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

Abstract

The present invention discloses a method for estimating the contribution rate of plant β-CAS and ST to KCN metabolism, including: short-term stress plant seedlings, the plant seedling hydroponic reaction system of each treatment group is equivalent to a plant hydroponic fully mixed bioreactor, a differential equation for KCN metabolism of the plant hydroponic fully mixed bioreactor is constructed, and then the relationship between the metabolic amount and time of the plant hydroponic fully mixed bioreactor of each treatment group to KCN is obtained, and then the material balance equation for estimating the contribution rate of plant β-CAS and ST to KCN metabolism is obtained, and according to the limiting conditions, the range of the contribution rate of plant β-CAS and ST to KCN metabolism is obtained. The method for estimating the contribution rate of plant β-CAS and ST to KCN metabolism of the present invention avoids the cumbersome time dynamics experiment in the process of plant restoration evaluation, and forms boundary conditions to evaluate the contribution rate of plant β-CAS and ST to KCN metabolism, which is conducive to evaluating the efficiency of plant restoration of KCN.

Description

Technical Field

[0001] The invention belongs to the technical field of plant crop science and relates to a method for estimating the contribution rates of plant beta-CAS and ST to KCN metabolism. Background Art

[0002] Simple cyanide (KCN) has always been the preferred leaching agent in the gold mining process. In addition, it is also an indispensable industrial chemical in the metal finishing and hardening, electroplating, steel and printed circuit board manufacturing industries. According to statistics, more than 100,000 tons of CN are produced from the mining industry each year. - It is transported to the catchment outlet through surface runoff and infiltration, contaminating soil, surface water, and groundwater. Due to its extremely toxic nature, a low-cost and sustainable remediation method is urgently needed to remove or detoxify CN in the environment. - .

[0003] Phytoremediation is a method of using green plants to transfer, contain or transform pollutants, thereby reducing the harm of pollutants to the environment. In the past two decades, a variety of plants have been shown to metabolize exogenous KCN, and only a small amount of residual CN can be detected in plant tissues. - This is mainly due to the contribution of β-cyanoalanine synthase (β-CAS) in plants. β-CAS can convert CN - It is converted into β-cyanoalanine, which can be further converted into asparagine or aspartic acid. In addition, it has been reported that sulfur transferases (ST) in Arabidopsis, sorghum and cassava can also metabolize CN - , but the specific metabolic pathway is still controversial.

[0004] 1-Amino-cyclopropane-1-carboxylic acid (ACC) has been shown to be involved in the biosynthesis of ethylene in plants. During the biosynthesis of ET, amino-cyclopropane-1-carboxylic acid oxidase (ACO) converts 1-amino-cyclopropane-1-carboxylic acid (ACC) into ET and simultaneously produces endogenous CN. - By adding exogenous ACC, the expression of β-CAS and ST in plants stressed by exogenous KCN can be promoted. - metabolism, regarded as an effective chemical regulator.

[0005] In summary, in the field of plant crop science, the contribution rate of β-CAS and ST to the metabolism of exogenous KCN in plants is still unclear. β-CAS and ST are related to the metabolic pathways and products of exogenous KCN in plants, and are directly related to the evaluation of plant 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

[0006] In order to achieve the above-mentioned purpose, the present invention provides a method for estimating the contribution rate of plant β-CAS and ST to KCN metabolism, and the plant seedling hydroponic reaction system is equivalent to a plant hydroponic fully mixed bioreactor, and the KCN metabolic rate curve in the plant hydroponic fully mixed bioreactor is obtained by fitting the concentration of KCN in the nutrient solution of the plant hydroponic fully mixed bioreactor with the stress time, and then the metabolic amount of KCN in the plant hydroponic fully mixed reactor at different times is simulated, thereby avoiding the cumbersome time dynamics experiment in the plant restoration evaluation process, and by comparing with the KCN treatment group without adding ACC, a "boundary condition" is formed to evaluate the contribution rate of plant β-CAS and ST to exogenous KCN metabolism, which is conducive to evaluating the KCN restoration efficiency of plants, and solves the problem of lack of a method for estimating the contribution rate of plant endogenous enzymes to the metabolism of exogenous stressors in the prior art.

[0007] The technical solution adopted by the present invention is a method for estimating the contribution rate of plant β-CAS and ST to KCN metabolism, comprising the following steps:

[0008] Step 1: placing the same number of plant seedlings in multiple nutrient solutions to form multiple treatment groups; adding different amounts of KCN to the nutrient solutions of some treatment groups to form multiple KCN treatment groups; adding a certain amount of ACC to the nutrient solutions of other treatment groups, and then adding different amounts of KCN to form multiple KCN+ACC treatment groups; in each treatment group, KCN was used to short-term stress the plant seedlings for 2 days;

[0009] Step 2: Determine the KCN content of the nutrient solution before and after short-term stress in each treatment group, as well as the CN in the plant seedlings after short-term stress - Content and SCN - content; determine the differential expression levels of genes related to regulating β-CAS and ST in plant seedlings after short-term stress in each treatment group;

[0010] Step 3: Based on the KCN content of the nutrient solution before and after short-term stress in each treatment group, the CN in the plant seedlings after short-term stress - The content was used to obtain the amount of KCN metabolism of the plant seedlings in each treatment group; according to the amount of KCN metabolism of the plant seedlings in each treatment group, the rate of KCN metabolism of the plant seedlings in each treatment group was obtained;

[0011] Step 4: The plant seedling hydroponic reaction system of each treatment group is equivalent to a plant hydroponic fully mixed bioreactor. According to the concentration of KCN in the nutrient solution of each treatment group, the transpiration of the plant seedlings, the decay rate constant of KCN in the plant hydroponic fully mixed bioreactor, and the volume of the plant hydroponic fully mixed bioreactor, a differential equation for KCN metabolism in the plant hydroponic fully mixed bioreactor without spatial variables is constructed;

[0012] Step 5: According to the differential equation of KCN metabolism in the plant hydroponic fully mixed bioreactor of each treatment group, the relationship between the KCN concentration in the nutrient solution of each treatment group and time is obtained, and then the relationship between the KCN metabolism amount and time of the plant hydroponic fully mixed bioreactor of each treatment group is obtained according to the mass conversion; according to the relationship between the KCN metabolism amount and time of the plant hydroponic fully mixed bioreactor of each KCN treatment group and each KCN+ACC treatment group, the material balance equation for estimating the contribution rate of plant β-CAS and ST to KCN metabolism is obtained;

[0013] Step 6: The amount of KCN metabolism in the seedlings of each treatment group is composed of the amount of KCN metabolism by β-CAS and the amount of KCN metabolism by ST; the amount of KCN metabolism by ST in the seedlings of each treatment group is calculated based on the SCN produced in the seedlings. - Total amount obtained; SCN produced in plant seedlings - SCN - Cumulative amount and seedling response to SCN - Metabolism composition; SCN in plant seedlings of each treatment group - The cumulative amount is the SCN in the plant seedlings after short-term stress measured in step 2 - content;

[0014] The SCN sensitivity of plant seedlings in each KCN treatment group was set in 0.5 μg increments. - The value of the metabolic amount is based on the SCN in plant seedlings. - The cumulative amount was the intermediate condition, and the calculation was stopped when the metabolic amount of β-CAS or ST to KCN in each KCN treatment group reached the maximum value;

[0015] The SCN sensitivity of plant seedlings in each KCN+ACC treatment group was set with a step size of 0.5 μg. - The value of the metabolic amount is based on the SCN in plant seedlings. - The cumulative amount was the intermediate condition, and the calculation was stopped when the metabolic amount of β-CAS or ST to KCN in each KCN+ACC treatment group reached the maximum value;

[0016] Based on the fact that the metabolism of KCN by β-CAS or ST in the KCN+ACC treatment group was always greater than that in the KCN treatment group, the range of the contribution rate of plant β-CAS and ST to KCN metabolism was obtained, and the contribution rate of plant β-CAS and ST to KCN metabolism was estimated.

[0017] Furthermore, step 2 also includes, based on the measured differential expression levels of genes related to regulating β-CAS and ST in plant seedlings of each treatment group after short-term stress, obtaining that the differential expression levels of genes related to regulating β-CAS and ST in plant seedlings of the KCN+ACC treatment group under the same stress concentration are higher than those of the KCN treatment group.

[0018] Furthermore, in step 3, the rate of KCN metabolism of the plant seedlings in each treatment group is the ratio of the amount of KCN metabolism of each treatment group during the short-term stress period to the fresh weight of the plant seedlings in each treatment group.

[0019] Furthermore, in step 4, the concentration differential equation of KCN metabolism in the plant hydroponic fully mixed bioreactor without spatial variables is as follows:

[0020]

[0021] In the formula, C (t) represents the concentration of KCN in the nutrient solution at time t, in μg / mL; β represents the correction factor; Q represents the transpiration of the plant seedling, in g / h; C i represents the initial concentration of KCN in the nutrient solution, in μg / mL; k represents the decay rate constant of KCN in the plant hydroponic fully mixed bioreactor, in h -1 ; V represents the volume of the plant hydroponic fully mixed bioreactor, the unit is mL; t represents time, the unit is h; e is a natural constant.

[0022] Furthermore, the calculation of the correction coefficient is shown in the following formula:

[0023]

[0024] Furthermore, the decay rate constant of KCN in the plant hydroponic fully mixed bioreactor is obtained by taking the initial concentration of KCN in the nutrient solution of each KCN treatment group or each KCN+ACC treatment group as the independent variable, performing a linear fit on the rate of KCN metabolism of plant seedlings in each KCN treatment group or each KCN+ACC treatment group, and the slope of the fitting curve is the decay rate constant of KCN in the KCN treatment group or the KCN+ACC treatment group in the plant hydroponic fully mixed bioreactor.

[0025] Furthermore, the transpiration of the plant seedlings is the change in the total weight of the plant hydroponic fully mixed bioreactor per unit stress time.

[0026] Further, in step 6, the amount of KCN metabolized by ST in each treatment group of plant seedlings is determined according to the SCN produced in the plant seedlings. - The total amount is obtained, specifically: SCN produced in plant seedlings -The stoichiometric ratio of total amount to KCN metabolism by ST in plant seedlings is 1:1. SCN produced in plant seedlings - The total molar amount was used to obtain the molar amount of the metabolism of ST to KCN in the plant seedlings. After mass conversion, the metabolism of ST to KCN in the plant seedlings of each treatment group was obtained.

[0027] Furthermore, in step 6, the contribution rate of β-CAS and ST to KCN metabolism is specifically: the mass percentage of the metabolic amount of β-CAS or ST to KCN in the metabolic amount of KCN in the plant seedling.

[0028] The beneficial effects of the present invention are as follows: in the embodiment of the present invention, ACC is selected as a chemical regulator, and a plant toxicology method is used to subject plant seedlings to short-term KCN stress; the plant seedling hydroponic reaction system is equivalent to a plant hydroponic fully mixed bioreactor, and the KCN metabolic rate curve in the plant hydroponic fully mixed bioreactor is obtained by fitting the concentration of KCN in the nutrient solution of the plant hydroponic fully mixed bioreactor with the stress time, and then the metabolic amount of KCN in the plant hydroponic fully mixed reactor at different times is simulated, thereby avoiding cumbersome time kinetic experiments in the process of plant restoration evaluation, and by comparing with a KCN treatment group without adding ACC, a "boundary condition" is formed to evaluate the contribution rate of plant β-CAS and ST to exogenous KCN metabolism, and the method for estimating the contribution rate of plant β-CAS and ST to KCN metabolism in the embodiment of the present invention is conducive to the subsequent evaluation of the plant's KCN restoration efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0030] Figure 1 It is a flow chart of a method for estimating the contribution rate of plant β-CAS and ST to KCN metabolism according to an embodiment of the present invention.

[0031] Figure 2 CN in rice seedlings of each treatment group in the present embodiment - Content results, Figure 2 a in the middle is CN in rice seedlings of each KCN treatment group - content, Figure 2 b shows CN in rice seedlings of each ACC+KCN treatment group - content.

[0032] Figure 3The SCN in the rice seedlings of each treatment group in the embodiment of the present invention is - Content results, Figure 3 a in the middle is the SCN in rice seedlings of each KCN treatment group - The content of Figure 3 Middle b shows the SCN in rice seedlings of each ACC+KCN treatment group - The content.

[0033] Figure 4a It is an analysis diagram of the differential expression levels of β-CAS and ST candidate genes in each KCN treatment group of the examples of the present invention.

[0034] Figure 4b It is an analysis diagram of the differential expression levels of β-CAS and ST candidate genes in each ACC+KCN treatment group of the examples of the present invention.

[0035] Figure 5 It is a schematic diagram of a rice hydroponic fully mixed bioreactor model according to an embodiment of the present invention.

[0036] Figure 6 is a graph of the decay rate constant of KCN in a rice hydroponic fully mixed bioreactor according to an embodiment of the present invention, Figure 6 Figure a is the decay rate constant of KCN in each KCN treatment group in the rice hydroponic fully mixed bioreactor. Figure 6 Middle (b) is the decay rate constant diagram of KCN in each ACC+KCN treatment group in the rice hydroponic fully mixed bioreactor.

[0037] Figure 7 This is a time kinetic characteristic diagram of KCN metabolism in a rice hydroponic fully mixed bioreactor according to an embodiment of the present invention. DETAILED DESCRIPTION

[0038] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] The method for estimating the contribution of plant β-CAS and ST to KCN metabolism is as follows: Figure 1 As shown, the following steps are included:

[0040] 1. Preparation of rice seedlings

[0041] The rice seeds were soaked in water for 12 hours, and then placed in disposable paper cups for sand culture, with a planting density of about 30-40 rice seeds / cup. The culture was carried out in an artificial climate chamber with a temperature of 25°C, a humidity of 65%, and a light intensity of 20,000 lux. The rice was irrigated with a modified version of ISO8692 nutrient solution, as shown in Table 1. After the rice had grown for 16 days, the sand at its roots was cleaned, and rice seedlings of uniform individual size were selected for use.

[0042] Table 1 Improved ISO8692 nutrient solution

[0043] Serial number Reagents concentration Serial number Reagents concentration 1 <![CDATA[KNO3]]> 2823.9μmol / L 7 <![CDATA[H3BO3]]> 2992.1nmol / L 2 <![CDATA[MgCl2·6H2O]]> 59.0μmol / L 8 <![CDATA[MnCl2·4H2O]]> 2097.0nmol / L 3 <![CDATA[CaCl2·2H2O]]> 122.4μmol / L 9 <![CDATA[Na2MoO4·2H2O]]> 28.9nmol / L 4 <![CDATA[MgSO4·7H2O]]> 60.9μmol / L 10 <![CDATA[CuSO4·2H2O]]> 0.1nmol / L 5 <![CDATA[KH2PO4]]> 246.0μmol / L 11 <![CDATA[ZnSO4]]> 22.0nmol / L 6 <![CDATA[NaHCO3]]> 1785.5μmol / L 12 <![CDATA[CoCl2·6H2O]]> 6.3nmol / L

[0044] 2. Experimental design

[0045] Selected rice seedlings were cultured in the following treatment solutions:

[0046] KCN treatment group: Four groups of rice seedlings, with fresh weights of 1.291 g, 1.285 g, 1.244 g, and 1.223 g, were placed in 50 mL conical flasks, and 50 mL of the modified ISO8692 nutrient solution as shown in Table 1 was added, and 0 mg / L, 1.0 mg / L, 2.0 mg / L, and 3.0 mg / L of KCN were added, respectively.

[0047] KCN+ACC treatment group: Four groups of rice seedlings, with fresh weights of 1.293 g, 1.29 g, 1.263 g, and 1.244 g, were placed in 50 mL conical flasks, and 50 mL of the modified ISO8692 nutrient solution as shown in Table 1 was added, and 10 μM ACC was added, and then 0 mg / L, 1.0 mg / L, 2.0 mg / L, and 3.0 mg / L KCN were added, respectively.

[0048] Each conical flask was wrapped in tin foil to avoid light, minimizing water loss and inhibiting algae growth. Each treatment group had 4 biological replicates, and the short-term exposure time was 2 days. All reagents used in this experiment were analytically pure.

[0049] 2.1. KCN content in nutrient solution and total CN in rice seedlings - Analysis of content

[0050] 2.1.1 Determination of KCN content in nutrient solution

[0051] The concentration of KCN in the nutrient solution was measured before and after exposure (2 days). A certain volume of solution sample (1 mL to 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 KH2PO4 and NaH2PO4 was added, and chloramine-T solution (0.2 mL, 1% [m / v]) was quickly added, and the mixture was shaken and placed for 3 min to 5 min, and then the mixture of isonicotinic acid and pyrazolone was added, and the volume was made up to 25 mL with MilliQ water (ultrapure water), mixed, and the colorimetric tube was placed in a water bath (32°C, 40 minutes). Finally, the KCN content in the nutrient solution was measured at 638 nm using a spectrophotometer.

[0052] 5.0 mL of the mixed solution of isonicotinic acid and pyrazolone is prepared by isonicotinic acid (1.5 g of isonicotinic acid is dissolved in 24 mL of 2% NaOH solution and diluted to 100 mL with water): pyrazolone (0.25 g of pyrazolone is dissolved in 20 mL of dimethylformamide solution).

[0053] 2.1.2 Total CN in rice seedlings - Content detection

[0054] Determination of Total CN in Rice Seedlings by Distillation - Content. Add 10 mL of NaOH (1%) solution to the collection bottle at the end of the distillation system. Place the accurately weighed chopped rice seedling root or leaf sample in a round-bottom flask (500 mL), add 200 mL of MilliQ water, then add 10 mL of EDTA (10%) solution and 10 mL of phosphoric acid (85%), start the distillation system, and when the solution in the collection bottle is close to 100 mL, turn off the distillation system and transfer the liquid in the collection bottle to a volumetric flask (100 mL) to be measured. CN in the collection liquid - Refer to 2.1.1 for content determination and analysis. The test results are as follows: Figure 2 shown.

[0055] 2.2 SCN in rice seedlings - Content detection

[0056] After 2 days of exposure, the roots and leaves of the rice seedlings were collected and weighed, and immediately ground into powder in liquid nitrogen. Subsequently, the powdered sample was mixed with 5 mL of citric acid (0.1 M), ultrasonically treated at 25 ° C and 40 kHz for 1 h, centrifuged at 10,000 × g (4 ° C) for 10 min, and the supernatant was collected. This process was repeated twice, and the supernatants of the two times were mixed. Finally, the sample was concentrated using a rotary evaporator (~2.0 mL). After filtering with a 0.45 μm microporous filter membrane, SCN was determined using an ion chromatograph. - Content, test results such as Figure 3shown.

[0057] 2.3 Analysis of differential expression of β-CAS and ST-related genes

[0058] The present invention uses three genes (OsCYS-C1, OsCYS-D1 and OsCYS-D2) that regulate β-CAS. It is reported that AtStr1 and AtStr2 in the Arabidopsis ST family genes are clearly involved in CN - Metabolism, so we screened the genes involved in CN in Arabidopsis from the rice database RGAP (http: / / rice.plantbiology.msu.edu / analyses_search_blast.shtm), the National Rice Data Center (http: / / www.ricedata.cn / gene / index.htm), and RAP-DB (http: / / rapdb.dna.affrc.go.jp / ). - Three genes with high metabolic homology (OsStr9, OsStr23 and OsStr24).

[0059] Real-time quantitative PCR (qRT-PCR) was used to determine the differential expression levels of β-CAS and ST genes, 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 ultramicrospectrophotometer between 1.8 and 2.0. DNase I was used to remove the contamination of gDNA, and then a reverse transcription kit was used for the first-strand synthesis of cDNA. The SYBR green I dye method was used for real-time fluorescence quantitative PCR (qRT-PCR); Primer5.0 was used to design primers for the selected 6 candidate genes, and the expression of genes was analyzed using an AB-7500 quantitative PCR instrument. The differential expression fold was 2 -ΔΔCT Calculation, the results are as follows Figure 4a and Figure 4b shown.

[0060] Table 2 Primer design for β-CAS and ST candidate genes in rice seedlings

[0061]

[0062] 3. Experimental results

[0063] 3.1. KCN content in nutrient solution and CN in rice seedlings - Analysis of content

[0064] At the beginning of the reaction, the KCN concentrations 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 added exogenous KCN was 50 μg, 100 μg, and 150 μg, respectively. After the 2-day exposure, the KCN concentration in the nutrient solution was measured, and the removal rate R of KCN in the nutrient solution was calculated according to formula (1). The specific calculation results are shown in Table 3:

[0065]

[0066] In the formula, C f Indicates the KCN concentration in the nutrient solution at the end of stress, V f Indicates the volume of nutrient solution at the end of stress, C i represents the KCN concentration in the nutrient solution before stress, V i Indicates the volume of nutrient solution before stress.

[0067] Table 3 Removal rate of KCN in nutrient solution

[0068]

[0069] From Table 3, it can be concluded that the KCN removal rate of the KCN+ACC treatment group is higher than that of the KCN treatment group.

[0070] 3.2 CN in rice seedlings of each treatment group - Analysis of the content results

[0071] from Figure 2 As can be seen from the figure, with the increase of KCN content in the nutrient solution, the CN in the roots and leaves of rice seedlings - The concentration increased significantly. Figure 2 As shown in a, CN in roots and leaves of the KCN treatment group - The concentrations of α-hydroxy-1-nitropropene are between 0.64 μg / g and 1.58 μg / g and between 0.38 μg / g and 1.14 μg / g respectively. Figure 2 As shown in middle b, in the KCN+ACC treatment group, CN in roots and leaves - The levels of KCN and ACC in the roots and leaves of rice seedlings were between 0.28 μg / g and 0.36 μg / g, respectively. - The concentration was significantly lower than that in the KCN treatment group, indicating that exogenous addition of ACC can reduce CN in rice seedlings under short-term stress of exogenous KCN. - of residues.

[0072] 3.3 Calculation of KCN metabolism rate in rice seedlings

[0073] According to the material balance of KCN in the rice hydroponic fully mixed bioreactor, the metabolism of KCN by rice seedlings = the absorption of KCN by rice seedlings - the CN in rice seedlings - Therefore, the rate of KCN metabolism in rice seedlings at 48 h can be calculated, as shown in Table 4.

[0074] Table 4 KCN metabolism rate of rice seedlings at 48 hours

[0075]

[0076] In Table 4, the 48h rice seedlings were sensitive to KCN. The unit is μg / g FW·h, where FW represents the fresh weight of rice seedlings (g). As shown in Table 4, exogenous addition of ACC can effectively increase the metabolic rate of KCN in rice seedlings.

[0077] 3.4 SCN in rice seedlings - Analysis of content

[0078] from Figure 3 It can be seen that with the increase of KCN addition, SCN in the roots and leaves of rice seedlings - The concentration of Figure 3 As shown in a, SCN in roots and leaves of the KCN treatment group - The concentration ranges are between 12.21μg / g and 37.69μg / g and 8.71μg / g and 26.69μg / g respectively. Figure 3 As shown in b, SCN in roots and leaves of the KCN+ACC treatment group - The concentrations of SCN in the roots and leaves of rice seedlings treated with KCN+ACC were between 8.65μg / g and 25.87μg / g and 5.98μg / g and 19.23μg / g, respectively. - The concentration was significantly lower than that of the KCN treatment group, indicating that the addition of exogenous ACC can reduce SCN in rice seedling tissues under KCN stress. - accumulation.

[0079] 3.5 Analysis of ST and β-CAS candidate gene expression

[0080] like Figure 4a and Figure 4bAs shown in the figure, 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 upregulated in roots and leaves, among which the differential expression levels of the three candidate ST genes in the KCN+ACC treatment group were significantly higher than those in the KCN treatment group. Similar to the differential expression of ST genes, in the KCN treatment group and the KCN+ACC treatment group, the differential expression levels of the three β-CAS candidate genes in rice seedlings were also upregulated in roots and leaves, among which the differential expression levels of the three β-CAS candidate genes in the KCN+ACC treatment group were significantly higher than those in the KCN treatment group. These results indicate that exogenous addition of ACC can positively increase the differential expression levels of β-CAS and ST genes in rice seedling tissues.

[0081] 4. Evaluation of the contribution of β-CAS and ST to KCN metabolism using a rice hydroponic fully mixed bioreaction model with combined boundary conditions

[0082] The treatment of the present invention is based on a rice hydroponic reaction system, such as Figure 5 As shown, the exogenous KCN added to the growth medium of rice seedlings follows the first-order kinetic absorption characteristics and enters the rice seedlings. Its metabolic process in the rice seedlings also follows the same reaction law. After the exogenous KCN enters the rice hydroponic fully mixed bioreactor, it is evenly mixed in three-dimensional space.

[0083] Rice hydroponic fully mixed bioreactor model assumptions:

[0084] KCN complies with the law of conservation of mass in rice hydroponic fully mixed bioreactor;

[0085] KCN follows first-order kinetic decay characteristics in a rice hydroponic fully mixed bioreactor;

[0086] In rice seedlings, only β-CAS and ST are involved in KCN metabolism and are regulated by related genes.

[0087] 4.1 Model Construction

[0088] Formulate differential equations without spatial variables based on material balance:

[0089]

[0090] Where V is the volume of the rice hydroponic mixed bioreactor, in mL; C is the concentration of KCN in the nutrient solution, in μg / mL; t is the time, in h; Q is the transpiration of the rice seedlings, in g / h; C i Indicates the initial concentration of KCN in the nutrient solution, in μg / mL; C frepresents the concentration of KCN in the nutrient solution at the end of stress, in μg / mL; k represents the decay rate constant of KCN in the rice hydroponic fully mixed bioreactor, in h -1 .

[0091] In the case of non-steady-state decay, According to formula (1), the concentration of KCN in the nutrient solution of the rice hydroponic fully mixed bioreactor at time t is obtained as C (t) , as shown in formula (3):

[0092]

[0093] In order to make the simulation results closer to the actual results, it is necessary to introduce a correction coefficient to correct formula (3), as shown in formula (4):

[0094]

[0095] Among them, β represents the correction coefficient and e is a natural constant.

[0096]

[0097] 4.2 Parameter Definition

[0098] 4.2.1 Calculation of volume of rice hydroponic fully mixed bioreactor

[0099] The volume of the rice hydroponic fully mixed bioreactor V = m / ρ, where m represents the fresh weight of the rice seedlings in each treatment group (g), the density of the rice seedlings ρ = 0.72 g / mL, and the calculation results of the volume of the rice hydroponic fully mixed bioreactor in each treatment group are shown in Table 5.

[0100] Table 5 Volume of rice hydroponic fully mixed bioreactor

[0101] 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

[0102] 4.2.2 Calculation of transpiration of rice seedlings

[0103] The transpiration of rice seedlings is equivalent to the total weight change of the rice hydroponic fully mixed bioreactor within unit stress time. The transpiration of rice seedlings in each treatment group is shown in Table 6.

[0104] Table 6 Transpiration of rice seedlings

[0105] 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

[0106] 4.2.3. Decay rate constant of KCN in rice hydroponic fully mixed bioreactor

[0107] According to Table 4, with KCN stress concentration as the X-axis and the rate of KCN metabolism of 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 fully mixed bioreactor is 0.6141h -1 The decay rate constant of KCN in the rice hydroponic fully mixed bioreactor was 0.7089h. -1 ,like Figure 6 shown.

[0108] 4.3 Result Output

[0109] Substituting the above results into formula (4) respectively, the relationship between the concentration of KCN in the nutrient solution and time in the rice hydroponic fully mixed bioreactor is obtained. Then, according to the mass conversion, the relationship between the metabolism of KCN in the rice hydroponic fully mixed bioreactor and time is obtained, as follows: Figure 7 As shown in the figure, it can be seen that exogenous KCN enters the metabolic plateau phase 12 hours after entering the rice seedlings. Therefore, one advantage of this model is that it simulates the metabolism of KCN in rice hydroponic fully mixed bioreactors at different times, avoiding the tedious time kinetic experiments in the rice restoration evaluation process.

[0110] 4.4 Simulation of the contribution of β-CAS and ST to KCN metabolism in rice seedlings

[0111] The amount of KCN metabolized by rice seedlings = the amount of KCN metabolized by β-CAS + the amount of KCN metabolized by ST. The following material balance equation was obtained based on the mass balance, which was used as the contribution rate model of β-CAS and ST to KCN metabolism in rice seedlings:

[0112]

[0113]

[0114] Where M β-CAS1 、M β-CAS2 、M β-CAS3 Respectively represent the metabolism of KCN by β-CAS in rice seedlings of the three KCN treatment groups, the unit is μg; M ST1 、M ST2 、M ST3 Respectively represent the metabolism of KCN by ST of rice seedlings in three KCN treatment groups, the unit is μg; M Total1 、M Total2 、M Total3 Respectively represent the total metabolism of KCN by β-CAS and ST in the three KCN treatment groups, the unit is μg;

[0115] M' β-CAS1 , M'β-CAS2 , M' β-CAS3 Respectively represent the metabolism of KCN by β-CAS in rice seedlings of the three ACC+KCN treatment groups, the unit is μg; M' ST1 , M' ST2 , M' ST3 Respectively represent the metabolism of KCN by ST in rice seedlings of the three ACC+KCN treatment groups, in μg; M' Total1 , M' Total2 , M' Total3 Respectively represent the total metabolism of KCN by β-CAS and ST in rice seedlings of the three ACC+KCN treatment groups, the unit is μg.

[0116] Formulas (5) to (7) simulate the contribution of β-CAS and ST to KCN metabolism in rice seedlings of each KCN treatment group; Formulas (8) to (10) simulate the contribution of β-CAS and ST to KCN metabolism in each ACC+KCN treatment group.

[0117] like Figure 3 As shown in the figure, 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 β-CAS and ST in rice seedlings to KCN metabolism, it was considered that the metabolism of β-CAS and ST in rice seedlings in the ACC+KCN treatment group was greater than that in the KCN treatment group, that is, M' was met under any treatment. β-CAS >M β-CAS and M' ST >M ST .

[0118] KCN produces SCN via the ST metabolic pathway - (Stoichiometric ratio CN - : SCN - =1:1), and SCN - During the generation process, part of the SCN - It is also metabolized by rice seedlings. Therefore, SCN produced in rice seedlings - Total amount = SCN - Metabolism + SCN - The cumulative amount. Therefore, equations (5) to (10) can be transformed into equations (11) to (16):

[0119]

[0120]

[0121] Among them, X ST1 , X ST2 , X ST3Respectively represent the effects of three KCN treatment groups on the SCN - Metabolism, in μg, X' ST1 , X' ST2 , X' ST3 Represents the effects of SCN on rice seedlings in the three ACC+KCN treatment groups. - The amount of metabolism is in μg.

[0122] Figure 7 It showed that the metabolism of KCN in the rice hydroponic fully mixed bioreactor had reached a stable limit state at around 12 hours, and SCN in rice seedlings - The cumulative amount of SCN no longer continues to increase. - The production and metabolism of ST The value of, such as 0.5, 1, 1.5..., n, the SCN measured at the 48th hour - The cumulative amount of KCN in the KCN treatment group is taken as the intermediate condition. When the calculated KCN metabolism of β-CAS or ST in the KCN treatment group is the maximum, the calculation is stopped. At this time, the M under each step length is obtained. β-CAS and M ST Set X' in steps of 0.5 μg. ST The value of, such as 0.5, 1, 1.5..., n, the SCN measured at the 48th hour - The cumulative amount of is taken as the intermediate condition to obtain M' under each step length β-CAS and M' ST The series value of .

[0123] The contribution of β-CAS and ST to KCN metabolism was evaluated according to the limiting conditions of (17) and (18). The KCN metabolism of β-CAS and ST at 2 h and 12 h was calculated according to these two limiting conditions, as shown in Table 7. The calculation formula is as follows:

[0124]

[0125] The above-mentioned limiting conditions indicate that under different exogenous KCN stress concentrations, the amount of KCN metabolized by β-CAS(ST) in the ACC+KCN treatment group (μg) must be greater than the amount of KCN metabolized by β-CAS(ST) in the KCN treatment group (μg).

[0126] Table 7 Estimated contribution of β-CAS and ST to KCN metabolism in rice seedlings

[0127]

[0128] From Table 7, we can see that: (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, the metabolic contribution rate of β-CAS and ST to KCN is basically similar; (3) As the stress time goes by, the metabolic efficiency of β-CAS to KCN increases while the metabolic efficiency of ST to KCN decreases, which should be due to the sensitivity of ST to KCN. Solving this problem will be beneficial to the subsequent evaluation of rice's KCN repair efficiency.

[0129] 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 any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the presence of other identical elements in the process, method, article or device including the elements.

[0130] Each embodiment in this specification is described in a related manner, and the same or similar parts between the embodiments can be referenced to each other, and each embodiment focuses on the differences from other embodiments.

[0131] The above description is only a preferred embodiment of the present invention and is 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

1. A method for estimating the contribution of plant β-CAS and ST to KCN metabolism, characterized in that: The following steps are involved: Step 1: placing the same number of plant seedlings in multiple nutrient solutions to form multiple treatment groups; adding different amounts of KCN to the nutrient solutions of some treatment groups to form multiple KCN treatment groups; adding a certain amount of ACC to the nutrient solutions of other treatment groups, and then adding different amounts of KCN to form multiple KCN+ACC treatment groups; in each treatment group, KCN was used to short-term stress the 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, as well as the CN in the plant seedlings after short-term stress - Content and SCN - content; determine the differential expression levels of genes related to regulating β-CAS and ST in plant seedlings after short-term stress in each treatment group; Step 3: Based on the KCN content of the nutrient solution before and after short-term stress in each treatment group, the CN in the plant seedlings after short-term stress - The content was used to obtain the amount of KCN metabolism of the plant seedlings in each treatment group; according to the amount of KCN metabolism of the plant seedlings in each treatment group, the rate of KCN metabolism of the plant seedlings in each treatment group was obtained; Step 4: The plant seedling hydroponic reaction system of each treatment group is equivalent to a plant hydroponic fully mixed bioreactor. According to the concentration of KCN in the nutrient solution of each treatment group, the transpiration of the plant seedlings, the decay rate constant of KCN in the plant hydroponic fully mixed bioreactor, and the volume of the plant hydroponic fully mixed bioreactor, a differential equation for KCN metabolism in the plant hydroponic fully mixed bioreactor without spatial variables is constructed; Step 5: According to the differential equation of KCN metabolism in the plant hydroponic fully mixed bioreactor of each treatment group, the relationship between the KCN concentration in the nutrient solution of each treatment group and time is obtained, and then the relationship between the KCN metabolism amount and time of the plant hydroponic fully mixed bioreactor of each treatment group is obtained according to the mass conversion; according to the relationship between the KCN metabolism amount and time of the plant hydroponic fully mixed bioreactor of each KCN treatment group and each KCN+ACC treatment group, the material balance equation for estimating the contribution rate of plant β-CAS and ST to KCN metabolism is obtained; Step 6: The amount of KCN metabolism in the seedlings of each treatment group is composed of the amount of KCN metabolism by β-CAS and the amount of KCN metabolism by ST; the amount of KCN metabolism by ST in the seedlings of each treatment group is calculated based on the SCN produced in the seedlings. - Total amount obtained; SCN produced in plant seedlings - SCN - Cumulative amount and seedling response to SCN - Metabolism composition; SCN in plant seedlings of each treatment group - The cumulative amount is the SCN in the plant seedlings after short-term stress measured in step 2 - content; The SCN sensitivity of plant seedlings in each KCN treatment group was set in 0.5 μg increments. - The value of the metabolic amount is based on the SCN in plant seedlings. - The cumulative amount was the intermediate condition, and the calculation was stopped when the metabolic amount of β-CAS or ST to KCN in each KCN treatment group reached the maximum value; The SCN sensitivity of plant seedlings in each KCN+ACC treatment group was set with a step size of 0.5 μg. - The value of the metabolic amount is based on the SCN in plant seedlings. - The cumulative amount was the intermediate condition, and the calculation was stopped when the metabolic amount of β-CAS or ST to KCN in each KCN+ACC treatment group reached the maximum value; Based on the fact that the metabolism of KCN by β-CAS or ST in the KCN+ACC treatment group was always greater than that in the KCN treatment group, the range of the contribution rate of plant β-CAS and ST to KCN metabolism was obtained, and the contribution rate of plant β-CAS and ST to KCN metabolism was estimated.

2. The method for estimating the contribution rate of plant β-CAS and ST to KCN metabolism according to claim 1, characterized in that: Step 2 also includes, based on the measured differential expression levels of genes related to regulation of β-CAS and ST in the plant seedlings of each treatment group after short-term stress, obtaining that the differential expression levels of genes related to regulation of β-CAS and ST in the plant seedlings of the KCN+ACC treatment group at the same stress concentration are higher than those of the KCN treatment group.

3. 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 3, the rate of KCN metabolism of the plant seedlings in each treatment group is the ratio of the amount of KCN metabolism of each treatment group during the short-term stress period to the fresh weight of the 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 concentration differential equation of KCN metabolism in the plant hydroponic fully mixed bioreactor without spatial variables is as shown below: In the formula, C (t) represents the concentration of KCN in the nutrient solution at time t, in μg / mL; β represents the correction factor; Q represents the transpiration of the plant seedling, in g / h; C i represents the initial concentration of KCN in the nutrient solution, in μg / mL; k represents the decay rate constant of KCN in the plant hydroponic fully mixed bioreactor, in h -1 ; V represents the volume of the plant hydroponic fully mixed bioreactor, the unit is mL; t represents time, the unit is 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, characterized in that: The calculation of the correction coefficient is shown in the following formula:

6. The method for estimating the contribution of plant β-CAS and ST to KCN metabolism according to claim 3 or 4, characterized in that: The attenuation rate constant of KCN in the plant hydroponic fully mixed bioreactor is obtained by taking the initial concentration of KCN in the nutrient solution of each KCN treatment group or each KCN+ACC treatment group as the independent variable, performing a linear fit on the rate of KCN metabolism of the plant seedlings in each KCN treatment group or each KCN+ACC treatment group, and the slope of the fitting curve is the attenuation rate constant of KCN in the KCN treatment group or the KCN+ACC treatment group in the plant hydroponic fully mixed bioreactor.

7. The method for estimating the contribution rate of plant β-CAS and ST to KCN metabolism according to claim 4, characterized in that: The transpiration of the plant seedlings is the change in the total weight of the plant hydroponic fully mixed bioreactor per unit stress time.

8. 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 6, the amount of KCN metabolized by ST in each treatment group of plant seedlings is determined according to the SCN produced in the plant seedlings. - The total amount is obtained by the following steps: SCN produced in plant seedlings - The total amount and the stoichiometric ratio of KCN metabolism by ST in plant seedlings are 1:1, and SCN produced in plant seedlings - The total molar amount was used to obtain the molar amount of the metabolism of ST to KCN in the seedlings, and then the mass conversion was used to obtain the metabolism of ST to KCN in the seedlings of each treatment group.

9. 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 6, the contribution rate of β-CAS and ST to KCN metabolism is specifically: the mass percentage of the metabolic amount of β-CAS or ST to KCN in the metabolic amount of KCN in the plant seedling.

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