CN114333988A - Estimation method for the contribution rate of plant β-CAS and ST to KCN metabolism - Google Patents

Abstract

The invention discloses a method for estimating contribution rate of plants beta-CAS and ST to KCN metabolism, which comprises the following steps: and (2) stressing the plant seedlings in a short period, wherein a plant seedling water culture reaction system of each treatment group is equivalent to a plant water culture fully-mixed bioreactor, constructing a differential equation of the plant water culture fully-mixed bioreactor on KCN metabolism, further obtaining the relation between the KCN metabolism amount and time of the plant water culture fully-mixed bioreactors of each treatment group, further obtaining a substance balance equation for estimating the contribution rate of the beta-CAS and ST to the KCN metabolism, and obtaining the range of the contribution rate of the beta-CAS and ST to the KCN metabolism. The method for estimating the contribution rate of the plants beta-CAS and ST to KCN metabolism avoids complicated time dynamics experiments in the process of plant repair evaluation, forms boundary conditions to evaluate the contribution rate of the plants beta-CAS and ST to KCN metabolism, and is favorable for evaluating the KCN repair efficiency of the plants.

Description

Estimation method of the contribution rate of plant β-CAS and ST to KCN metabolism

technical field

The invention belongs to the technical field of plant crop science, and relates to a method for estimating the contribution rate of plant β-CAS and ST to KCN metabolism.

Background technique

Simple cyanide (KCN) has been the leaching agent of choice in gold mining. In addition, it is an indispensable industrial chemical for the metal finishing and hardening, electroplating, steel and printed circuit board manufacturing industries. According to statistics, more than 100,000 tons of CN from the mining industry are transported to the outlet ^of the catchment area through surface runoff and infiltration each year, contaminating soil, surface water and groundwater. Due to its extreme toxicity, a low-cost and sustainable remediation method to remove or detoxify CN- ⁱⁿ the environment is urgently needed.

Phytoremediation is the use of green plants to transfer, contain or transform pollutants in order to reduce the harm of pollutants to the environment. In the past two decades, various plants have been shown to metabolize exogenous KCN, and only a small amount of residual CN ^- can be detected in plant tissues, which is mainly attributed to β-cyanoalanine synthase ( β-CAS). β-CAS can convert CN- to β ^- cyanoalanine, which can further be converted to asparagine or aspartic acid. In addition, it has been reported in the literature that thiotransferase (ST) in Arabidopsis, sorghum and cassava has been confirmed to metabolize CN ^- , but the specific metabolic pathway is still controversial.

1-Amino-cyclopropane-1-carboxylic acid (ACC) has been confirmed to be involved in the synthesis of ethylene in plants. During the synthesis of ET, aminocyclopropane-1-carboxylic acid oxidase (ACO) converts 1-amino-cyclopropane-1- Carboxylic acid (ACC) is converted into ET and synchronously produces endogenous CN ^- . By adding exogenous ACC, it can promote the metabolism of exogenous CN ^- by β-CAS and ST in plants stressed by exogenous KCN, which is regarded as an efficient chemical regulator.

Based on the above, in the field of plant crops, the contribution rate of β-CAS and ST to exogenous KCN metabolism in plants is not clear, and β-CAS and ST are related to the metabolic pathways and products of exogenous KCN in plants, and are directly related to plants. For the evaluation of KCN repair efficiency, an effective method for estimating the contribution rate of plant β-CAS and ST to KCN metabolism is urgently needed.

SUMMARY OF THE INVENTION

In order to achieve the above purpose, the present invention provides a method for estimating the contribution rate of plant β-CAS and ST to KCN metabolism. The plant seedling hydroponic reaction system is equivalent to a plant hydroponic total hybrid bioreactor. The KCN metabolism rate curve in the plant hydroponic total mixed bioreactor was obtained by fitting the concentration of KCN in the nutrient solution of the reactor and the stress time, and then the metabolism of KCN in the plant hydroponic total mixed reactor at different times was simulated to avoid plant hydroponic total mixing. The tedious time kinetic experiments in the repair evaluation process, and the comparison with the KCN treatment group without ACC, thus forming a "boundary condition" to evaluate the contribution rate of plant β-CAS and ST to exogenous KCN metabolism, which is conducive to evaluating plants For the KCN repair efficiency, the problem of lack of estimation methods for the contribution rate of plant endogenous enzymes to metabolism of exogenous stress substances in the prior art is solved.

The technical solution adopted in the present invention is that the method for estimating the contribution rate of plant β-CAS and ST to KCN metabolism includes the following steps:

Step 1: Put the same number of plant seedlings into multiple nutrient solutions respectively to form multiple treatment groups; a part of the nutrient solutions of the treatment groups are added with different contents of KCN to form multiple KCN treatment groups; the other part of the nutrient solutions of the treatment groups are A certain quality of ACC was added first, and then different contents of KCN were added to form multiple KCN+ACC treatment groups; KCN in each treatment group was subjected to short-term stress on plant seedlings for 2 days;

Step 2: Determine the KCN content of the nutrient solution before and after short-term stress in each treatment group, and the CN ^- content and SCN ^- content in plant seedlings after short-term stress; determine the regulation of β-CAS and SCN-content in plant seedlings after short-term stress in each treatment group. Differential expression levels of ST-related genes;

Step 3: According to the KCN content of the nutrient solution before and after the short-term stress in each treatment group, and the CN ^- content in the plant seedlings after the short-term stress, the metabolic amount of KCN in the plant seedlings of each treatment group was obtained; Metabolic amount, the rate of KCN metabolism by plant seedlings in each treatment group was obtained;

Step 4: The hydroponic reaction system of plant seedlings in each treatment group is equivalent to a plant hydroponic total mixed bioreactor. The decay rate constant in the mixed bioreactor, the volume of the plant hydroponic total mixed bioreactor, and the differential equation of KCN metabolism in the plant hydroponic total mixed bioreactor without spatial variables;

Step 5: According to the differential equation of KCN metabolism in the plant hydroponic total mixed bioreactor of each treatment group, the relationship between the concentration of KCN in the nutrient solution of each treatment group and time is obtained, and then according to the mass conversion, the plant hydroponic total mixed organism of each treatment group is obtained The relationship between the amount of KCN metabolism in the reactor and time; according to the relationship between the amount of KCN metabolism in the hydroponic total mixed bioreactor of each KCN treatment group and each KCN+ACC treatment group, the estimated plant β-CAS and ST were obtained. The mass balance equation of the contribution rate to KCN metabolism;

Step 6: The amount of metabolism of KCN by plant seedlings in each treatment group is composed of the amount of metabolism of β-CAS to KCN and the amount of metabolism of ST to KCN; the amount of metabolism of plant seedlings of ST to KCN in each treatment group is based on the amount of ^The total amount of SCN- is obtained; the total amount of SCN- produced in plant seedlings is composed ^of the cumulative amount of SCN- ⁱⁿ plant seedlings ^and the amount of SCN- metabolized in plant seedlings; the cumulative amount of SCN- ⁱⁿ plant seedlings of each treatment group is step 2 Measured SCN ^- content in plant seedlings after short-term stress;

The value of SCN ^- metabolism in plant seedlings of each KCN treatment group was set with 0.5 μg as a step unit, and the cumulative amount of SCN ^- in plant seedlings was used as an intermediate condition. When calculating the amount of β-CAS or ST in each KCN treatment group The metabolic amount of KCN is calculated at the maximum value;

The value of SCN ^- metabolism in plant seedlings in each KCN+ACC treatment group was set with 0.5 μg as a step unit, and the cumulative amount of SCN ^- in plant seedlings was used as the intermediate condition. The metabolism of CAS or ST to KCN is the maximum value and the calculation is stopped;

According to the metabolism of β-CAS or ST to KCN in the KCN+ACC treatment group is always greater than the metabolism of β-CAS or ST to KCN in the KCN treatment group, the range of the contribution rate of plant β-CAS and ST to KCN metabolism was obtained. , to complete the estimation of the contribution rate of plant β-CAS and ST to KCN metabolism.

Further, step 2 also includes, according to the determined differential expression levels of the genes involved in regulating β-CAS and ST in the plant seedlings of each treatment group after short-term stress, to obtain the in vivo regulation of plant seedlings in the KCN+ACC treatment group under the same stress concentration. The differential expression levels of related genes of β-CAS and ST were higher than those in the KCN-treated group.

Further, in step 3, the rate of KCN metabolism by plant seedlings in each treatment group is the ratio of the amount of KCN metabolism in each treatment group to the fresh weight of plant seedlings in each treatment group within a short-term stress period.

Further, in step 4, the concentration differential equation of KCN metabolism in the plant hydroponic total mixed bioreactor that does not contain spatial variables is shown in the following formula:

In the formula, C _(t) represents the concentration of KCN in the nutrient solution at t, in μg/mL; β represents the correction coefficient; Q represents the transpiration of plant seedlings, in g/h; C _i represents the concentration of KCN in the nutrient solution. Initial concentration, the unit is μg/mL; k is the decay rate constant of KCN in the plant hydroponic total mixed bioreactor, the unit is h ^-1 ; V is the volume of the plant hydroponic total mixed bioreactor, the unit is mL ; t represents time in h; e is a natural constant.

Further, the calculation of the correction coefficient is as follows:

Further, the acquisition of the decay rate constant of KCN in the plant hydroponic total mixed bioreactor is specifically: taking the initial concentration of KCN in the nutrient solution of each KCN treatment group or each KCN+ACC treatment group as an independent variable, The rate of KCN metabolism in each KCN treatment group or each KCN+ACC treatment group was linearly fitted, and the slope of the fitting curve was the KCN in the KCN treatment group or the KCN+ACC treatment group in the plant hydroponic total mixed bioreactor. The decay rate constant in .

Furthermore, the transpiration of plant seedlings is the change in the total weight of the plant hydroponic total mixed bioreactor per unit stress time.

Further, in step 6, the amount of metabolism of plant seedling ST to KCN in each treatment group is obtained according to the SCN ^- total amount produced in the plant seedling, specifically: the SCN ^- total amount produced in the plant seedling and the amount of the plant seedling ST to KCN. The stoichiometric ratio of the metabolic amount is 1:1, and the molar amount of the SCN ^- total amount of the plant seedlings produced in the body is obtained to obtain the molar amount of the plant seedling ST to the metabolic amount of KCN, and after mass conversion, the ST of each treatment group is obtained. Metabolism of KCN.

Further, in step 6, the contribution rate of β-CAS and ST to KCN metabolism is specifically: the mass percentage of the metabolism amount of β-CAS or ST to KCN to the amount of metabolism of KCN by plant seedlings.

The beneficial effects of the present invention are: in the embodiment of the present invention, ACC is selected as a chemical regulator, and plant toxicology methods are used to carry out short-term KCN stress on plant seedlings; the hydroponic reaction system of plant seedlings is equivalent to a plant hydroponic total mixed bioreactor , the KCN metabolism rate curve in the plant hydroponic total mixed bioreactor was obtained by fitting the concentration of KCN in the nutrient solution of the plant hydroponic total mixed bioreactor and the stress time, and then simulated the plant hydroponic total mixed reactor at different times. The metabolism of KCN avoids the tedious time kinetic experiments in the phytoremediation evaluation process, and is compared with the KCN treatment group without ACC to form a "boundary condition" to evaluate the metabolism of plant β-CAS and ST to exogenous KCN The method for estimating the contribution rate of plant β-CAS and ST to KCN metabolism in the embodiment of the present invention is beneficial to the subsequent evaluation of plant restoration efficiency of KCN.

Description of drawings

In order to explain the embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only These are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained according to these drawings without creative efforts.

Fig. 1 is a flow chart of the method for estimating the contribution rate of plant β-CAS and ST to KCN metabolism according to the embodiment of the present invention.

Fig. 2 is the result of CN ^{-content in the rice seedlings of each treatment group according to the embodiment of the present invention, in Fig. 2 a is the CN- content} in the rice seedlings of each KCN treatment group, and b in Fig. 2 is the in vivo content of the rice seedlings of each ACC+KCN treatment group CN ^- content.

Fig. 3 is the result of SCN ^- content in rice seedlings of each treatment group according to the embodiment of the present invention, in Fig. 3 a is the content of SCN ^- in rice seedlings of each KCN treatment group, and b in Fig. 3 is the in vivo content of rice seedlings of each ACC+KCN treatment group SCN ^- content.

Figure 4a is an analysis diagram of the differential expression levels of β-CAS and ST candidate genes in each KCN treatment group according to the embodiment of the present invention.

Figure 4b is an analysis diagram of the differential expression levels of β-CAS and ST candidate genes in each ACC+KCN treatment group according to the embodiment of the present invention.

Fig. 5 is a schematic diagram of a model of a rice hydroponic total mixed bioreactor according to an embodiment of the present invention.

Fig. 6 is the decay rate constant diagram of KCN in the rice hydroponic total mixed bioreactor according to the embodiment of the present invention, in Fig. 6 a is the decay rate constant diagram of KCN in each KCN treatment group in the rice hydroponic total mixed bioreactor, In Figure 6, b is the decay rate constant diagram of KCN in each ACC+KCN treatment group in the rice hydroponic total mixed bioreactor.

FIG. 7 is a time kinetic characteristic diagram of KCN metabolism in a rice hydroponic total mixed bioreactor according to an embodiment of the present invention.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

The method for estimating the contribution rate of plant β-CAS and ST to KCN metabolism, as shown in Figure 1, includes the following steps:

1. Preparation of rice seedlings

The rice seeds are soaked in water for 12 hours, and then the seeds are put into disposable paper cups for sand cultivation. The planting density is about 30-40 rice per cup. Cultivation was carried out in an artificial climate box at a temperature of 25° C., a humidity of 65%, and a light intensity of 20,000 lux. The rice was irrigated with an improved version of ISO8692 nutrient solution, as shown in Table 1. After the rice was grown for 16 days, the sandy soil at its roots was cleaned, and rice seedlings with the same individual size were selected for use.

Table 1 Improved version of ISO8692 nutrient solution

serial number reagent concentration serial number reagent concentration 1 KNO₃ 2823.9μmol/L 7 H₃BO₃ 2992.1nmol/L 2 MgCl₂·6H₂O 59.0μmol/L 8 MnCl₂·4H₂O 2097.0nmol/L 3 CaCl₂·2H₂O 122.4μmol/L 9 Na₂MoO₄·2H₂O 28.9nmol/L 4 MgSO₄·7H₂O 60.9μmol/L 10 CuSO₄·2H₂O 0.1nmol/L 5 KH₂PO₄ 246.0μmol/L 11 ZnSO₄ 22.0nmol/L 6 NaHCO₃ 1785.5μmol/L 12 CoCl₂·6H₂O 6.3nmol/L

2. Experimental design

Selected rice seedlings were grown in the following treatment solutions:

KCN treatment group: Four groups of rice seedlings with fresh weights of 1.291g, 1.285g, 1.244g and 1.223g were placed in 50mL conical flasks, and 50mL of the improved version of ISO8692 nutrition as shown in Table 1 was added. solution, and added 0 mg/L, 1.0 mg/L, 2.0 mg/L, and 3.0 mg/L of KCN, respectively.

KCN+ACC treatment group: Four groups of rice seedlings with fresh weights of 1.293g, 1.29g, 1.263g, and 1.244g were placed in 50mL conical flasks, and 50mL of the improved version shown in Table 1 was added. ISO8692 nutrient solution was added with 10 μM ACC, and then 0 mg/L, 1.0 mg/L, 2.0 mg/L and 3.0 mg/L KCN were added respectively.

Each Erlenmeyer flask is wrapped in foil to protect from light, minimizing water loss and inhibiting algae growth. Each treatment group had 4 biological replicates with a short-term exposure of 2 days. All reagents used in this experiment were of analytical grade.

2.1. Analysis of KCN content in nutrient solution and total CN ^- content in rice seedlings

2.1.1. Determination of KCN content in nutrient solution

The concentration of KCN in the nutrient solution was measured before and after exposure (2 days), respectively. A certain volume of solution sample (1 mL-5 mL) was collected in a colorimetric tube, and NaOH (0.1%) was added to the 10 mL mark. Then, a buffer solution (5.0 mL) containing KH ₂ PO ₄ and NaH ₂ PO ₄ was added, and chloramine-T solution (0.2 mL, 1% [m/v]) was added quickly, shaken for 3 min to 5 min, and then added The mixture of isonicotinic acid and pyrazolone is made up to 25mL with MilliQ water (ultrapure water), mixed well, and the colorimetric tube is placed in a water bath (32°C, 40 minutes). The KCN content in the nutrient solution was determined by a photometer.

Among them, 5.0 mL of isonicotinic acid and pyrazolone mixed solution is composed of isonicotinic acid (1.5 g of isonicotinic acid is dissolved in 24 mL of 2% NaOH solution, diluted with water to 100 mL): pyrazolone (0.25 g of pyrazole citronone dissolved in 20 mL of dimethylformamide solution).

2.1.2 Detection of total CN ^- content in rice seedlings

Determination of total CN ^- content in rice seedlings by distillation. 10 mL of NaOH (1%) solution was added to the collection flask at the end of the distillation system. Place an accurately weighed sample of chopped rice seedling roots or leaves into a round bottom flask (500 mL), add 200 mL of MilliQ water, then 10 mL of EDTA (10%) solution and 10 mL of phosphoric acid (85%), start Distillation system, when the solution in the collection bottle is close to 100mL ^, close the distillation system, and transfer the liquid in the collection bottle to a volumetric flask (100mL) to constant volume to be measured. The test results are shown in Figure 2.

2.2. Detection of SCN ^- content in rice seedlings

After 2-day exposure of rice seedlings, the roots and leaves of rice seedlings were collected and weighed, and immediately ground into powder in liquid nitrogen. Subsequently, the powdered samples were mixed with 5 mL of citric acid (0.1 M) at 25 ℃, 40kHz ultrasonic treatment for 1h, 10,000×g low temperature (4℃) centrifugation for 10min, and the supernatant was collected. This process was repeated 2 times, and the supernatants of the 2 times were mixed. Finally, the sample (~2.0 mL) was concentrated using a rotary evaporator. After filtration with a 0.45 μm microporous membrane, the SCN ^- content was measured by an ion chromatograph. The test results are shown in Figure 3.

2.3. Analysis of differential expression of β-CAS and ST-related genes

The present invention adopts three genes (OsCYS-C1, OsCYS-D1 and OsCYS-D2) that regulate β-CAS. It has been reported that AtStr1 and AtStr2 in Arabidopsis ST family genes are explicitly involved in CN ^- metabolism, thus obtained from the Rice Database RGAP (http://rice.plantbiology.msu.edu/analyses_search_blast.shtm), National Rice Data Center (http: //www.ricedata.cn/gene/index.htm), and RAP-DB (http://rapdb.dna.affrc.go.jp/) screened out the homology with Arabidopsis involved in CN ^- metabolism The higher 3 genes (OsStr9, OsStr23 and OsStr24).

Quantitative real-time PCR (qRT-PCR) was used to determine the gene differential expression levels of β-CAS and ST, as shown in Table 2. After 2 days of exposure treatment, 0.2 g of rice seedling tissue was weighed and immediately ground in liquid nitrogen, the total RNA of the sample was extracted using an RNA extraction kit, and the A260/A280 of the extracted RNA was detected by an ultra-microspectrophotometer between 1.8 and 2.0. DNase I was used to remove the contamination of gDNA, then reverse transcription kit was used to synthesize the first strand of cDNA, and SYBR green I dye method was used to perform real-time quantitative PCR (qRT-PCR). Primer 5.0 was used to design primers, and AB-7500 quantitative PCR instrument was used to analyze gene expression. The differential expression fold was calculated by 2 ^-ΔΔCT . The measurement results are shown in Figure 4a and Figure 4b.

Table 2 Primer design of β-CAS and ST candidate genes in rice seedlings

3. Experimental results

3.1. Analysis ^of KCN content in nutrient solution and CN- content in rice seedlings

At the beginning of the reaction, the concentrations of KCN added to the nutrient solution of the 50 mL reactor were 1.0 mg/L, 2.0 mg/L, and 3.0 mg/L, respectively. Therefore, the calculated exogenous KCN added was 50 μg, 100 μg, and 150 μg, respectively; 2 After the end of day exposure, measure the KCN concentration in the nutrient solution, and calculate the removal rate R of KCN in the nutrient solution according to formula (1), and the specific calculation results are shown in Table 3:

In the formula, C _f represents the KCN concentration in the nutrient solution at the end of the stress, V _f represents the volume of the nutrient solution at the end of the stress, _Ci represents the KCN concentration in the nutrient solution before the stress, and _Vi represents the volume of the nutrient solution before the stress.

Table 3 Removal rate of KCN in nutrient solution

It can be concluded from Table 3 that the KCN removal rate of the KCN+ACC treatment group was higher than that of the KCN treatment group.

3.2. Analysis of CN ^- content in rice seedlings in each treatment group

As can be seen from Figure 2, with the increase of KCN content in the nutrient solution, the CN ^- concentration in the roots and leaves of rice seedlings increased significantly. As shown in a in Fig. 2, the concentrations of CN ^- in the roots and leaves of the KCN-treated groups were between 0.64 μg/g and 1.58 μg/g and 0.38 μg/g and 1.14 μg/g, respectively. As shown in b in Fig. 2, in the KCN+ACC treatment group, the levels of CN ⁻ in roots and leaves ranged from 0.28 μg/g to 0.36 μg/g and 0.23 μg/g to 0.31 μg/g, respectively. Obviously, the residual CN ^- concentrations in the roots and leaves of rice seedlings in the KCN+ACC treatment group were significantly lower than those in the KCN treatment group, indicating that exogenous addition of ACC could reduce the residual CN ^- in rice seedlings under short-term exogenous KCN stress.

3.3. Rate calculation of KCN metabolism by rice seedlings

According to the material balance of KCN in the rice hydroponic total mixed bioreactor, the metabolism of KCN by rice seedlings = the absorption of KCN by rice seedlings - the accumulation of CN ^- in rice seedlings. Therefore, the rate of KCN metabolism by rice seedlings at 48h can be calculated, as shown in Table 4.

Table 4 Metabolism rate of KCN by 48h rice seedlings

单位是μg/g FW·h，FW表示水稻幼苗鲜重(g)。从表4可知，外源添加ACC能有效提高水稻幼苗对KCN的代谢速率。In Table 4, the effect of 48h rice seedlings on KCN

The unit is μg/g FW·h, and FW represents the fresh weight of rice seedlings (g). It can be seen from Table 4 that exogenous addition of ACC can effectively increase the metabolic rate of KCN in rice seedlings.

3.4. Analysis of SCN ^- content in rice seedlings

It can be seen from Fig. 3 that with the increase of KCN addition, the concentration of SCN ^- in the roots and leaves of rice seedlings increased significantly. As shown in a in Fig. 3, the concentrations of SCN- ⁱⁿ roots and leaves in the KCN-treated group ranged from 12.21 μg/g to 37.69 μg/g and 8.71 μg/g to 26.69 μg/g, respectively. As shown in b in Fig. 3, the concentrations of SCN- ⁱⁿ roots and leaves in the KCN+ACC treatment group ranged from 8.65 μg/g to 25.87 μg/g and 5.98 μg/g to 19.23 μg/g, respectively. Obviously, the SCN ^- concentration in the roots and leaves of rice seedlings in the KCN+ACC treatment group was significantly lower than that in the KCN treatment group, indicating that the addition of exogenous ACC could reduce the accumulation of SCN ^- in the tissues of rice seedlings under KCN stress.

3.5. Analysis of ST and β-CAS candidate gene expression

As shown in Figure 4a and Figure 4b, in the KCN treatment group and the KCN+ACC treatment group, the differential expression levels of the three ST candidate genes in rice seedlings were up-regulated in both roots and leaves, among which the three candidate ST genes in the KCN+ACC treatment group were The differential expression levels were significantly higher than those in the KCN-treated group. Similar to the differential expression of ST genes, the differential expression levels of three β-CAS candidate genes in rice seedlings were also up-regulated in roots and leaves in the KCN treatment group and the KCN+ACC treatment group, among which 3 in the KCN+ACC treatment group The differential expression levels of β-CAS candidate genes were significantly higher than those in the KCN-treated group. These results indicated that exogenous addition of ACC could positively increase the differential expression levels of β-CAS and ST genes in rice seedling tissues.

4. Evaluation of the contribution rate of β-CAS and ST to KCN metabolism using a rice hydroponic total hybrid biological response model combined with boundary conditions

The treatment of the present invention is based on the rice hydroponic reaction system. As shown in Figure 5, the exogenous KCN added to the growth medium of rice seedlings enters into the rice seedlings following the first-order kinetic absorption characteristics, and its metabolic process in the rice seedlings is also Following the same reaction law, the exogenous KCN can achieve uniform mixing in three-dimensional space after entering the rice hydroponic total mixing bioreactor.

Hybrid bioreactor model assumptions for rice hydroponics:

KCN conforms to the law of conservation of mass in the rice hydroponic total mixed bioreactor;

KCN follows first-order kinetic decay characteristics in a rice hydroponic total mixed bioreactor;

In rice seedlings, only β-CAS and ST are involved in the metabolism of KCN and are regulated by associated genes.

4.1. Model construction

Set up a differential equation with no spatial variables from the mass balance:

Among them, V is the volume of the rice hydroponic total mixed bioreactor, the unit is mL; C is the concentration of KCN in the nutrient solution, the unit is μg/mL, t is the time, the unit is h; Q is the transpiration of the rice seedling, The unit is g/h; C _i is the initial concentration of KCN in the nutrient solution, the unit is μg/mL; C _f is the concentration of KCN in the nutrient solution at the end of the stress, the unit is μg/mL; k is the KCN in the rice hydroponic culture Decay rate constant in a fully mixed bioreactor in h ^-1 .

根据式(1)得出t时水稻水培全混生物反应器营养液中KCN的浓度C_(t)，如式(3)所示：When the non-steady state decays,

According to the formula (1), the concentration C _(t) of KCN in the nutrient solution of the rice hydroponic total mixed bioreactor at t is obtained, as shown in the formula (3):

In order to make the simulation results more similar to the actual results, it is necessary to introduce a correction coefficient to correct equation (3), as shown in equation (4):

Among them, β represents a correction coefficient, and e is a natural constant.

4.2, parameter definition

4.2.1. Calculation of volume of rice hydroponic total mixed bioreactor

The volume of the rice hydroponic total hybrid bioreactor V=m/ρ, where m represents the fresh weight (g) of the rice seedlings in each treatment group, the density of the rice seedlings ρ=0.72g/mL, and the rice hydroponic total hybrid organisms in each treatment group The calculated results of the reactor volume are shown in Table 5.

Table 5 Rice hydroponic total mixed bioreactor volume

KCN concentration (mg CN/L) KCN treatment group ACC+KCN treatment group 0.0 1.7932mL 1.7957mL 1.0 1.7845mL 1.7922mL 2.0 1.7287mL 1.7547mL 3.0 1.6983mL 1.7278mL

4.2.2. Calculation of transpiration of rice seedlings

The transpiration of rice seedlings is equivalent to the change in the total weight of the rice hydroponic total mixed bioreactor per unit stress time. The transpiration of rice seedlings in each treatment group is shown in Table 6.

Table 6 Transpiration of rice seedlings

KCN concentration (mg CN/L) KCN treatment group ACC+KCN treatment group 0.0 0.246g/h 0.254g/h 1.0 0.238g/h 0.250g/h 2.0 0.183g/h 0.200g/h 3.0 0.129g/h 0.171g/h

4.2.3. Decay rate constant of KCN in rice hydroponic total mixed bioreactor

According to Table 4, taking the KCN stress concentration as the X-axis and the rate of KCN metabolism by rice seedlings as the Y-axis, a linear fit was performed, and the slope was the decay rate constant (h ^-1 ). The fitting results show that the decay rate constant of KCN in the rice hydroponic total mixed bioreactor in the KCN treatment group is 0.6141h ^-1 , and the decay rate constant of KCN in the ACC+KCN treatment group in the rice hydroponic total mixed bioreactor is 0.7089h ^-1 , as shown in Figure 6.

4.3. Result output

Substitute the above results into formula (4) to obtain the relationship between the concentration and time of KCN in the nutrient solution in the rice hydroponic total-mixed bioreactor, and then obtain the metabolic amount and time of KCN in the rice hydroponic total-mixed bioreactor according to the mass conversion. relationship, as shown in Figure 7. It can be seen that the exogenous KCN entered the metabolic plateau 12h after entering the rice seedlings. Therefore, one of the advantages of this model is to simulate the metabolism of KCN in the rice hydroponic total mixed bioreactor at different times, avoiding the tedious time kinetic experiments in the process of rice restoration evaluation.

4.4. Simulation of the contribution rate of β-CAS and ST to KCN metabolism in rice seedlings

The amount of metabolism of rice seedlings to KCN = the amount of metabolism of β-CAS to KCN + the amount of metabolism of ST to KCN, according to the mass balance, the following material balance equation is obtained, as the contribution rate model of rice seedlings β-CAS and ST to KCN metabolism:

In the formula, M _β-CAS1 , M _β-CAS2 , and M _β-CAS3 represent the metabolism of β-CAS to KCN in three KCN-treated rice seedlings, respectively, in μg; M _ST1 , M _ST2 , and M _ST3 represent 3 Metabolism of KCN by ST of rice seedlings in each KCN treatment group, the unit is μg; M _Total1 , M _Total2 , M _Total3 represent the total metabolism of β-CAS and ST to KCN in the three KCN treatment groups, and the unit is μg;

M' _β-CAS1 , M' _β-CAS2 , M' _β-CAS3 represent the metabolism of β-CAS to KCN in three ACC+KCN treatment groups, respectively, the unit is μg; M' _ST1 , M' _ST2 , M ' _ST3 represents the metabolic capacity of rice seedlings ST to KCN in the three ACC+KCN treatment groups, respectively, and the unit is μg; M' _Total1 , M' _Total2 , and M' _Total3 represent the three ACC+KCN treatment groups, respectively, the β-CAS and Total metabolism of KCN by ST, in μg.

Equations (5) to (7) are the simulations of the contribution of β-CAS and ST to KCN metabolism in rice seedlings of each KCN treatment group; Contribution simulation of KCN metabolism.

As shown in Figure 3, the differential expression levels of β-CAS and ST genes in rice seedlings in the ACC+KCN treatment group were higher than those in the KCN treatment group. Therefore, when constructing the contribution rate model of rice seedlings β-CAS and ST to KCN metabolism, it is considered that the metabolism of β-CAS and ST to KCN in rice seedlings in ACC+KCN treatment group is greater than that in KCN treatment group, that is, under any treatment, it is consistent with M' _β-CAS >M _β-CAS and M' _ST >M _ST .

The pathway of KCN metabolism through ST will produce SCN ^- (stoichiometric ratio CN ^- : SCN ^- = 1:1), and in the process of SCN ^- production, part of SCN ^- will also be metabolized by rice seedlings. Therefore, the total amount of SCN ^- produced in rice seedlings = the metabolic amount of SCN ^- + the accumulated amount of SCN ^- . Thus, equations (5) to (10) can be transformed into equations (11) to (16):

Among them, X _ST1 , X _ST2 , and X _ST3 represent the metabolism of SCN ^- in rice seedlings of the three KCN treatment groups, respectively, and the unit is μg, and X' _ST1 , X' _ST2 , and X' _ST3 represent the three ACC+KCN treatment groups, respectively. Metabolism ^of SCN- by medium rice seedlings, in μg.

Figure 7 shows that the metabolism of KCN in the rice hydroponic total mixed bioreactor has reached a stable limit at about 12 h, and the accumulation of SCN ^- in rice seedlings does not continue to increase, that is, the difference between the production and metabolism of SCN ^- reaches the limit. balance. Set the value of X _ST with a step size of 0.5 μg, such as 0.5, 1, 1.5...,n, and take the accumulated amount of SCN ^- measured at the 48th hour as an intermediate condition, when the β-CAS or β-CAS in the KCN treatment group is calculated. The calculation was stopped when the metabolic amount of ST to KCN reached the maximum value. At this time, the series values of M _β-CAS and M _ST at each step were obtained. Set the value of X′ _ST with a step size of 0.5μg, such as 0.5, 1, 1.5...,n, and use the accumulated amount of SCN ^- measured at the 48th h as an intermediate condition to obtain M′ _β-CAS under each step size and series values of _M'ST .

According to the qualifications of (17) and (18), the contribution rate of β-CAS and ST to KCN metabolism was evaluated, and the metabolism of β-CAS and ST to KCN at 2h and 12h was calculated according to these 2 qualifications, as shown in Table 7. As shown, the calculation formula is as follows:

The above limiting conditions indicate that under different exogenous KCN stress concentrations, the metabolic amount (μg) of β-CAS(ST) to KCN in the ACC+KCN treatment group is greater than that of β-CAS(ST) in the KCN treatment group. KCN metabolism (μg).

Table 7 Estimation of contribution rate of β-CAS and ST to KCN metabolism in rice seedlings

It can be obtained from Table 7: (1) Under different concentrations of exogenous KCN stress, the metabolic efficiency of β-CAS to KCN is always higher than that of ST; (2) Under different concentrations of exogenous KCN stress, β-CAS and ST The metabolic contribution rate range of KCN was basically similar; (3) With the passage of stress time, the metabolic efficiency of β-CAS to KCN increased while that of ST decreased, which should be due to the sensitivity of ST to KCN. Solving this problem is beneficial to the subsequent evaluation of the KCN repair efficiency of rice.

It should be noted that in this application, relational terms such as first, second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply these entities or operations There is no such actual relationship or order between them. Moreover, the terms "comprising", "comprising" or any other variation thereof are intended to encompass a non-exclusive inclusion such that a process, method, article or device comprising a list of elements includes not only those elements, but also includes not explicitly listed or other elements inherent to such a process, method, article or apparatus. Without further limitation, an element qualified by the phrase "comprising a..." does not preclude the presence of additional identical elements in a process, method, article or apparatus that includes the element.

Each embodiment in this specification is described in a related manner, and the same and similar parts between the various embodiments may be referred to each other, and each embodiment focuses on the differences from other embodiments.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention are included in the protection scope of the present invention.

Claims (9)

1. A method for estimating the contribution rate of plant β-CAS and ST to KCN metabolism, comprising the following steps: Step 1: Put the same number of plant seedlings into multiple nutrient solutions respectively to form multiple treatment groups; a part of the nutrient solutions of the treatment groups are added with different contents of KCN to form multiple KCN treatment groups; the other part of the nutrient solutions of the treatment groups are A certain quality of ACC was added first, and then different contents of KCN were added to form multiple KCN+ACC treatment groups; KCN in each treatment group was subjected to short-term stress on plant seedlings for 2 days; Step 2: Determine the KCN content of the nutrient solution before and after short-term stress in each treatment group, and the CN ^- content and SCN ^- content in plant seedlings after short-term stress; determine the regulation of β-CAS and SCN-content in plant seedlings after short-term stress in each treatment group. Differential expression levels of ST-related genes; Step 3: According to the KCN content of the nutrient solution before and after the short-term stress in each treatment group, and the CN ^- content in the plant seedlings after the short-term stress, the metabolic amount of KCN in the plant seedlings of each treatment group was obtained; Metabolic amount, the rate of KCN metabolism by plant seedlings in each treatment group was obtained; Step 4: The hydroponic reaction system of plant seedlings in each treatment group is equivalent to a plant hydroponic total mixed bioreactor. The decay rate constant in the mixed bioreactor, the volume of the plant hydroponic total mixed bioreactor, and the differential equation of KCN metabolism in the plant hydroponic total mixed bioreactor without spatial variables; Step 5: According to the differential equation of KCN metabolism in the plant hydroponic total mixed bioreactor of each treatment group, the relationship between the concentration of KCN in the nutrient solution of each treatment group and time is obtained, and then according to the mass conversion, the plant hydroponic complete mixed organism of each treatment group is obtained The relationship between the amount of KCN metabolism in the reactor and time; according to the relationship between the amount of KCN metabolism in the hydroponic total mixed bioreactor of each KCN treatment group and each KCN+ACC treatment group, the estimated plant β-CAS and ST were obtained. The mass balance equation of the contribution rate to KCN metabolism; Step 6: The amount of metabolism of KCN by plant seedlings in each treatment group is composed of the amount of metabolism of β-CAS to KCN and the amount of metabolism of ST to KCN; the amount of metabolism of plant seedlings of ST to KCN in each treatment group is based on the amount of ^The total amount of SCN- is obtained; the total amount of SCN- produced in plant seedlings is composed ^of the cumulative amount of SCN- ⁱⁿ plant seedlings ^and the amount of SCN- metabolized in plant seedlings; the cumulative amount of SCN- ⁱⁿ plant seedlings of each treatment group is step 2 Measured SCN ^- content in plant seedlings after short-term stress; The value of SCN ^- metabolism in plant seedlings of each KCN treatment group was set with 0.5 μg as a step unit, and the cumulative amount of SCN ^- in plant seedlings was used as an intermediate condition. When calculating the amount of β-CAS or ST in each KCN treatment group The metabolic amount of KCN is calculated at the maximum value; The value of SCN ^- metabolism in plant seedlings in each KCN+ACC treatment group was set with 0.5 μg as a step unit, and the cumulative amount of SCN ^- in plant seedlings was used as the intermediate condition. The metabolism of CAS or ST to KCN is the maximum value and the calculation is stopped; According to the metabolism of β-CAS or ST to KCN in the KCN+ACC treatment group is always greater than the metabolism of β-CAS or ST to KCN in the KCN treatment group, the range of the contribution rate of plant β-CAS and ST to KCN metabolism was obtained. , to complete the estimation of the contribution rate of plant β-CAS and ST to KCN metabolism. 2. The method for estimating the contribution rate of plant β-CAS and ST to KCN metabolism according to claim 1, wherein step 2 further comprises, according to the determined short-term stress of each treatment group in the plant seedling body The differential expression levels of related genes regulating β-CAS and ST were obtained, and the differential expression levels of related genes regulating β-CAS and ST in plant seedlings in KCN+ACC treatment group were higher than those in KCN treatment group under the same stress concentration. 3. the estimation method of the contribution rate of plant β-CAS and ST to KCN metabolism according to claim 1, is characterized in that, in step 3, the rate of each described treatment group plant seedling to KCN metabolism is short-term stress time The ratio of KCN metabolism in each treatment group to the fresh weight of plant seedlings in each treatment group. 4. The method for estimating the contribution rate of plant β-CAS and ST to KCN metabolism according to claim 1, characterized in that, in step 4, the plant hydroponic total mixed bioreactor that does not contain spatial variables is to KCN The concentration differential equation for metabolism is as follows:

In the formula, C _(t) represents the concentration of KCN in the nutrient solution at t, in μg/mL; β represents the correction coefficient; Q represents the transpiration of plant seedlings, in g/h; C _i represents the concentration of KCN in the nutrient solution. Initial concentration, the unit is μg/mL; k is the decay rate constant of KCN in the plant hydroponic total mixed bioreactor, the unit is h ^-1 ; V is the volume of the plant hydroponic total mixed bioreactor, the unit is mL ; t represents time in h; e is a natural constant. 5. The method for estimating the contribution rate of plant β-CAS and ST to KCN metabolism according to claim 4, wherein the calculation of the correction coefficient is as shown in the following formula:

6. The method for estimating the contribution rate of plant β-CAS and ST to KCN metabolism according to claim 3 or 4, characterized in that, the acquisition of the decay rate constant of the KCN in the plant hydroponic total mixed bioreactor , specifically: taking the initial concentration of KCN in the nutrient solution of each KCN treatment group or each KCN+ACC treatment group as an independent variable, the rate of KCN metabolism by plant seedlings in each KCN treatment group or each KCN+ACC treatment group was linearly fitted. The slope of the fitted curve is the decay rate constant of KCN in the plant hydroponic total mixed bioreactor in the KCN treatment group or the KCN+ACC treatment group. 7. The method for estimating the contribution rate of plant β-CAS and ST to KCN metabolism according to claim 4, wherein the transpiration of the plant seedling is the total amount of the plant hydroponic complete mixed bioreactor per unit stress time. The amount of change in weight. 8. The method for estimating the contribution rate of plant β-CAS and ST to KCN metabolism according to claim 1, wherein in step 6, the metabolic amount of plant seedling ST to KCN in each treatment group is based on the plant seedling The SCN ^- total amount produced in the body is obtained, and the specific steps are: the stoichiometric ratio of the SCN ^- total amount produced in the plant seedling body and the metabolic amount of the plant seedling ST to KCN is 1:1, and the SCN ^- total amount produced in the plant seedling body is first obtained. The molar amount of the amount was obtained as the molar amount of the metabolic amount of plant seedling ST to KCN, and then converted by mass to obtain the metabolic amount of plant seedling ST to KCN in each treatment group. 9. The method for estimating the contribution rate of plant β-CAS and ST to KCN metabolism according to claim 1, wherein in step 6, the contribution rate of described β-CAS and ST to KCN metabolism is specifically: The mass percentage of the amount of KCN metabolized by β-CAS or ST to the amount of KCN metabolized by plant seedlings.

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