labeling method in which 15 N-urea and 13 C-glucose solutions are introduced into plant stems through sewing needles and absorbent cotton thread has a high recovery rate, but it is not easy to mark a large number of plants.
- in order to quickly obtain 13 C and 15 N dual-labeled biomass char it is necessary to first develop a relatively simple, fast and efficient method for dual-labeling plants.
- the technical problem to be solved by the present invention is to provide a method for obtaining 13 C and 15 N double-labeled plants.
- the labeling method is relatively flexible and simple, the labeling process has a short cycle and high efficiency, and is not limited by the growth stage of plants. In situ experiments are all applicable, and get rid of the limitation that the dual labeling of 13 C and 15 N isotopes is difficult to implement in plant samples.
- the second technical problem to be solved by the present invention is to provide a 13 C and 15 N double-labeled plant.
- the third technical problem to be solved by the present invention is to provide an application of 13 C and 15 N double-labeled plants in the preparation of biomass charcoal.
- a method for obtaining 13 C and 15 N double-labeled plants adopts 13 C pulse labeling and 15 N foliar spray labeling to double-label the plants to obtain 13 C and 15 N double-labeled plants.
- 15 N labeling is performed first, and then 13 C labeling is performed during the labeling process.
- This labeling sequence is adopted because: (1) If the 13 C labeling of plants is carried out first, the plants cannot be killed in time, and a part of the 13 C assimilation of plants can be used through respiration, resulting in a decrease in the abundance of 13 C in plants; (2) If the plant 13 C labeling is performed first, the plant's standing time is prolonged, the plant 13 C will be transported downwards, and it will be enriched in the roots of the plant and may be excreted in the form of root exudates, which is not conducive to increasing the 13 C.
- the 15 N-labeled nitrogen fertilizer solution is a 15 N-urea solution with a concentration of 1 to 3 g/L.
- a surfactant is added to the 15 N-labeled nitrogen fertilizer solution.
- the surfactant is Triton X-100, and the volume of the 15 N-labeled nitrogen fertilizer solution is 0.02% to 0.08%.
- the 13 C-carbonate is Ba 13 CO 3 and the 12 C-carbonate is Ba 12 CO 3 .
- the number of 13 C-carbonate and 12 C-carbonate is 2 parts respectively.
- the plants are herbaceous plants or low woody plants.
- the height of herbaceous plants or low woody plants should not be greater than the height of the plexiglass box.
- the herbaceous plants are rice, wheat, corn or soybeans, and the low woody plants are shrubs or saplings.
- the double-labeled plants are isolated from air and put into a muffle furnace for high-temperature pyrolysis to obtain biomass charcoal; the heating rate of the muffle furnace It is 4 ⁇ 20°C/min, and lasts for 3 ⁇ 6h after reaching the target temperature of 250 ⁇ 700°C.
- the beneficial effects of the present invention include:
- the method of the present invention for quickly obtaining 13 C and 15 N double-labeled plants has the characteristics of strong timeliness; continuous labeling is not required, and the labeling of plants can be completed within one day.
- the device required for the dual marking method of the present invention is simple and easy to operate; it does not require a steel cylinder for storing 13 CO 2 gas, does not require an additional power system (such as an electric pump), and does not require the installation of complex devices.
- the method for quickly obtaining 13 C and 15 N double-labeled plants of the present invention has the characteristics of wide applicability; it is not limited to pot experiments, but can also be applied to in-situ experiments in the field; it can be applied to a variety of plants, including rice Herbs such as wheat, corn, soybeans, etc., can also be low shrubs or saplings; they are not limited by the growth stage of the plant.
- the plant material used is rice.
- the rice pot experiment was carried out at Xiashu Forest Farm (119°13′28′′E, 32°7′32′′N) of Nanjing Forestry University, Zhenjiang City, Jiangsu province.
- the local climate belongs to the subtropical monsoon climate, the annual precipitation is 1055.6mm, the annual average temperature is 15.2°C, and the annual average sunshine hours is 2157h.
- Rice 13 C marking materials Ba 13 CO 3 ( 13 C atom 98%, Shanghai Research Institute of Chemical Industry), Ba 12 CO 3 , hydrochloric acid, transparent plexiglass box, gas generator, 704 silicone rubber, small battery fan, ice box, Thermometer and hygrometer, black cloth;
- Transparent plexiglass box when performing 13 C marking, put potted rice in the plexiglass box (the height of the plant to be marked should not be greater than the height of the plexiglass box) to ensure that the marking process is carried out in a sealed condition.
- the box body is made of plexiglass with a thickness of about 0.50cm, length ⁇ width ⁇ height is 106cm ⁇ 71cm ⁇ 100cm, and the bottom of the box is made of a 111cm ⁇ 76cm PVC panel. There is a sink with a height of about 8cm and a width of 4cm around the panel.
- the bottom edge of the plexiglass box is placed in the sink, and the box is sealed by adding water to the sink; four holes with a diameter of about 2.30cm are evenly designed on the top of the plexiglass box to install the separatory funnel; one on each side of the box Handle to facilitate the movement of the transparent plexiglass box;
- Gas generating device consists of a 250mL separatory funnel, a 250mL beaker and a silicone rubber tube with an outer diameter of 6mm; add Ba 13 CO 3 or Ba 12 CO 3 powder into the beaker, and add hydrochloric acid to the separatory funnel, After opening the separatory funnel, the hydrochloric acid enters the beaker through the silicone rubber tube and reacts with the BaCO 3 powder to generate CO 2 gas; there are 4 gas generators, 2 generating 13 CO 2 , and 2 generating 12 CO 2 ;
- Small battery fan and ice box The small battery fan is suspended on the upper and bottom surface of the transparent plexiglass box to mix the CO 2 gas generated; when the temperature in the plexiglass box is too high, use an ice box to cool it down;
- Thermometer and hygrometer used to monitor the temperature and humidity in the plexiglass box in real time;
- Black cloth Cover the plexiglass box before the CO 2 is produced by the gas generator to promote the absorption of the CO 2 produced by the rice.
- the size should just cover the entire plexiglass box.
- Atomization bottle (Amway Company): marked with a scale, with a capacity of 100mL, used for foliar spray;
- Surfactant Triton X-100, with a concentration of 0.05% (v/v). Surfactant can reduce the surface tension of the solution, inhibit capillary adsorption, and slow down the dissolution and recrystallization process of fertilizers. Cell permeability promotes better absorption of 15 N-urea solution.
- the early stage of reproductive growth of rice was selected, and the isotope labeling of rice was carried out on September 27, 2019.
- Using this labeling sequence has the following advantages: (1) If the plant 13 C labeling is carried out first, the plant cannot be killed in time, and a part of the 13 C assimilation of the plant can pass Respiration is used to reduce the 13 C abundance of plants; (2) If the plant 13 C labeling is carried out first, the plant’s standing time will be prolonged, and the plant 13 C will be transported downwards and accumulated in the roots of the plant.
- step (5) add HCl solution to the second beaker containing Ba 13 CO 3 to complete the second 13 C pulse mark;
- step (5) In order to improve the absorption of 13 CO 2 by rice, repeat step (5) twice, and add HCl solution to the two beakers containing Ba 12 CO 3 to produce 12 CO 2 for rice absorption and utilization;
- the abundance of the double-labeled rice obtained in this example is shown in Table 1.
- Table 1 There is a large difference in the abundance of 13 C between the aboveground and underground parts of rice , which indicates that the 13 C absorbed by photosynthesis of plants is limited in transportation to the roots in a relatively short period of time.
- 13 C abundance the difference in 15 N abundance above ground and underground is smaller.
- the method of the present invention has successfully obtained 13 C and 15 N double-labeled rice.
- Example 1 On October 25, 2019, in the Xiashu Forest Farm of Nanjing Forestry University, the 13 C and 15 N double-labeled rice obtained in Example 1 was used to prepare biomass char, and the abundance of 13 C and 15 N of the biomass char was measured.
- the 13 C and 15 N abundances of the prepared biomass charcoal are shown in Table 2.
- the 13 C abundance of biomass charcoal prepared from rice decreased, especially in the underground parts of rice, which decreased by 69.2% and 71.3% at 300°C and 500°C, respectively.
- the abundance of 15 N also decreased, but the degree of decrease was lower than that of 13 C.
- the 15 N abundance of biomass charcoal prepared from the aboveground part of rice hardly changed, while the 15 N abundance of biomass charcoal prepared from the underground part decreased by 40.5%.
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Abstract
Disclosed are a method for obtaining a 13C and 15N dual-labeled plant and the use thereof in the preparation of biomass charcoal. The present invention belongs to the technical field of isotope labeling. The method involves dual-labeling a plant using a method of 13C pulse labeling and 15N foliage spraying to obtain a 13C and 15N dual-labeled plant. The dual-labeled plant is used for preparing 13C and 15N dual-labeled biomass charcoal. The method of the present invention is relatively flexible, simple and convenient, has a short period for the labeling process and a high efficiency, is not limited by the growth stage of the plant, is applicable to both potting and in situ field experiments, and gets rid of the limitation that the dual-labeling of a plant sample with 13C and 15N isotopes is hard to implement. In addition, 13C and 15N dual-labeled biomass charcoal is prepared from the obtained dual-labeled plant as a raw material.
Description
本发明属于同位素标记技术领域,具体涉及一种获得
13C和
15N双标记植物的方法及其在制备生物质炭中的应用。
The invention belongs to the technical field of isotope labeling, and specifically relates to a method for obtaining 13 C and 15 N double-labeled plants and its application in the preparation of biomass charcoal.
稳定同位素示踪技术能够区分外源物质和土壤本身物质,从而为土壤元素循环的研究提供重要的技术手段。生物质炭是由生物质材料在无氧或缺氧条件下热解而产生。由于其较高的稳定性,生物质炭成为一种新型的固碳减排材料。研究添加到土壤中的生物质炭的稳定性及其对土壤本身元素循环的影响成为一个研究热点,而获得同位素标记的生物质炭是开展这项研究的基础。Stable isotope tracing technology can distinguish between exogenous materials and soil itself, thereby providing important technical means for the study of soil element cycle. Biomass charcoal is produced by the pyrolysis of biomass materials under anaerobic or anaerobic conditions. Due to its high stability, biomass charcoal has become a new type of carbon sequestration and emission reduction material. Research on the stability of biomass charcoal added to the soil and its influence on the element cycle of the soil itself has become a research hotspot, and obtaining isotope-labeled biomass charcoal is the basis for this research.
植物是制备生物质炭的重要原料之一。植物
13C或
15N的单同位素标记的方法比较常见。然而,对植物同时进行
13C和
15N同位素双标记的方法还比较有限。现有公开技术包括:利用缝衣针和脱脂棉绳进行植物茎杆
13C和
15N双标记(CN 104142382 A)、注射器注入
13CO
2气体和在石英砂基质中施加
15N溶液获取高丰度双标记植物样品(CN 107047265 A)、利用高压气瓶通入
13CO
2气体和在土壤中滴灌
15N溶液连续标记植物的装置和方法(CN 104221740 B)、利用高压气瓶通入
13CO
2气体和在水培箱中添加
15N溶液对水生植物进行连续
13C和
15N双标记的装置(CN 207318164 U)。现有方法主要是通过直接使用
13CO
2气体对植物进行
13C连续标记,这在实施过程中需要使用钢瓶,其运输不方便,并存在一定的安全风险。滴灌和施肥的方法是将
15N溶液或肥料添加到土壤等基质中,植物通过根系吸收
15N并进行同化而实现标记。该两种方法中的营养液和肥料需要重复添加,并且时间周期长。通过缝衣针和脱脂棉线将
15N-尿素和
13C-葡萄糖溶液导入植物茎秆的标记方法,回收率较高,但不容易实现对大量植物的标记。综上所述,为了快速获取
13C和
15N双标记的生物质炭,需要首先开发一种相对简单、快速高效的对植物进行双标记的方法。
Plants are one of the important raw materials for the preparation of biomass charcoal. The method of monoisotopic labeling of plants with 13 C or 15 N is relatively common. However, the methods for dual-labeling plants with 13 C and 15 N isotopes are still relatively limited. Existing published technologies include: using sewing needles and absorbent cotton ropes for 13 C and 15 N double marking of plant stems (CN 104142382 A), syringe injection of 13 CO 2 gas and application of 15 N solution in quartz sand matrix to obtain high abundance Dual-labeled plant samples (CN 107047265 A), using a high-pressure gas cylinder to pass 13 CO 2 gas and drip irrigation 15 N solution in the soil to continuously mark plants and methods (CN 104221740 B), using a high-pressure gas bottle to pass 13 CO 2 A device (CN 207318164 U) for continuous 13 C and 15 N double labeling of aquatic plants by adding 15 N solution to gas and a hydroponic tank. The existing method mainly uses 13 CO 2 gas to perform 13 C continuous labeling on plants, which requires the use of steel cylinders during the implementation process, which is inconvenient to transport and poses a certain safety risk. The method of drip irrigation and fertilization is to add 15 N solution or fertilizer to the soil and other substrates. The plant absorbs 15 N through roots and assimilates to achieve marking. The nutrient solution and fertilizer in the two methods need to be added repeatedly, and the time period is long. The labeling method in which 15 N-urea and 13 C-glucose solutions are introduced into plant stems through sewing needles and absorbent cotton thread has a high recovery rate, but it is not easy to mark a large number of plants. In summary, in order to quickly obtain 13 C and 15 N dual-labeled biomass char, it is necessary to first develop a relatively simple, fast and efficient method for dual-labeling plants.
发明内容Summary of the invention
本发明要解决的技术问题是提供一种获得
13C和
15N双标记植物的方法,该标记方法相对灵活、简便,标记过程周期短、效率高,不受植物生长阶段的限制,盆栽和野外原位实验均适用,摆脱了植物样品
13C和
15N同位素双标记难以实施的局限。本发明要解决的第2个技术问题是提供一种
13C和
15N双标记植物。本发明要解决的第3个技术问题是提供一种
13C和
15N双标记植物在制备生物质炭中的应用。
The technical problem to be solved by the present invention is to provide a method for obtaining 13 C and 15 N double-labeled plants. The labeling method is relatively flexible and simple, the labeling process has a short cycle and high efficiency, and is not limited by the growth stage of plants. In situ experiments are all applicable, and get rid of the limitation that the dual labeling of 13 C and 15 N isotopes is difficult to implement in plant samples. The second technical problem to be solved by the present invention is to provide a 13 C and 15 N double-labeled plant. The third technical problem to be solved by the present invention is to provide an application of 13 C and 15 N double-labeled plants in the preparation of biomass charcoal.
为了解决上述问题,本发明所采用的技术方案如下:In order to solve the above problems, the technical solutions adopted by the present invention are as follows:
一种获得
13C和
15N双标记植物的方法,采用
13C脉冲标记和
15N叶面喷施标记对植物进行双标记,获得
13C和
15N双标记植物。
A method for obtaining 13 C and 15 N double-labeled plants, adopts 13 C pulse labeling and 15 N foliar spray labeling to double-label the plants to obtain 13 C and 15 N double-labeled plants.
所述的获得
13C和
15N双标记植物的方法,在标记过程中先进行
15N的标记,再进行
13C的标记。采用这样的标记顺序是因为:(1)若先进行植物
13C标记,则不能及时对植物进行杀青,植物同化的一部分
13C能够通过呼吸作用被利用,从而造成植物
13C丰度的降低;(2)若先进行植物
13C标记,植物静置时间延长,植物
13C会发生向下运输,并在植物的根部富集并有可能以根系分泌物的形式排出,这样不利于提高
13C回收率;(3)
15N标记完成后仍然需要一段时间使植物吸收同化
15N,恰好在这段时间内进行
13C标记可以节省标记时间,而且会避免上面(1)和(2)所提到的弊端。所以,采用先进行植物
15N的标记,再进行
13C的标记方法,才能在短时间内得到本发明预期的双标记样品。具体包括以下步骤:
In the method for obtaining 13 C and 15 N double-labeled plants, 15 N labeling is performed first, and then 13 C labeling is performed during the labeling process. This labeling sequence is adopted because: (1) If the 13 C labeling of plants is carried out first, the plants cannot be killed in time, and a part of the 13 C assimilation of plants can be used through respiration, resulting in a decrease in the abundance of 13 C in plants; (2) If the plant 13 C labeling is performed first, the plant's standing time is prolonged, the plant 13 C will be transported downwards, and it will be enriched in the roots of the plant and may be excreted in the form of root exudates, which is not conducive to increasing the 13 C. Recovery rate: (3) After 15 N labeling is completed, it still takes a while for plants to absorb and assimilate 15 N. It happens that 13 C labeling during this period can save labeling time and avoid the above (1) and (2). To the drawbacks. Therefore, the 15 N labeling of plants and then the 13 C labeling method can be adopted to obtain the double-labeled sample expected by the present invention in a short time. It includes the following steps:
(1)去除待标记植物衰老枯黄的叶片,采用雾化瓶喷施
15N标记的氮素肥料溶液;
(1) Remove the senescent and yellow leaves of the plants to be marked, and spray a 15 N-labeled nitrogen fertilizer solution with an atomized bottle;
(2)将植物置于有机玻璃箱,在密闭条件下,首先通过盐酸和
13C-碳酸盐反应产生
13CO
2进行脉冲标记,然后通过盐酸和
12C-碳酸盐反应产生
12CO
2,每份
13C-碳酸盐或
12C-碳酸盐所产生CO
2的体积浓度为300~800ppm;
(2) Put the plant in a plexiglass box, under airtight conditions, firstly generate 13 CO 2 by the reaction of hydrochloric acid and 13 C-carbonate for pulse labeling, and then generate 12 CO 2 by the reaction of hydrochloric acid and 12 C-carbonate , The volume concentration of CO 2 produced by each 13 C-carbonate or 12 C-carbonate is 300~800ppm;
(3)标记结束后,取下有机玻璃箱,通风,完成标记过程。(3) After marking, remove the plexiglass box, ventilate, and complete the marking process.
所述的获得
13C和
15N双标记植物的方法,所述
15N标记的氮素肥料溶液为
15N-尿素溶液,浓度为1~3g/L。
In the method for obtaining 13 C and 15 N dual-labeled plants, the 15 N-labeled nitrogen fertilizer solution is a 15 N-urea solution with a concentration of 1 to 3 g/L.
所述的获得
13C和
15N双标记植物的方法,所述
15N标记的氮素肥料溶液中加入表面活性剂。
In the method for obtaining 13 C and 15 N double-labeled plants, a surfactant is added to the 15 N-labeled nitrogen fertilizer solution.
所述的获得
13C和
15N双标记植物的方法,所述表面活性剂为曲拉通TritonX-100,加入体积为
15N标记的氮素肥料溶液体积的0.02%~0.08%。
In the method for obtaining 13 C and 15 N double-labeled plants, the surfactant is Triton X-100, and the volume of the 15 N-labeled nitrogen fertilizer solution is 0.02% to 0.08%.
所述的获得
13C和
15N双标记植物的方法,所述
13C-碳酸盐为Ba
13CO
3,所述
12C-碳酸盐为Ba
12CO
3。
In the method for obtaining 13 C and 15 N dual-labeled plants, the 13 C-carbonate is Ba 13 CO 3 and the 12 C-carbonate is Ba 12 CO 3 .
所述的获得
13C和
15N双标记植物的方法,所述
13C-碳酸盐和
12C-碳酸盐的份数分别为2份。
In the method for obtaining 13 C and 15 N dual-labeled plants, the number of 13 C-carbonate and 12 C-carbonate is 2 parts respectively.
所述的获得
13C和
15N双标记植物的方法,所述植物为草本植物或低矮的木本植物。草本植物或低矮的木本植物的株高应不大于有机玻璃箱的高度。
In the method for obtaining 13 C and 15 N double-labeled plants, the plants are herbaceous plants or low woody plants. The height of herbaceous plants or low woody plants should not be greater than the height of the plexiglass box.
所述的获得
13C和
15N双标记植物的方法,所述草本植物为水稻、小麦、玉米或大豆,所述低矮的木本植物为灌木或树苗。
In the method for obtaining 13 C and 15 N double-labeled plants, the herbaceous plants are rice, wheat, corn or soybeans, and the low woody plants are shrubs or saplings.
上述获得
13C和
15N双标记植物的方法制备得到的
13C和
15N双标记植物。
13 C and 15 N double-labeled plants prepared by the above method for obtaining 13 C and 15 N double-labeled plants.
上述
13C和
15N双标记植物在制备生物质炭中的应用。
Application of the above-mentioned 13 C and 15 N double-labeled plants in the preparation of biomass charcoal.
所述的
13C和
15N双标记植物在制备生物质炭中的应用,将双标记植物与空气隔绝后放入马弗炉中高温热解得到生物质炭;所述马弗炉的升温速率为4~20℃/min,达到目标温度250~700℃后持续3~6h。
The application of the 13 C and 15 N double-labeled plants in the preparation of biomass charcoal, the double-labeled plants are isolated from air and put into a muffle furnace for high-temperature pyrolysis to obtain biomass charcoal; the heating rate of the muffle furnace It is 4~20℃/min, and lasts for 3~6h after reaching the target temperature of 250~700℃.
与现有的技术相比,本发明的有益效果包括:Compared with the existing technology, the beneficial effects of the present invention include:
(1)本发明快速获得
13C和
15N双标记植物的方法,具有时效性强的特点;不需要连续标记,1天内能够完成对植物的标记。
(1) The method of the present invention for quickly obtaining 13 C and 15 N double-labeled plants has the characteristics of strong timeliness; continuous labeling is not required, and the labeling of plants can be completed within one day.
(2)本发明双标记方法所需装置简单、易于操作;不需要贮存
13CO
2气体的钢瓶、不需要额外的动力系统(如电泵)、不需要复杂装置的安装。
(2) The device required for the dual marking method of the present invention is simple and easy to operate; it does not require a steel cylinder for storing 13 CO 2 gas, does not require an additional power system (such as an electric pump), and does not require the installation of complex devices.
(3)本发明快速获得
13C和
15N双标记植物的方法,具有适用性广的特点;不局限于盆栽实验,也可以应用于野外原位实验;可应用于多种植物,可以为水稻、小麦、玉米、大豆等草本植物,也可以为低矮的灌木或树苗;不受植物生长阶段的限制。
(3) The method for quickly obtaining 13 C and 15 N double-labeled plants of the present invention has the characteristics of wide applicability; it is not limited to pot experiments, but can also be applied to in-situ experiments in the field; it can be applied to a variety of plants, including rice Herbs such as wheat, corn, soybeans, etc., can also be low shrubs or saplings; they are not limited by the growth stage of the plant.
为使本发明的上述目的、特征和优点能够更加明显易懂,下面结合具体实施例对本发明的具体实施方式做详细的说明。In order to make the above objectives, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention will be described in detail below in conjunction with specific embodiments.
实施例1Example 1
使用的植物材料为水稻。2019年5月份在江苏省镇江市南京林业大学下蜀林场(119°13′28″E,32°7′32″N)开展了水稻盆栽实验。当地气候属于亚热带季风气候,年降水量为1055.6mm,年平均气温为15.2℃,年平均日照时数为2157h。The plant material used is rice. In May 2019, the rice pot experiment was carried out at Xiashu Forest Farm (119°13′28″E, 32°7′32″N) of Nanjing Forestry University, Zhenjiang City, Jiangsu Province. The local climate belongs to the subtropical monsoon climate, the annual precipitation is 1055.6mm, the annual average temperature is 15.2℃, and the annual average sunshine hours is 2157h.
1、实验材料1. Experimental materials
水稻
13C标记材料:Ba
13CO
3(
13C atom 98%,上海化工研究院)、Ba
12CO
3、盐酸、透明有机玻璃箱、气体发生装置、704硅橡胶、小型蓄电池风扇、冰盒、温度计与湿度计、黑布;
Rice 13 C marking materials: Ba 13 CO 3 ( 13 C atom 98%, Shanghai Research Institute of Chemical Industry), Ba 12 CO 3 , hydrochloric acid, transparent plexiglass box, gas generator, 704 silicone rubber, small battery fan, ice box, Thermometer and hygrometer, black cloth;
(1)透明有机玻璃箱:进行
13C标记时,将盆栽的水稻放入有机玻璃箱(待标记植物的株高应不大于有机玻璃箱的高度),保证标记过程在密封条件下进行。箱体由厚度约0.50cm的有机玻璃制成,长×宽×高为106cm×71cm×100cm,箱底由111cm×76cm的PVC面板制成,面板四周设有高度约为8cm、宽4cm的水槽,有机玻璃箱下缘放置在水槽中,通过向水槽中加水保证箱体密封;有机玻璃箱上方均匀设计四个直径约2.30cm的孔洞,用于安装分液漏斗;箱体两侧各设计1个提手,方便透明有机玻璃箱的移动;
(1) Transparent plexiglass box: when performing 13 C marking, put potted rice in the plexiglass box (the height of the plant to be marked should not be greater than the height of the plexiglass box) to ensure that the marking process is carried out in a sealed condition. The box body is made of plexiglass with a thickness of about 0.50cm, length×width×height is 106cm×71cm×100cm, and the bottom of the box is made of a 111cm×76cm PVC panel. There is a sink with a height of about 8cm and a width of 4cm around the panel. The bottom edge of the plexiglass box is placed in the sink, and the box is sealed by adding water to the sink; four holes with a diameter of about 2.30cm are evenly designed on the top of the plexiglass box to install the separatory funnel; one on each side of the box Handle to facilitate the movement of the transparent plexiglass box;
(2)气体发生装置:由250mL的分液漏斗和250mL的烧杯以及外径6mm的硅橡胶管组成;将Ba
13CO
3或Ba
12CO
3粉末加入烧杯中,将盐酸加入分液漏斗中,打开分液漏斗后盐酸通过硅橡胶管进入烧杯,与BaCO
3粉末反应生成CO
2气体;气体发生装置有4个,2个产 生
13CO
2,2个产生
12CO
2;
(2) Gas generating device: consists of a 250mL separatory funnel, a 250mL beaker and a silicone rubber tube with an outer diameter of 6mm; add Ba 13 CO 3 or Ba 12 CO 3 powder into the beaker, and add hydrochloric acid to the separatory funnel, After opening the separatory funnel, the hydrochloric acid enters the beaker through the silicone rubber tube and reacts with the BaCO 3 powder to generate CO 2 gas; there are 4 gas generators, 2 generating 13 CO 2 , and 2 generating 12 CO 2 ;
(3)704硅橡胶:涂抹于有机玻璃箱孔洞和分液漏斗接缝处,保证气密性;(3) 704 silicone rubber: apply to the plexiglass box holes and the joints of the separatory funnel to ensure air tightness;
(4)小型蓄电池风扇与冰盒:小型蓄电池风扇悬挂在透明有机玻璃箱的上底面,用于混匀产生的CO
2气体;有机玻璃箱中温度过高时,用冰盒为其降温;
(4) Small battery fan and ice box: The small battery fan is suspended on the upper and bottom surface of the transparent plexiglass box to mix the CO 2 gas generated; when the temperature in the plexiglass box is too high, use an ice box to cool it down;
(5)温度计与湿度计:用于实时监测有机玻璃箱内的温度和湿度;(5) Thermometer and hygrometer: used to monitor the temperature and humidity in the plexiglass box in real time;
(6)黑布:在气体发生装置产生CO
2前遮盖有机玻璃箱以促进水稻对所产生CO
2的吸收,尺寸大小以刚好遮住整个有机玻璃箱体为宜。
(6) Black cloth: Cover the plexiglass box before the CO 2 is produced by the gas generator to promote the absorption of the CO 2 produced by the rice. The size should just cover the entire plexiglass box.
水稻
15N标记的材料:
Rice 15 N-labeled materials:
(1)
15N-尿素(
15N atom 10%,上海化工研究院):溶液浓度为0.2%(w/v),即2g/L;
(1) 15 N-urea ( 15 N atom 10%, Shanghai Research Institute of Chemical Industry): the solution concentration is 0.2% (w/v), which is 2g/L;
(2)雾化瓶(安利公司):标有刻度,容量为100mL,用于叶面喷施;(2) Atomization bottle (Amway Company): marked with a scale, with a capacity of 100mL, used for foliar spray;
(3)表面活性剂:曲拉通TritonX-100,浓度为0.05%(v/v),表面活性剂能够减小溶液表面张力,抑制毛细吸附,使肥料的溶解和重结晶进程减慢,增加细胞通透性,促进
15N-尿素溶液更好的吸收。
(3) Surfactant: Triton X-100, with a concentration of 0.05% (v/v). Surfactant can reduce the surface tension of the solution, inhibit capillary adsorption, and slow down the dissolution and recrystallization process of fertilizers. Cell permeability promotes better absorption of 15 N-urea solution.
2、植物
13C和
15N双标记
2. Plant 13 C and 15 N double labeling
选择水稻生殖生长初期,2019年9月27日进行水稻的同位素标记。先进行
15N的标记,接着进行
13C的标记,采用这样的标记顺序有以下优点:(1)若先进行植物
13C标记,则不能及时对植物进行杀青,植物同化的一部分
13C能够通过呼吸作用被利用,从而造成植物
13C丰度的降低;(2)若先进行植物
13C标记,植物静置时间延长,植物
13C会发生向下运输,并在植物的根部富集并有可能以根系分泌物的形式排出,这样不利于提高
13C回收率;(3)
15N标记完成后仍然需要一段时间使植物吸收同化
15N,恰好在这段时间内进行
13C标记可以节省标记时间,而且会避免上面(1)和(2)所提到的弊端。具体操作过程如下:
The early stage of reproductive growth of rice was selected, and the isotope labeling of rice was carried out on September 27, 2019. First carry out the 15 N labeling, and then carry out the 13 C labeling. Using this labeling sequence has the following advantages: (1) If the plant 13 C labeling is carried out first, the plant cannot be killed in time, and a part of the 13 C assimilation of the plant can pass Respiration is used to reduce the 13 C abundance of plants; (2) If the plant 13 C labeling is carried out first, the plant’s standing time will be prolonged, and the plant 13 C will be transported downwards and accumulated in the roots of the plant. It may be excreted in the form of root exudates, which is not conducive to improving the 13 C recovery rate; (3) After the 15 N labeling is completed, it still takes a while for the plant to absorb and assimilate 15 N. It happens that the 13 C labeling during this period can save labeling. Time, and will avoid the drawbacks mentioned in (1) and (2) above. The specific operation process is as follows:
(1)标记前去除盆栽水稻的衰老枯黄的叶片;(1) Remove the senescent and yellow leaves of potted rice before marking;
(2)使用雾化瓶进行
15N溶液的喷施,将喷头调节至雾形(spray),调节好喷头喷雾量,确保每棵水稻的叶片都能均匀喷洒,且避免喷洒过量形成叶片径流而造成
15N-尿素的浪费;
(2) Use an atomizing bottle to spray the 15 N solution, adjust the nozzle to spray, adjust the spray amount of the nozzle to ensure that the leaves of each rice plant can be sprayed evenly, and avoid excessive spraying to form leaf runoff. Cause a waste of 15 N-urea;
(3)喷施完成后,期间不要浇水,将3盆水稻放入透明有机玻璃箱中,进行
13C标记;标记前将2份Ba
13CO
3粉末和2份Ba
12CO
3粉末分别放入气体发生装置的4个烧杯,每份Ba
13CO
3或Ba
12CO
3质量均为2.65g;
(3) After spraying, do not water during the period. Put 3 pots of rice in a transparent plexiglass box and mark with 13 C; before marking, put 2 parts of Ba 13 CO 3 powder and 2 parts of Ba 12 CO 3 powder separately Into the 4 beakers of the gas generating device, the mass of each portion of Ba 13 CO 3 or Ba 12 CO 3 is 2.65g;
(4)将有机玻璃箱放在底座槽内,向底座槽内注水密封,启动风扇;如果箱内温度过高,可以提前在箱内放置冰盒降温;(4) Put the plexiglass box in the base trough, pour water into the base trough to seal, and start the fan; if the temperature in the box is too high, you can put an ice box in the box to cool down in advance;
(5)标记时先用黑布遮住整个有机玻璃箱,向气体发生装置的分液漏斗中注入200mL 2mol/L HCl溶液,与第1个烧杯中的Ba
13CO
3反应产生
13CO
2气体,5min后,将黑布打开, 水稻在有机玻璃箱内进行光合作用,光合45min后完成第1次脉冲标记;每次标记开始前用黑布将装有水稻的有机玻璃箱体罩住,是为了进行饥饿处理,促使水稻吸收更多CO
2气体;
(5) When marking, first cover the entire plexiglass box with a black cloth, and pour 200mL 2mol/L HCl solution into the separatory funnel of the gas generator, and react with the Ba 13 CO 3 in the first beaker to produce 13 CO 2 gas After 5 minutes, the black cloth was opened, the rice was photosynthesized in the plexiglass box, and the first pulse marking was completed after 45 minutes of photosynthesis; the plexiglass box containing the rice was covered with a black cloth before each marking, yes In order to deal with starvation, encourage rice to absorb more CO 2 gas;
(6)重复步骤(5),向第2个盛放Ba
13CO
3的烧杯添加HCl溶液,完成第2次
13C的脉冲标记;
(6) Repeat step (5), add HCl solution to the second beaker containing Ba 13 CO 3 to complete the second 13 C pulse mark;
(7)为了提高水稻对
13CO
2的吸收,重复步骤(5)2次,分别向盛放Ba
12CO
3的2个烧杯添加HCl溶液,产生
12CO
2供水稻吸收利用;
(7) In order to improve the absorption of 13 CO 2 by rice, repeat step (5) twice, and add HCl solution to the two beakers containing Ba 12 CO 3 to produce 12 CO 2 for rice absorption and utilization;
(8)标记结束后,取下有机玻璃箱,通风,收获植物。(8) After the marking is over, remove the plexiglass box, ventilate, and harvest the plants.
3、同位素丰度测定3. Determination of isotope abundance
将收获的水稻洗净后,放入105℃烘箱杀青30min,接着放入70℃烘箱中烘至恒重。烘干后取出水稻,冷却,密封保存。取水稻各器官的等量样品,充分磨碎使其颗粒度达0.149mm,混匀,使用稳定性同位素比例质谱仪(DELTA V Advantage,Thermofisher公司,美国)对水稻样品的
13C和
15N丰度进行测定。
After washing the harvested rice, put it in an oven at 105°C for 30 minutes, and then put it in an oven at 70°C to bake to constant weight. After drying, the rice is taken out, cooled, and sealed for storage. Take an equal amount of samples from each organ of rice, fully grind them to a particle size of 0.149 mm, mix them, and use a stable isotope ratio mass spectrometer (DELTA V Advantage, Thermofisher, USA) to determine the 13 C and 15 N abundance of the rice samples. Degree is measured.
4、结果分析4. Result analysis
本实施例中所获得双标记水稻的丰度见表1。水稻地上部分和地下部分
13C的丰度差距较大,说明较短的时间内植物光合作用吸收的
13C向根部运输有限。与
13C丰度相比,水稻地上和地下
15N丰度的差异较小。综上所述,采用本发明方法成功获得
13C和
15N双标记水稻。
The abundance of the double-labeled rice obtained in this example is shown in Table 1. There is a large difference in the abundance of 13 C between the aboveground and underground parts of rice , which indicates that the 13 C absorbed by photosynthesis of plants is limited in transportation to the roots in a relatively short period of time. Compared with 13 C abundance, the difference in 15 N abundance above ground and underground is smaller. In summary, the method of the present invention has successfully obtained 13 C and 15 N double-labeled rice.
表1水稻地上部分和地下部分
13C和
15N丰度
Table 1 13 C and 15 N abundances in the aboveground and underground parts of rice
实施例2Example 2
2019年10月25日,在南京林业大学下蜀林场,利用实施例1所获得的
13C和
15N双标记水稻制备生物质炭,并测定生物质炭的
13C和
15N丰度。
On October 25, 2019, in the Xiashu Forest Farm of Nanjing Forestry University, the 13 C and 15 N double-labeled rice obtained in Example 1 was used to prepare biomass char, and the abundance of 13 C and 15 N of the biomass char was measured.
1、生物质炭的制备过程1. The preparation process of biomass charcoal
(1)将烘干后的
13C和
15N双标记的水稻植株剪成小段,并用粉碎机粉碎;
(1) Cut the dried 13 C and 15 N double-labeled rice plants into small sections and crush them with a pulverizer;
(2)将混匀的双标记水稻植株放入坩埚,尽量将坩埚填满压实,保证内部无空隙;(2) Put the mixed double-labeled rice plants into the crucible, fill the crucible as much as possible, and ensure that there are no voids inside;
(3)用铝箔将坩埚密封,确保铝箔表面无裂缝或破洞,以保证坩埚内的植物隔绝空气;(3) Seal the crucible with aluminum foil to ensure that there are no cracks or holes on the surface of the aluminum foil to ensure that the plants in the crucible are isolated from air;
(4)将密封好后的坩埚放入Carbolite马弗炉(GPC12/131),目标温度设置为300℃和500℃,升温程序设置为10℃/min,达到目标温度后持续4h;(4) Put the sealed crucible into the Carbolite muffle furnace (GPC12/131), set the target temperature to 300°C and 500°C, set the heating program to 10°C/min, and continue for 4 hours after reaching the target temperature;
(5)关闭马弗炉,冷却至室温,取出坩埚,收集所制备的生物质炭。(5) Turn off the muffle furnace, cool to room temperature, take out the crucible, and collect the prepared biomass charcoal.
2、生物质炭
13C和
15N丰度的测定
2. Determination of 13 C and 15 N abundance of biomass charcoal
制备成的生物质炭
13C和
15N丰度见表2。与植物原材料相比,水稻制备成的生物质炭
13C丰度下降,尤其是水稻地下部位,300℃和500℃下分别降低69.2%和71.3%。
15N丰度也有所下降,但下降程度低于
13C丰度。500℃下,与植物原材料相比,水稻地上部分制备成的生物质炭
15N丰度几乎没有变化,而地下部分制备成的生物质炭
15N丰度下降了40.5%。
The 13 C and 15 N abundances of the prepared biomass charcoal are shown in Table 2. Compared with plant raw materials, the 13 C abundance of biomass charcoal prepared from rice decreased, especially in the underground parts of rice, which decreased by 69.2% and 71.3% at 300°C and 500°C, respectively. The abundance of 15 N also decreased, but the degree of decrease was lower than that of 13 C. At 500°C, compared with plant raw materials, the 15 N abundance of biomass charcoal prepared from the aboveground part of rice hardly changed, while the 15 N abundance of biomass charcoal prepared from the underground part decreased by 40.5%.
表2水稻地上和地下部分制备成的生物质炭的
13C和
15N丰度
Table 2 13 C and 15 N abundances of biomass char prepared from aboveground and underground parts of rice
Claims (13)
- 一种获得 13C和 15N双标记植物的方法,其特征在于,采用 13C脉冲标记和 15N叶面喷施标记的方法对植物进行双标记,获得 13C和 15N双标记植物。 A method for obtaining 13 C and 15 N double-labeled plants is characterized in that the 13 C pulse labeling and 15 N foliar spraying labeling methods are used to double-label the plants to obtain 13 C and 15 N double-labeled plants.
- 根据权利要求1所述的获得 13C和 15N双标记植物的方法,其特征在于,在标记过程中先进行 15N的标记,再进行 13C的标记。 The method for obtaining 13 C and 15 N double-labeled plants according to claim 1, characterized in that, during the labeling process, 15 N labeling is performed first, and then 13 C labeling is performed.
- 根据权利要求2所述的获得 13C和 15N双标记植物的方法,其特征在于,具体包括以下步骤: The method for obtaining 13 C and 15 N double-labeled plants according to claim 2, characterized in that it specifically comprises the following steps:(1)去除待标记植物衰老枯黄的叶片,采用雾化瓶喷施 15N标记的氮素肥料溶液; (1) Remove the senescent and yellow leaves of the plants to be marked, and spray a 15 N-labeled nitrogen fertilizer solution with an atomized bottle;(2)将植物置于有机玻璃箱,在密闭条件下,首先通过盐酸和 13C-碳酸盐反应产生 13CO 2进行脉冲标记,然后通过盐酸和 12C-碳酸盐反应产生 12CO 2,每份 13C-碳酸盐或 12C-碳酸盐所产生CO 2的体积浓度为300~800ppm; (2) Put the plant in a plexiglass box, under airtight conditions, firstly generate 13 CO 2 by the reaction of hydrochloric acid and 13 C-carbonate for pulse labeling, and then generate 12 CO 2 by the reaction of hydrochloric acid and 12 C-carbonate , The volume concentration of CO 2 produced by each 13 C-carbonate or 12 C-carbonate is 300~800ppm;(3)标记结束后,取下有机玻璃箱,通风,完成标记过程。(3) After marking, remove the plexiglass box, ventilate, and complete the marking process.
- 根据权利要求3所述的获得 13C和 15N双标记植物的方法,其特征在于,所述 15N标记的氮素肥料溶液为 15N-尿素溶液,浓度为1~3g/L。 The method for obtaining 13 C and 15 N dual-labeled plants according to claim 3 , wherein the 15 N-labeled nitrogen fertilizer solution is a 15 N-urea solution with a concentration of 1 to 3 g/L.
- 根据权利要求3或4所述的获得 13C和 15N双标记植物的方法,其特征在于,所述 15N标记的氮素肥料溶液中加入表面活性剂。 The method for obtaining 13 C and 15 N dual-labeled plants according to claim 3 or 4, wherein a surfactant is added to the 15 N-labeled nitrogen fertilizer solution.
- 根据权利要求5所述的获得 13C和 15N双标记植物的方法,其特征在于,所述表面活性剂为曲拉通TritonX-100,加入体积为 15N标记的氮素肥料溶液体积的0.02%~0.08%。 The method for obtaining 13 C and 15 N dual-labeled plants according to claim 5, wherein the surfactant is Triton X-100, and the added volume is 0.02 of the volume of the 15 N-labeled nitrogen fertilizer solution. %~0.08%.
- 根据权利要求3所述的获得 13C和 15N双标记植物的方法,其特征在于,所述 13C-碳酸盐为Ba 13CO 3,所述 12C-碳酸盐为Ba 12CO 3。 The method for obtaining 13 C and 15 N dual-labeled plants according to claim 3 , wherein the 13 C-carbonate is Ba 13 CO 3 and the 12 C-carbonate is Ba 12 CO 3 .
- 根据权利要求3或7所述的获得 13C和 15N双标记植物的方法,其特征在于,所述 13C-碳酸盐和 12C-碳酸盐的份数分别为2份。 The method for obtaining 13 C and 15 N dual-labeled plants according to claim 3 or 7, characterized in that the parts of 13 C-carbonate and 12 C-carbonate are 2 parts respectively.
- 根据权利要求1所述的获得 13C和 15N双标记植物的方法,其特征在于,所述植物为草本植物或低矮的木本植物。 The method for obtaining 13 C and 15 N double-labeled plants according to claim 1, wherein the plants are herbaceous plants or low woody plants.
- 根据权利要求9所述的获得 13C和 15N双标记植物的方法,其特征在于,所述草本植物为水稻、小麦、玉米或大豆;所述低矮的木本植物为灌木或树苗。 The method for obtaining 13 C and 15 N double-labeled plants according to claim 9, wherein the herbaceous plant is rice, wheat, corn or soybean; and the low woody plant is a shrub or sapling.
- 权利要求1所述的获得 13C和 15N双标记植物的方法制备得到的 13C和 15N双标记植物。 Preparation method for obtaining 13 C and 15 N double-labeled plant obtained 13 C and 15 N double-labeled plant according to claim 1.
- 权利要求11所述的 13C和 15N双标记植物在制备生物质炭中的应用。 The application of the 13 C and 15 N dual-labeled plants as claimed in claim 11 in the preparation of biomass charcoal.
- 根据权利要求12所述的 13C和 15N双标记植物在制备生物质炭中的应用,其特征在于,将双标记植物隔绝空气后放入马弗炉中高温热解得到生物质炭;所述马弗炉的升温速率为4~20℃/min,达到目标温度250~700℃后持续3~6h。 The application of 13 C and 15 N dual-labeled plants in the preparation of biomass charcoal according to claim 12, characterized in that the dual-labeled plants are isolated from air and placed in a muffle furnace for high-temperature pyrolysis to obtain biomass char; The heating rate of the muffle furnace is 4-20°C/min, and it lasts for 3-6 hours after reaching the target temperature of 250-700°C.
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