CN113201330A

CN113201330A - Magnesium-nitrogen doped carbon dot, preparation method thereof and application of magnesium-nitrogen doped carbon dot in improvement of plant photosynthesis

Info

Publication number: CN113201330A
Application number: CN202110435563.3A
Authority: CN
Inventors: 刘应亮; 李亚东; 胡超凡; 庄健乐; 雷炳富; 张学杰; 许晓凯; 郑明涛; 李唯; 张浩然
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2021-04-22
Filing date: 2021-04-22
Publication date: 2021-08-03
Anticipated expiration: 2041-04-22
Also published as: CN113201330B

Abstract

The invention belongs to the field of research on biological effects of nano materials, and discloses a magnesium-nitrogen doped carbon dot, a preparation method thereof and application thereof in improving plant photosynthesis. The preparation method comprises the following steps: dissolving citric acid, ethanolamine and magnesium hydroxide in ultrapure water, performing ultrasonic treatment, pouring into a reaction kettle, and reacting at 200 ℃ for 6 hours; cooling to room temperature after the reaction is finished, filtering the product by using a filter head with the pore diameter of 0.22 mu m, and then dialyzing for 12 hours; and (5) freeze-drying to obtain the magnesium-nitrogen doped carbon dots. After being compounded with chloroplasts, the excitation energy of the composite material can be absorbed by the chloroplasts for photosynthesis, so that the photosynthetic activity is improved. After the foliar fertilizer is applied to rice plants by foliar spraying, Mg and N-CDs can be uniformly distributed in leaf cells, expression of related enzyme genes is regulated and controlled, synthesis and metabolism of chlorophyll in leaves are promoted, and the activity of chlorophyll molecules is kept at a higher level, so that the photosynthetic activity of the leaves is improved. Eventually, plant growth is significantly improved.

Description

Magnesium-nitrogen doped carbon dot, preparation method thereof and application of magnesium-nitrogen doped carbon dot in improvement of plant photosynthesis

Technical Field

The invention belongs to the field of research on biological effects of nano materials, and particularly relates to a magnesium-nitrogen doped carbon dot, a preparation method thereof and application thereof in improving plant photosynthesis.

Background

Photosynthesis is an important physiological process in the growth process of plants and has important significance on grain yield. Increasing crop yields by regulating photosynthesis has been a hot topic of interest to many researchers. At present, the photosynthesis efficiency of plants is far lower than the theoretical value, and the main limiting factors comprise: (1) the limited light harvesting capability of the plant itself and (2) the limited activity of the photosynthetic system. As light traps, photosynthetic pigments are involved in almost all physiological processes of plant photosynthesis, including: light capture, energy transfer, energy conversion, and the like. Magnesium and nitrogen are important components of plant photosynthetic pigments, photosynthetic proteins, and phototransducers. The contents of chlorophyll, magnesium and nitrogen in plant leaves have been widely used as main reference indicators for evaluating plant photosynthesis.

The rapid development of nanomaterials provides better solutions to many social problems, including agricultural and environmental fields. Carbon Dots (CDs) are fluorescent carbon nano materials, have the particle size of less than 10nm, and have the advantages of excellent optical performance, good water solubility, low toxicity, environmental friendliness, biocompatibility, wide raw material source, low preparation cost and the like. CDs are electron donors and electron acceptors, and can be used as good energy transfer intermediates. In the photosynthesis of plants, researchers find that when the fluorescence emission spectrum of CDs is coincident with the light absorption spectrum of chloroplasts, the excitation energy of CDs can be captured by chloroplasts, so that the transmission rate of photosynthetic electrons on a thylakoid membrane is increased, and the photosynthesis and growth rate of plants are promoted. In addition, CDs are very easy to modify and can be functionalized by specific substances.

Disclosure of Invention

In order to overcome the defects and shortcomings in the prior art, the invention mainly aims to provide a preparation method of magnesium nitrogen doped carbon dots (Mg, N-CDs).

The invention also aims to provide the magnesium-nitrogen doped carbon dot prepared by the preparation method and a preparation method thereof.

The invention further aims to provide application of the magnesium nitrogen-doped carbon dots. The invention can not only play the role of energy transfer in photosynthesis, but also promote the photosynthetic efficiency and growth and development of the plant by improving the activity of the plant photosynthetic system by doping the elements of magnesium and nitrogen required by the plant photosynthetic system into CDs.

The purpose of the invention is realized by the following technical scheme:

a preparation method of a magnesium nitrogen doped carbon dot comprises the following operation steps: dissolving citric acid, ethanolamine and magnesium hydroxide in ultrapure water, pouring mixed liquor obtained after ultrasonic treatment into a reaction kettle, and reacting for 6 hours at 200 ℃; cooling to room temperature after the reaction is finished, filtering the product by using a filter head with the aperture of 0.22 mu m, and dialyzing for 12 hours by using a dialysis bag; and (4) after freeze drying, obtaining magnesium nitrogen doped carbon dots (Mg, N-CDs).

Preferably, the molar ratio of the citric acid to the ethanolamine to the magnesium hydroxide is 1:0.2: 0.35.

Preferably, the time of the ultrasonic treatment is 30 min.

Preferably, the cut-off molecular weight MW of the dialysis bag is 100-500; the dialysis adopts ultrapure water as dialysate.

The magnesium-nitrogen doped carbon dot prepared by the preparation method.

The application of the magnesium-nitrogen doped carbon dots in improving the photosynthesis of plants.

The mechanism of the Mg, N-CDs for improving the photosynthesis of the plants is as follows: (1) the metabolic rate of chlorophyll is accelerated, and the activity is improved; (2) improving the light absorption capacity of chloroplasts (higher and wider); (3) photosynthetic electron transfer rate; (4) CO 2₂And (4) assimilating. Chlorophyll molecules participate in a number of important physiological processes in photosynthesis, including: light capture, energy transfer, and conversion of light energy. There are two key intermediate processes in the biosynthesis of chlorophyll: (1) magnesium chelatases (also known as magnesium-protoporphyrin IX chelatases) catalyze the binding of magnesium ions to protoporphyrin IX; (2) the chlorophyll synthetase catalyzes chlorophyllin ester to react with geranyl pyrophosphate to generate chlorophyll a, and then the chlorophyll a is converted into chlorophyll b. Chlorophyllase is responsible for the catalytic degradation of chlorophyll. In the research of the invention, the expression quantities of genes of chlorophyll synthetase Ch1G, two subunits of magnesium chelatase Ch1I and CH1D and chlorophyllase-2 can be obviously improved by Mg and N-CDs, so that the synthesis and metabolism of chlorophyll molecules in rice leaves can be accelerated. Therefore, chlorophyll can exert higher activity in photosynthesis, improve the capture capacity of light energy and promote the conversion of energy. The content ratio of chlorophyll a to chlorophyll b is reduced along with the increase of the treatment concentration of Mg and N-CDs, which shows that the blue light absorption capacity of rice leaves is gradually enhanced, so that the blue fluorescence of Mg and N-CDs can be reabsorbed by chloroplasts for photosynthesis, the transmission rate of photosynthetic electrons is further accelerated, and the photosynthetic activity is improved. The Calvin cycle in photosynthesis can utilize ATP and NADPH generated in photoreaction to convert CO₂Is converted into a carbohydrate. RuBisCO is a key enzyme in the RuBisCO, and directly determines CO₂The rate of assimilation of (a). Mg, N-CDs improve the enzyme activity of RuBisCO in rice, and further can accelerate CO₂Assimilation of (1). Under the simultaneous action of all these functions, the photosynthesis rate of the plant is accelerated, and the growth of the plant is promoted. However, it was found in the experiments that the carbohydrate content in young rice plants was suppressed by the Mg, N-CDs treatment and decreased with increasing treatment concentration. Therefore, we conclude that Mg, N-CDs may still be presentThe subsequent conversion process of carbohydrates into other substances in plants can be regulated, which needs further research and demonstration in the future.

Compared with the prior art, the invention has the following advantages and beneficial effects:

in the magnesium nitrogen doped carbon dots, Mg and N respectively exist in Mg, N-CDs in structures similar to magnesium carbonate and graphite nitrogen. The doping of Mg changes the transition path of excited electrons of Mg and N-CDs, so that the fluorescence emission of the excited electrons is red-shifted. After being compounded with chloroplasts, the excitation energy of Mg and N-CDs can be absorbed by the chloroplasts for photosynthesis, so that the photosynthetic activity is improved. After the fertilizer is applied to rice plants by foliar spraying, Mg and N-CDs can be uniformly distributed in leaf cells. At 300. mu.g.mL^-1Under the treatment concentration, Mg and N-CDs can regulate and control the expression of related enzyme genes, promote the synthesis and metabolism of chlorophyll in the leaves, and keep the activity of chlorophyll molecules at a higher level, thereby improving the photosynthetic activity of the leaves. Meanwhile, Mg, N-CDs can also up-regulate the activity of RuBisCO enzyme and promote CO₂Assimilation of (1). Finally, the growth of the rice plants treated by Mg, N-CDs is accelerated.

Drawings

FIG. 1 is a photograph (A) of an aqueous Mg, N-CDs solution in sunlight and under 365nm light irradiation; UV-Vis light absorption spectrum, optimal fluorescence excitation spectrum and emission spectrum (B) of Mg, N-CDs; a 3D plot of the fluorescence emission of Mg, N-CDs (C); fluorescence lifetime decay curves (D) for Mg, N-CDs.

FIG. 2 is a TEM image of Mg, N-CDs with inset particle size distribution (A), high resolution TEM image (B and C), XRD profile (D), AFM profile and corresponding particle height profile (E and F) and Zeta potential result (G).

FIG. 3 is a high resolution spectrum of FTIR spectrum (A), XPS total spectrum (B), C1s (C), O1s (D), N1s (E), Mg1s (F), Mg2s (G) and Mg2p (H) of Mg, N-CDs.

FIG. 4 is a thermogravimetric curve (TG) (A), a thermogravimetric integration curve (DTG) (B) and a differential scanning calorimetry curve (DSC) (C) of Mg, N-CDs; (ii) mass spectrometry (D) of the Mg, N-CDs thermo gravimetric analysis of the overflow gases; XRD patterns (E) of Mg, N-CDs under different temperature conditions.

FIG. 5 shows control group and 50, 100 and 300. mu.g.mL^-1Of rice seedlings in the Mg, N-CDs treatment groupPhotograph (a) and the corresponding plant height (B), fresh weight (C), chlorophyll content (D) and carbohydrate content (E). The length of the white bar in FIG. A is 5 cm. The error bars in B-E are standard deviations (n.gtoreq.5). With different letter designations representing differences (p) between processes<0.05)。

FIG. 6 is control and 50, 100 and 300. mu.g.mL^-1The Mg, N-CDs-treated rice leaves of (A and B) the respective expression levels of the magnesium chelatase subunits ChlI and ChlD, (C) chlorophyll synthase and (D) chlorophyllase genes. Data are presented as mean. + -. standard deviation (n.gtoreq.5) and different letters are used to indicate differences (p) between treatments<0.05)。

FIG. 7 is a TEM image of rice leaves of the control group (A) and Mg, N-CDs-treated (B); control and rice leaf treated with different concentrations of Mg, N-CDs NPQ (C), qP (D), ETR (E), Y (NPQ) (F), Y (NO) (G), and Y (II) (H). Error bars represent standard deviations of the data (n.gtoreq.5, p < 0.05).

FIG. 8 shows (A) 300. mu.g.mL^-1The leaf of the rice treated with Mg, N-CDs was 461. mu. mol. m^-2·s^-1NPQ, qP, Y (NPQ), Y (NO), Y (II), and ETR. (B) Mg, N-CDs (50, 100 and 300. mu.g.mL)^-1) RuBisCO activity of treated rice. Data are presented as mean. + -. standard deviation (n.gtoreq.5) and different letters are used to indicate differences (p) between treatments<0.05)。

FIG. 9 shows control group (A) and 300. mu.g.mL^-1SEM picture of Mg, N-CDs complex chloroplast (B); fluorescence map of Mg, N-CDs complex chloroplasts: a superposition graph (F) of a bright field (C), 361-389nm excitation (D), 515-565nm excitation (E), D and E; fluorescence lifetime (G) of Mg, N-CDs and Mg, N-CD complex chloroplasts; chloroplast and Mg, N-CD complex chloroplast Hill reaction under 1000LUX (H) and 9000LUX (J) xenon lamp irradiation, and reduction activity (I) against cyanide under 3000LUX xenon lamp irradiation. Error bars represent the difference in labeling of data (n-3, p)<0.05)。

FIG. 10 is a diagram of the mechanism by which Mg, N-CDs regulate plant photosynthesis.

Detailed description of the invention

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.

Example 1

Preparation of magnesium nitrogen-doped carbon dots (Mg, N-CDs): preparing Mg, N-CDs by using citric acid, ethanolamine and magnesium hydroxide as raw materials by a one-step hydrothermal method, dissolving the citric acid, the ethanolamine and the magnesium hydroxide in a molar ratio of 1:0.2:0.35 in ultrapure water, and performing ultrasonic treatment for 30 min. Magnesium hydroxide, which is slightly soluble in water, can be completely dissolved due to the chelation of citric acid. Then pouring the mixed solution obtained by ultrasonic treatment into a lining of a 50mL polytetrafluoroethylene reaction kettle, putting the mixture into the reaction kettle, and reacting for 6h at 200 ℃. And (3) after the reaction kettle is cooled to room temperature, filtering the brown product by using a filter head with the aperture of 0.22 mu m, and dialyzing for 12 hours by using a dialysis bag (MW 100-500), wherein the dialysate is ultrapure water. After freeze drying, the magnesium nitrogen doped carbon dots (Mg, N-CDs) can be obtained.

To investigate the presence of Mg in Mg, N-CDs and the effect of Mg doping on their fluorescence emission, N-doped CDs alone (N-CDs) were prepared in the same manner as above using equal amounts of citric acid and ethanolamine. The relative QY of the CDs obtained was determined with quinine sulfate (dissolved in 0.1M sulfuric acid, QY 54%) as a reference (assay methods reference: RSC Advances,2017,7,70, 44144-.

Example 2

The magnesium nitrogen-doped carbon dots (Mg, N-CDs) obtained in example 1 were characterized:

the characterization method comprises the following steps: the morphology of Mg, N-CDs was observed by JEM-2100F field emission projection electron microscope (TEM) and SPA-400 Atomic Force Microscope (AFM). Characterization was performed using a Thermo Scientific Nicolet 6700FTIR spectrometer and Thermo Scientific K-Alpha X-ray photoelectron spectroscopy (XPS). The fluorescence emission spectrum and fluorescence lifetime decay curve of Mg, N-CDs were measured by F-7000Hitachi and FLS1000-stm fluorescence spectrometers, respectively. A Perkin Elmer UV-Vis Spectrophotometer (Lambda 750) was used to determine the UV-Vis light absorption curve of Mg, N-CDs. Meanwhile, the thermal decomposition curve and the product composition of Mg, N-CDs from room temperature to 800 ℃ are measured by using a TGA-DSC thermogravimetry (Labsys STA) and mass spectrum (LC-DZOOM Pro) (TG-MS) combined system. Finally, XRD profiles of Mg, N-CDs at different temperatures were recorded using an Xpert Pro MPD X-ray diffractometer (XRD) for analysis of the composition of the decomposition residues.

Optical characterization of Mg, N-CDs: under UV light (365nm, 16W), an aqueous solution of Mg, N-CDs fluoresces blue (A in FIG. 1). The UV/Vis line in B of FIG. 1 shows the UV-Vis light absorption spectrum of Mg, N-CDs, from which two absorption peaks are observed, located at 320nm and 370nm, respectively, corresponding to N- π electronic transitions between the Mg, N-CDs surface functional groups and the conjugated structures. The maximum fluorescence excitation spectrum and the emission spectrum of Mg, N-CDs are respectively positioned at 374nm and 485nm (FL excitation and FL emission lines in B of FIG. 1), and both can be subjected to peak-splitting fitting to form two small peaks respectively positioned at 343nm, 378nm (excitation peak) and 483nm and 519nm (emission peak). As can be seen from C in FIG. 1, the fluorescence emission peak position of Mg, N-CDs is red-shifted from 400nm to 550nm with the red-shift of the excitation light wavelength. The two fluorescence lifetimes of Mg, N-CDs were calculated from the fluorescence decay curve (D in FIG. 1) to be 2.41ns (96.50%) and 20.49ns (3.50%), respectively, with a mean fluorescence lifetime of 3.04 ns.

Morphology characterization of Mg, N-CDs: a in FIG. 2 is a TEM image of Mg, N-CDs, which are uniformly distributed nearly spherical particles having an average particle diameter of 2.53 nm. High resolution TEM images (B and C in fig. 2) show the lattice spacings of Mg, N-CDs of 0.21nm and 0.24nm, corresponding to the (100) and (1120) crystal planes of graphite, respectively. D in FIG. 2 is the XRD pattern of Mg, N-CDs with two broad diffraction peaks at 2 θ 18.8 ° and 34.5 °, demonstrating the presence of amorphous carbon in Mg, N-CDs. AFM of Mg, N-CDs illustrates that the particle height is about 2nm (E and F in FIG. 2), again illustrating that Mg, N-CDs are nearly spherical particles. By surface potential analysis, Mg, N-CDs were found to be negatively charged (-5.8. + -. 0.66mV) (G in FIG. 2) on their surface, facilitating their entry into organisms and biological cells in biological applications.

Chemical composition characterization of Mg, N-CDs: the chemical composition of Mg, N-CDs was analyzed by FTIR and XPS. A in FIG. 3 is an FTIR spectrum of Mg, N-CDs. At 3390cm^-1Peak at position corresponding to stretching vibration of hydroxyl group or N-H bond, 1585cm^-1The peak at (B) indicates that the surface of Mg, N-CDs has alkyl primary amide, 1417cm^-1The peak at (A) is due to in-plane bending vibration of hydroxyl group or stretching vibration of C-N in primary amide, 1278cm^-1And 1076cm^-1The peak is respectively attributed to C-O and C-N stretching vibrationAnd (4) moving characteristic peaks. The FTIR result shows that the surface of Mg, N-CDs has abundant hydrophilic functional groups, which is beneficial to the application of the Mg, N-CDs in the biological field.

And characterizing the element composition and the element chemical state of Mg, N-CDs by XPS. From B in FIG. 3, it can be seen that the XPS total spectrum of Mg, N-CDs has C1s (284.4eV), N1s (400.6eV), O1s (531.86eV), Mg1s (1304.25eV), Mg2s (89.52eV), Mg2p (50.79eV) and the helical peaks of Mg (MgKll, 306.2 eV). The high resolution plot of C1s shows the presence of three C elements: C-C at 284.8eV, C-O-C at 286.3eV, and O-C ═ O at 288.7eV (C of fig. 3). The high resolution of O1s indicates that the oxygen element in Mg, N-CDs exists as a metal carbonate (531.8eV) and a C ═ O bond (533.3eV) (D in fig. 3), indicating that Mg is doped into Mg, N-CDs and exists in a structure similar to magnesium carbonate. The results are consistent with those of FTIR. The characteristic peak of N1s was peak-only fit to one form of graphite nitrogen (fig. 3E). While the Mg1s and Mg2p peaks are attributed to the oxide of magnesium and Mg-CO, respectively₃Structure (F-H of fig. 3). In addition, the mgkl peak accompanying Mg1s indicates that a portion of Mg is encapsulated in the carbon core. From XPS data, it was calculated that the Mg and N contents in Mg, N-CDs were 6.39% and 3, 83%, respectively.

To further study the structure and composition of Mg, N-CDs, their thermal decomposition processes and products were tested using thermogravimetry coupled with mass spectrometry and high temperature XRD. FIGS. 4A-C show that as the temperature increases, Mg, N-CDs exhibit a total of three decomposition events, respectively: (1) endothermic reaction at 25-220 deg.C with mass loss of 27.92%; (2)220 ℃ and 500 ℃, the exothermic reaction is carried out, and the mass loss is 38.26 percent; (3)500 ℃ and 545 ℃ and has 24.75 percent of mass loss due to exothermic reaction. D in FIG. 4 shows that the mass loss of Mg, N-CDs at 25-220 ℃ and 220-500 ℃ is due to the evolution of NH by evaporation of water molecules and decomposition of amide groups₃、CO₂And NO₂And the result is that. The 500-545 ℃ thermal decomposition process is mainly the decomposition process of the carbon skeleton structure of Mg, N-CDs and doped graphite nitrogen to release NH₃、H₂O、CO₂、NO₂And a small amount of NO gas. E in FIG. 4 is the XRD curve of Mg, N-CDs under different temperature conditions. When Mg, N-CDs are completely decomposed at 800 ℃, the residue is MgO crystal, and the characteristic peak is opened at 550 DEG CInitially, this temperature corresponds to MgCO₃Again indicating that Mg is present in Mg, N-CDs in magnesium-like form. From the 8.96% residue in the thermogravimetric analysis, it was calculated that the Mg content in the Mg, N-CDs was about 5.58%, similar to the result of the XPS analysis.

According to the characterization results, the generation process of Mg and N-CDs and the influence of Mg doping on the fluorescence emission can be analyzed. During the reaction, ethanolamine and magnesium hydroxide are first combined with citric acid to form primary polymer via intermolecular dehydration condensation, and as the reaction proceeds and the reaction temperature is raised, the dehydration condensation reaction between these primary products continues to occur to produce larger polymer. When the carbonization is finished, Mg and N-CDs are obtained, and structures similar to magnesium carbonate and graphite nitrogen are formed in the surface and the inner part of the Mg and N-CDs. Suitable graphite nitrogen can increase the QY of CDs. The QY of N-CDs in this study was as high as 57.37%, while Mg, N-CDs were only 19.58%. The Mg-doped Mg and N-CDs have the advantages of enhanced light absorption capacity, reduced fluorescence lifetime and red shift of the maximum fluorescence emission peak. According to these results, the doping of Mg introduces a lower energy level by combining with the oxygen-containing structure, and lowers the original energy level, thereby changing the transition of electrons. When excited by light, most of the excited electrons are captured by the newly formed Mg level, and a small portion is captured by the relatively high N level, thereby emitting blue fluorescence with a longer wavelength. Due to the strong vibration relaxation of the Mg energy level, energy is dissipated in a thermal mode, and the QY value is reduced.

Example 3

Effect of magnesium nitrogen-doped carbon dots (Mg, N-CDs) obtained in example 1 on growth of Rice plants:

the method for treating the rice plants by the magnesium-nitrogen doped carbon dots comprises the following steps: firstly, disinfecting rice seeds (Hua navigation No. 31) by using a 75% ethanol solution, thoroughly washing the rice seeds by using deionized water, uniformly spreading the rice seeds in a germination disc, adding a 50% nutrient solution, and culturing the rice seeds at 30 ℃ in a dark place. After 3 days, the germination plates were uncovered and the seeds germinated essentially completely, placed in a greenhouse (22-30 ℃, 70% relative humidity, 400 μmol m)^-2·s^-1The light period is 16h light/8 h dark)Medium culture was continued until day 7. Transplanting the rice seedlings with uniform growth state into black square plastic boxes, wherein 12 plants are transplanted in each box, and the nutrient solution in each box is replaced 2 times per week. Mg, N-CDs at different concentrations (50, 100 and 300. mu.g.mL)^-1) Treating rice seedlings by a foliar spray mode, starting the first treatment on the next day after transplantation, spraying 5mL of the fertilizer in each box, and spraying the fertilizer every two days later for 16 days in total. Each concentration treatment was set to 3 replicates and rice seedlings sprayed with an equal amount of deionized water were used as control treatment. After the treatment is finished, collecting rice samples according to the requirements of subsequent determination indexes, and recording the plant height and fresh weight of rice seedlings to compare the growth difference between treatments with different concentrations. The chlorophyll content in rice leaves was determined by methods reported in the literature (Journal of Hazardous Materials,2020,394,122551). By using TEM, the distribution of Mg, N-CDs in rice leaves was observed after sampling by a standard method (RSC Advances,2017,7,62, 38853-.

Influence of the magnesium nitrogen-doped carbon point on the growth of rice plants: treatment by foliar spray of 50, 100 and 300. mu.g.mL^-1After 16 days of Mg, N-CDs, the growth parameters of the rice seedlings were recorded. As can be seen from A of FIG. 5, Mg, N-CDs significantly promoted the growth of rice seedlings compared to the control. 50. 100 and 300. mu.g.mL^-1The Mg and N-CDs respectively improve the plant height of the rice seedlings by 19.87 percent, 22.55 percent and 22.34 percent (B of figure 5) and the fresh weight by 33.90 percent, 55.25 percent and 70.60 percent (C of figure 5). In addition, Mg, N-CDs increased the content of chlorophyll a (11.52%, 12.06% and 14.39%) and chlorophyll b (15.57%, 20.31% and 26.54%) in rice leaves (fig. 5D). The ratio of chlorophyll a to b content decreased from 4.39 in the control group to 3.95 with increasing treatment concentration (fig. 5D). The content of carbohydrate in rice plants is measured to find that the content of carbohydrate in the rice plants is 50, 100 and 300 mu g/mL^-1The Mg, N-CDs reduced it significantly by 6.50%, 21.37% and 34.12% (E of fig. 5). In consideration of the important role of chlorophyll in plant photosynthesis, we measured the gene expression level of chlorophyll metabolism-related enzymes in rice seedlings.

Example 4

Effect of magnesium nitrogen-doped carbon dots (Mg, N-CDs) obtained in example 1 on photosynthesis of rice plants:

the expression quantity test method of chlorophyll synthesis and metabolism related genes comprises the following steps: the gene sequences of rice chlorophyll synthesis and metabolism related enzymes including two Mg chelatase subunits (ChlD: LOC4334537 and ChlI: LOC4333259), chlorophyll synthase (ChlG: LOC4338498) and chlorophyllase-2 (LOC4348648) were searched through the National Center for Biotechnology Information (NCBI) website, and the expression amount of the genes was determined using the real-time fluorescent quantitative PCR (qRT-PCR) technique. Primers were designed by Primer Premier 5 software (table 1). The treated rice leaves are immediately put into liquid nitrogen for preservation after being sampled. Extraction of RNA from the sample and reverse transcription of cDNA were performed using the Plant Total RNA Isolation Kit (BioBase, Chengdu, China) and TURescript 1st Stand cDNA SYNTHESIS Kit (Aidlab, Beijing, China) kits, respectively. The qRT-PCR test was performed using the QTOWER2.2 Real Time PCR system (ANALYTIKJENA, germany) with the reaction system: 5 μ L

Green Supermix, 0.5. mu.L forward primer, 1. mu.L reverse primer, 1. mu.L cDNA and 3. mu.L RNase-free water). The amount of gene expression is 2^-ΔΔCT method (Methods,2001,25,4, 402-.

TABLE 1 primer Table in qRT-PCR amplification

A chlorophyll fluorescence test method of rice plants comprises the following steps: the photosynthesis parameters of the treated rice seedlings were determined using a chlorophyll fluorescence apparatus (Phyto-PAM ED, Walz, Germany). All rice seedlings were left overnight in the dark before the test started to allow their photosynthetic response to reach equilibrium. During testing, the second leaf on the top of the rice seedling is selected, soaked absorbent cotton is used for wrapping the cut, and then the cut is placed on a test bench for testing. The measured photosynthetically active radiation intensities were 0, 36, 81, 146, 231, 396, 461, 611, 801, 1076 and 1251. mu. mol. m^-2·s^-1.6 rice leaves were tested per treatment,10 test points were selected for each leaf. The test parameters include: non-photochemical quenching (NPQ) (indicating photoprotective ability of leaves), photochemical fluorescence quenching (qP) (indicating photosynthetic activity), regulated non-photochemical energy loss in PS II [ Y (NPQ)]Unregulated non-photochemical energy loss [ Y (NO) ]]Photochemical energy conversion [ Y (II) ]](representing the actual photosynthetic efficiency of the leaves) and the Electron Transfer Rate (ETR).

The method for testing the enzyme activity of RuBisCO comprises the following steps: the collected rice samples were ground to a powder with liquid nitrogen. 0.1g of rice sample was weighed, added with 1ml of 0.04M Tris-HCl (pH7.6) (containing 10mM MgCl2, 0.25mM EDTA and 5mM glutathione), subjected to ultrasonic treatment in ice bath for 10min, centrifuged at 10000g and 4 ℃ for 10min, and the supernatant was collected as an enzyme extract. mu.L of the enzyme extract, 7. mu.L of glyceraldehyde-3-phosphate dehydrogenase, 7. mu.L of phosphoglycerate kinase, and 180. mu.L of the reaction solution (containing: 1mM ATP, 4mM NADH, 10mM RuBP, and 100mM creatine phosphate) were mixed well, and the absorbance of the mixture at 340nm was immediately measured, and the degree of oxidation of NADH in the mixture was recorded. 1 unit of RuBisCO enzyme activity is defined as the nanomolar number of NADH oxidized in 1min for 1g of rice samples.

The method for determining the composition of Mg, N-CDs and chloroplasts and the photosynthetic activity comprises the following steps: lettuce (Lactuca sativa L.) leaves with larger leaves and rich chloroplast content are selected for extracting the chloroplast. And (3) placing 10g of fresh leaves in a grinder, adding 20mL of sucrose buffer solution, grinding a sample into slurry, filtering the slurry by 4 layers of gauze, centrifuging the filtrate at 1000rpm for 10min, discarding the precipitate, centrifuging the supernatant at 3000rpm for 10min, and suspending the precipitate in the sucrose buffer solution to obtain the chloroplast suspension. All the above operations were carried out in an ice bath or at 4 ℃. The concentration of chlorophyll in the chloroplast is determined by extraction of the liquid (Advanced Functional Materials,2018,28,44, 1804004). Mixing chloroplast suspension containing 10 μ g chlorophyll per mL with Mg, N-CDs solution (final concentration of 10, 50 and 100 μ g/mL)^-1) Mix and incubate at 4 ℃ for 1h in the dark. The mixture was centrifuged at 3000rpm at 4 ℃ for 3min to collect the complex of Mg, N-CDs and chloroplasts (Mg, N-CDs @ Chl). The morphology and fluorescence emission distribution of Mg, N-CDs @ Chl were observed using a FEI Verios 460 Scanning Electron Microscope (SEM) and an inverted fluorescence microscope (Nikon, ECLIPSE Ti 2-U). Followed byAnd the UV-Vis light absorption spectrum, the fluorescence emission spectrum and the fluorescence lifetime of Mg, N-CDs @ Chl are measured.

The Hill reaction activity of Mg, N-CDs @ Chl and independent chloroplasts is measured by using DCPIP and potassium ferricyanide, and the photosynthetic activity of the Mg, N-CDs @ Chl and the independent chloroplasts is compared. A final concentration of 60. mu.M DCPIP solution or 400. mu.M potassium ferricyanide solution was mixed with Mg, N-CDs @ Chl and chloroplast suspension alone. And then placing the mixed solution under a xenon lamp for irradiation to initiate the reduction reaction of the DCPIP and the potassium ferricyanide. Recording the reduction degree of the mixed solution by measuring the absorbance of the mixed solution at 600nm (DCPIP) or 420nm (potassium ferricyanide), and further reacting Mg, N-CDs @ Chl and the photosynthetic activity of the chloroplast alone. Three parallel replicates per treatment setup.

The influence of the magnesium nitrogen doped carbon point on the expression quantity of genes related to synthesis and metabolism of chlorophyll of rice plants is as follows: the genes assayed included: two Mg chelatase subunits (ChlD: LOC4334537 and ChlI: LOC4333259), chlorophyll synthase (ChlG: LOC4338498) and chlorophyllase-2 (LOC 4348648). Respectively responsible for the chelation of Mg, the assembly of chlorophyll molecules and the degradation of chlorophyll molecules in the chlorophyll synthesis process. As can be seen from FIG. 6, 300. mu.g.mL^-1The desired expression levels of the ChlI, ChlD, ChlG and chlorophyllase genes of (1) were increased by 93.55%, 15.26%, 115.02% and 29.75%, respectively. But 50 and 100. mu.g.mL^-1The Mg, N-CDs have no obvious influence on the expression level of Ch1I, and the expression levels of Ch1D (33.86% and 34.42%) and chlorophyll synthase (33.33% and 33.10%) are reduced. For the expression level of Ch1G, 50. mu.g/mL^-1The content of Mg, N-CDs in the alloy is reduced by 31.58 percent, 100 mu g/mL^-1The Mg, N-CDs of (A) have no significant influence. These data show that under the condition of proper treatment concentration, Mg, N-CDs can promote synthesis and metabolism of chlorophyll in rice leaves, so as to accelerate the renewal of chlorophyll and keep higher activity.

Effect of Mg, N-CDs on photosynthesis of rice seedlings: the distribution of Mg, N-CDs in rice leaves was observed by TEM. A large number of evenly distributed black particles, including cytoplasm and chloroplasts, were observed in the leaves of Mg, N-CDs treated rice compared to the control treatment (A of FIG. 7), indicating that Mg, N-CDs can be incorporated by foliar spray applicationEnter the inside of rice leaf cells. The photosynthesis parameters of the treated rice leaves were tested using a chlorophyll fluorometer. qP and NPQ represent the photosynthetic activity of the leaf and the ability to dissipate excess energy for self-protection under intense light, respectively. Y (NPQ), Y ((NO) and Y (II) respectively represent the quantum efficiency of the regulated non-photochemical transformation, the quantum efficiency of the non-regulated non-photochemical transformation and the quantum efficiency of the actual photochemical energy conversion for photosynthesis in the plant leaf, ETR represents the photosynthetic electron transfer rate of the PS II system in the leaf, and the results show that the effect of Mg, N-CDs on photosynthesis in rice leaf has concentration dependence and photosynthetic radiation intensity dependence (C-H in FIG. 7). when the photosynthetic radiation intensity is less than 81. mu. mol. m.m.^-2·s^-1All treatment concentrations of Mg, N-CDs decreased NPQ, qP, ETR, Y (NPQ) and Y (II) of rice leaf, and increased Y (NO). Indicating that Mg, N-CDs are not beneficial to the photosynthesis of plant leaves under low illumination intensity. As the intensity of photosynthetic radiation increases, Mg, N-CDs show concentration dependence on photosynthesis of rice leaves. 50. mu.g/mL^-1The intensity of the photosynthetic radiation of the Mg, N-CDs reaches and is higher than 146 mu mol.m^-2·s^-1The NPQ of the rice leaf is obviously improved, and the photosynthetic radiation intensity reaches and is higher than 611 and 461 mu mol · m^-2·s^-1Then, the qP and ETR can be significantly improved. In the photosynthetic energy conversion, 50. mu.g.mL^-1The intensity of photosynthetic radiation of Mg, N-CDs is 231-611 mu mol.m^-2·s^-1While y (npq) was significantly increased and y (no) was decreased, y (ii) was not significantly affected. But when the intensity of the photosynthetic radiation reaches or exceeds 146 mu mol.m^-2·s^-1At 100 and 300. mu.g.mL^-1The Mg, N-CDs can obviously improve the qP, the ETR and the Y (II) of the rice leaf blade and reduce the Y (NPQ) and the Y (NO). For NPQ, 100 and 300. mu.g.mL^-1The Mg and N-CDs can respectively reach the photosynthetic irradiation intensity of 231-611 and 461-611 mu mol · m^-2·s^-1It has obvious improvement effect on the surface of the steel. Synthesis of all photosynthetic parameters, 300. mu.g.mL^-1The photosynthetic radiation intensity of Mg, N-CDs is 461 mu mol.m^-2·s^-1The effect of promoting photosynthesis of rice leaf is optimal, and qP, ETR and Y (II) are improvedThe rates reached 109.54%, 104.48% and 127.16% (a of fig. 8). And 100. mu.g/mL^-1The intensity of photosynthetic radiation of Mg, N-CDs is 611 mu mol.m^-2·s^-1The effect of promoting the NPQ of the rice leaves is optimal. Based on the above results, Mg, N-CDs can increase the photosynthetic activity (qP), photoprotective ability (NPQ) and Electron Transfer Rate (ETR) of rice leaves, thereby reducing the non-photosynthetic energy conversion in photosynthesis [ Y (NPQ) and Y (NO)]Increasing the energy conversion of photosynthesis [ Y (II)]。

The light reaction is CO₂Supply energy (ATP and NADPH) and supply CO (Raines, C A,2003)₂Conversion to carbohydrates directly determines the rate of photosynthesis. RuBisCO is responsible for CO₂One key enzyme in the calvin cycle of assimilation. B of FIG. 8 shows that Mg, N-CDs can significantly improve the RuBisCO enzyme activity of rice seedlings and have concentration dependence. 300. mu.g/mL^-1The Mg, N-CDs of the alloy increases the Mg, N-CDs by 46.62 percent. Thus, Mg, N-CDs treatment can increase rice CO₂The rate of assimilation of (a).

Effect of Mg, N-CDs on chloroplast photosynthetic activity: comparing the SEM images of chloroplasts and Mg, N-CDs @ Chl alone (A and B of FIG. 9), it was found that Mg, N-CDs had no significant effect on the morphology and structure of chloroplasts. The fluorescent microscope observation shows that Mg, N-CDs @ Chl emits blue fluorescence under 361-389nm excitation, and emits red fluorescence under 515-565nm excitation, and the distribution positions of the two are consistent (FIGS. 9C-F). The Mg and N-CDs enter the chloroplast, are distributed on the thylakoid membrane and are in direct contact with chlorophyll molecules, so that the possibility of energy transfer between the Mg and N-CDs is provided. G of FIG. 9 shows that the fluorescence lifetime of Mg, N-CDs in chloroplasts decreased from 3.04ns to 2.00ns, demonstrating that the excitation energy of Mg, N-CDs may be reabsorbed by chloroplasts. Changes in photosynthetic activity of Mg, N-CDs @ Chl were measured using DCPIP and potassium ferricyanide reduction. It was found that at the appropriate complex concentration, Mg, N-CDs @ Chl exhibited higher reducing activity on dcppip and potassium ferricyanide than chloroplast alone (H and I of fig. 9). And under saturated illumination (9000LUX), Mg, N-CDs @ Chl can still show higher reduction activity on DCPIP (J of FIG. 9). Both DCPIP and potassium ferricyanide can be reduced by receiving photosynthetic electrons generated by the PS II system, and the reduction degree is related to the transfer rate of the photosynthetic electrons, reflecting the photosynthetic activity of the PS II system. Based on the data, the chloroplast can capture the excitation energy of Mg and N-CDs, the transfer rate of electrons in the photosynthetic system is accelerated, the photosynthetic activity is further improved, and the Mg and N-CDs can break the limitation of chloroplast photosynthesis light saturation.

Example 5

Example 1 mode of the obtained magnesium-nitrogen-doped carbon dots (Mg, N-CDs) for regulating plant photosynthesis

Taken together with all the data herein, we can derive the mechanism by which Mg, N-CDs enhance plant photosynthesis: (1) the metabolic rate of chlorophyll is accelerated, and the activity is improved; (2) improving the light absorption capacity of chloroplasts (higher and wider); (3) photosynthetic electron transfer rate; (4) CO 2₂Assimilation (FIG. 10).

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

SEQUENCE LISTING

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Claims

1. A preparation method of a magnesium nitrogen doped carbon dot is characterized by comprising the following steps: dissolving citric acid, ethanolamine and magnesium hydroxide in ultrapure water, pouring mixed liquor obtained after ultrasonic treatment into a reaction kettle, and reacting for 6 hours at 200 ℃; cooling to room temperature after the reaction is finished, filtering the product by using a filter head with the aperture of 0.22 mu m, and dialyzing for 12 hours by using a dialysis bag; and (4) after freeze drying, obtaining the magnesium-nitrogen doped carbon dots.

2. The method of claim 1, wherein the method comprises: the molar ratio of the citric acid to the ethanolamine to the magnesium hydroxide is 1:0.2: 0.35.

3. The method of claim 1, wherein the method comprises: the time of the ultrasonic treatment is 30 min.

4. The method of claim 1, wherein the method comprises: the cut-off molecular weight MW of the dialysis bag is 100-500; the dialysis adopts ultrapure water as dialysate.

5. A magnesium nitrogen-doped carbon dot prepared by the preparation method of any one of claims 1 to 4.

6. Use of the magnesium nitrogen doped carbon dot of claim 5 for increasing plant photosynthesis.