Uses of chitosan in reducing carbofuran residues in crops

Abstract

This invention discloses the use of chitosan in reducing carbofuran residues in crops. It involves exogenously applying carboxylated chitosan to crops treated with carbofuran. For hydroponically grown crops, the method of exogenously applying carboxylated chitosan is as follows: dissolve the carboxylated chitosan into a stock solution, then add it directly to the nutrient solution or dilute it to a set concentration and spray it onto the crop leaves. For soil-grown crops, the method of exogenously applying carboxylated chitosan is as follows: dissolve the carboxylated chitosan into a stock solution, dilute it to a set concentration, and then apply it directly to the crop roots or spray it onto the crop leaves. This invention discovers the effect of exogenously applying carboxylated chitosan on promoting the metabolism of carbofuran in maize, and explores the possible mechanism by which carboxylated chitosan promotes the metabolism of carbofuran in maize through transcriptome sequencing analysis, providing a reference method for the regulation of pesticide residues in plants.

Description

Technical Field

[0001] This invention relates to the field of plant pesticide residue technology, specifically to the use of chitosan in reducing carbofuran residues in plants. Background Technology

[0002] The structural formula of carbofuran is shown in formula (1). It is a low-toxicity alternative to the highly toxic pesticide carbofuran, and is an insecticide and nematicide. It acts quickly and efficiently. In my country, it is mainly used for the control of underground pests in grain crops such as corn, rice and wheat through soil treatment and seed treatment. It has now been banned from use on vegetables, fruits, tea, fungi and Chinese medicinal herbs.

[0003] (1).

[0004] Carbofuran is primarily applied in the field via underground application, easily remaining in the soil. It can be absorbed, translocated, and accumulated in edible parts of crops, thus being transferred to humans through the food chain along with pesticide residues in agricultural products, posing a threat to human health. More importantly, carbofuran (LD50 in rats...) 50 (250 mg / kg, the same below) itself has low toxicity, but after application, it is easily metabolized in crops and the environment to form the highly toxic pesticide carbofuran (LD50). 50 =7.8 mg / kg), carbofuran's most readily metabolized product is 3-hydroxycarbofuran (LD50). 50 = 21.9 mg / kg) and 3-ketocarbofuran (LD50) 50 =108 mg / kg), the possible metabolic process is as follows:

[0005]

[0006] Therefore, carbofuran and its metabolites pose certain risks after use, such as toxicity to mammals (Cui et al., 2019), environmental risks (Fegrouche et al., 2014), and dietary risks from agricultural products (Yue et al., 2018; Wang et al., 2020).

[0007] Currently, technologies for removing carbofuran residues from agricultural products and their growth-related samples include physical, chemical, and biological methods. Physical methods typically involve food processing and the use of adsorption or ultrasonic cavitation effects to remove pesticide residues. For example, drying (Li et al., 2020a) and biochar treatment (Tang et al., 2021) have been shown to promote the degradation of carbofuran in plant-based foods and soil to some extent. However, physical methods often involve transferring pesticides from food or the environment to other places rather than eliminating them (Zhang Yajia and Li Zhonghai, 2016). Chemical methods for removing pesticide residues utilize the chemical reactions of pesticides, mainly including photocatalytic degradation and reduction reactions. For example, treatment with a strong oxidant, potassium ferrate, combined with a surfactant for 15-16 min resulted in a 72.59% removal efficiency of carbofuran residue in purple cabbage and lettuce (Wei Xiaoyi et al., 2022). Treatment with 2.0 mg / L ozone water effectively removed carbofuran residue from Chinese cabbage (Wang et al., 2021; Li et al., 2021). Chemical methods have the advantages of broad spectrum, high efficiency, and simple operation, but the reaction process may produce more toxic metabolites, and strong oxidants may destroy the nutritional components of food, adversely affecting human health or production equipment. Biological methods utilize enzymatic reactions and biological metabolic pathways to degrade pesticides under mild conditions, primarily through microbial degradation. For example, white rot fungi... Phlebia sp. and Irpex lacteus Both can effectively promote the degradation of carbofuran, the main metabolite of thiocarbofuran, in soil (Li et al., 2020b). *Pseudomonas putida* strains ( Pseudomonas putida JJ-1, Pichia pastoris ( Pichia pastoris ) strain JJ-4, fluorescent pseudomonocyte strain ( Pseudomonas fluorescens JJ-5 and Penicillium citrinum ( Penicillium citrinum Strains JJ-8 can significantly accelerate the degradation of carbofuran in fermented soybeans (Guo Weidan et al., 2020). The most significant advantage of biological methods is that they are pollution-free and easy to use on a large scale. However, specific strains are only effective against specific pesticide molecules, and microorganisms or enzymes are easily inactivated in vitro, so the effectiveness cannot be guaranteed. Summary of the Invention

[0008] This invention provides a method for reducing carbofuran residues in crops using carboxylated chitosan (CTS). By applying chitosan exogenously, the metabolism of carbofuran in maize is promoted. Transcriptome sequencing analysis is used to propose a possible mechanism by which chitosan promotes the metabolism of carbofuran in maize, providing a reference method for the regulation of pesticide residues in plants.

[0009] This invention discovers that exogenous application of chitosan can promote the metabolism of carbofuran in corn, thereby reducing carbofuran residues in corn. Based on this, this invention proposes the following application:

[0010] This invention provides the use of chitosan in reducing carbofuran residues in crops.

[0011] The carboxylated chitosan has a molecular weight of 1526.5 g / mol and a molecular formula of C2. 56 H 103 N9O 39 .

[0012] The crop is corn, wheat, rice, or peanut; carboxylated chitosan is exogenously applied to the crop treated with thiocarbamate.

[0013] The present invention also provides a method for reducing carbofuran residues in crops, comprising: exogenously applying carboxylated chitosan to crops treated with carbofuran.

[0014] The crop is either hydroponically grown or soil-grown.

[0015] For hydroponically grown crops, the method for exogenously applying carboxylated chitosan is as follows: dissolve the carboxylated chitosan into a stock solution, and then add it directly to the nutrient solution to the set concentration, or dilute the stock solution with water to the set concentration and then spray it on the crop leaves until the leaves are moist.

[0016] For soil-grown crops, the method for applying carboxylated chitosan exogenously is as follows: dissolve the carboxylated chitosan into a stock solution and apply it directly into the soil to the set concentration, or dilute the stock solution with water to the set concentration and then spray it on the crop leaves.

[0017] Preferably, for hydroponically grown crops, the amount of carboxylated chitosan stock solution added is based on a concentration of 1-100 mg / L of carboxylated chitosan in the nutrient solution, or by spraying a carboxylated chitosan aqueous solution diluted to 1-100 mg / L onto the crop leaves; further, the amount of carboxylated chitosan stock solution added is based on a concentration of 1-20 mg / L of carboxylated chitosan in the nutrient solution, or by spraying a carboxylated chitosan aqueous solution diluted to 1-20 mg / L onto the crop leaves; even further, the amount of carboxylated chitosan stock solution added is based on a concentration of 10 mg / L of carboxylated chitosan in the nutrient solution, or by spraying a carboxylated chitosan aqueous solution diluted to 10 mg / L onto the crop leaves.

[0018] Preferably, for soil-grown crops, the chitosan stock solution is directly applied to the soil until the chitosan content in the soil is 1-20 mg / kg, or a carboxylated chitosan aqueous solution diluted to 1-20 mg / L is sprayed onto the crop stems and leaves; further, the chitosan stock solution is directly applied to the soil until the chitosan content in the soil is 5-15 mg / kg, or a carboxylated chitosan aqueous solution diluted to 5-15 mg / L is sprayed onto the crop stems and leaves; even further, the chitosan stock solution is directly applied to the soil until the chitosan content in the soil is 10 mg / kg, or a carboxylated chitosan aqueous solution diluted to 10 mg / L is sprayed onto the crop stems and leaves.

[0019] Optionally, the hydroponic process is as follows: after transferring the crop seedlings to a nutrient solution containing carbofuran for 2-4 days, the roots of the crop are washed and dried, and then transferred to a nutrient solution without carbofuran. Then, carboxylated chitosan is applied exogenously. The concentration of carbofuran in the nutrient solution containing carbofuran is 5-6 mg / kg.

[0020] Optionally, the soil cultivation process is as follows: plant the crop seeds coated with thiocarbamate in the soil and water them once in the morning and once in the evening; when the seedlings grow to the stage of two leaves and one bud, apply carboxylated chitosan exogenously.

[0021] Preferably, the crop is corn, wheat, rice, or peanut; the crop is corn. The hydroponic process is as follows: after transferring the crop seedlings to a nutrient solution containing carbofuran for 3 days, the roots are washed and dried, and then transferred to a nutrient solution without carbofuran; the concentration of carbofuran in the nutrient solution containing carbofuran is 5.7 mg / L.

[0022] Optionally, the hydroponic cultivation conditions are as follows: a photoperiod of 16 h light / 8 h darkness, a temperature of 25 / 20℃ (light / dark), and a light intensity of 250 μmol / m². 2 / s, relative humidity maintained at 70%.

[0023] The soil cultivation conditions are the same as those for hydroponics.

[0024] Compared with the prior art, the present invention has at least one of the following advantages:

[0025] (1) In both hydroponic and soil-based systems, the application of chitosan through root treatment and spraying effectively promoted the metabolism of carbofuran in maize plants of both varieties, thereby reducing residual levels. In the hydroponic system, after 5 days of root treatment with chitosan at concentrations of 1–100 mg / L, the concentration of carbofuran in maize plants was 57.6%–61.1% of that in the control group; after 5 days of spraying treatment with the same concentration of chitosan, the concentration was 47.3%–59.4% of that in the control group; in the soil-based system, after 14 days of root treatment and spraying treatment with chitosan at concentrations of 10 mg / kg, the concentration of carbofuran was 78.3% and 73.9% of that in the control group, respectively.

[0026] (2) The promoting effect of chitosan on the metabolism of thiocarbofuran in maize plants is due to the increase in the activity of peroxidase POD, cytochrome P450 and GST enzyme in plants after chitosan treatment.

[0027] (3) Transcriptome sequencing analysis showed that chitosan promotes the metabolism of carbofuran in maize plants by upregulating the expression of genes encoding peroxidase and activating the antioxidant system. Chitosan promotes the synthesis of secondary metabolites and activates the phenylpropane pathway by upregulating the expression of genes encoding peroxidase POD, cytochrome P450 and GST enzymes, effectively enhancing the carbon, nitrogen and energy metabolism processes of maize plant roots induced by carbofuran, and ultimately promoting the metabolism of carbofuran. Attached Figure Description

[0028] Figure 1 The figure shows the effect of adding different concentrations of CTS directly to maize plants on the total concentration (μmol / kg, FW) of carbofuran.

[0029] Figure 2 The figure shows the effect of different concentrations of CTS spray treatment on the total concentration (μmol / kg, FW) of carbofuran in maize plants.

[0030] Figure 3 Figure showing the effects of soil treatment and spray application of CTS on the total concentration (μmol / kg, FW) of carbofuran in maize plants.

[0031] Figure 4 Net photosynthetic rate of maize leaves after 7 days for different treatments P N (a) and transpiration rate Tr (b) results.

[0032] Figure 5 Figure 1 shows the changes in the activity of POD (a), cytochrome P450 (b), and GST (c) enzymes in maize roots under different treatments.

[0033] Figure 6Volcano plots showing differential genes between CK and CAS, CK and CTS, and CK and CAS+CTS (where blue represents downregulated genes, red represents upregulated genes, and gray represents genes with no significant difference).

[0034] Figure 7 Venn diagram of DEGs for the three treatment groups and CK group.

[0035] Figure 8 Details of the detoxifying enzymes DEGs common or unique to different treatment groups (a) and shared detoxifying enzymes DEGs (b) (where blue indicates downregulated genes and red indicates upregulated genes).

[0036] Figure 9 The graph shows the GO enrichment analysis results of differentially expressed genes in maize roots for CAS (a), CTS (b), and CAS+CTS (c). (The enrichment factor is the ratio of the number of differentially expressed genes enriched in a specific GO entry to the total number of genes annotated in that entry; the higher the value, the greater the enrichment.)

[0037] Figure 10 The KEGG enrichment factor map of DEGs in maize roots of CAS (a) and CAS+CTS (b) is shown. (The enrichment factor refers to the ratio of the number of differentially expressed genes annotated to a specific KEGG metabolic pathway to the total number of genes annotated to that pathway. The higher the value, the higher the enrichment level.)

[0038] Figure 11 The main transcription factors of differentially regulated genes in different treatments relative to the control group are shown (where bars without pattern filling represent upregulated transcription factors, and bars with pattern filling represent downregulated transcription factors). Detailed Implementation

[0039] The reagents and chemicals used in the following examples:

[0040] Carboxylated chitosan (CTS, carboxylation degree ≥ 80%, CAS No.: 9012-76-4), Shanghai Yuanye Biotechnology Co., Ltd.; PBS buffer, Shanghai Sangon Biotech Co., Ltd.; maize seeds (Meiyu 7), Ningbo Jiangdong Luye Vegetable Seed Co., Ltd., for hydroponic experiments; maize seeds (Funongyu 008), Hubei Kangnong Seed Co., Ltd., for soil planting experiments; glutathione S-transferase (GST) activity assay kit, Shanghai Fantai Biotechnology Co., Ltd.; cytochrome P450 enzyme (P450) activity assay kit, Shanghai Fantai Biotechnology Co., Ltd.; glutathione reductase (POD) activity assay kit, Shanghai Fantai Biotechnology Co., Ltd.

[0041] The instruments and equipment used in the following embodiments:

[0042] AMR-100 microplate reader, Hangzhou Aosheng Instrument Co., Ltd.; Portable LI-6400 photosynthesis system, Lincoln Corporation, USA; HH series digital display constant temperature water bath, Jintan Kexi Instrument Co., Ltd.; DEPC water, Shanghai Sangon Biotech Co., Ltd.; Other equipment is conventional experimental equipment.

[0043] Example 1: The promoting effect of carboxylated chitosan on the metabolism of thiocarbamate in nutrient-cultured maize.

[0044] Corn seedlings were transferred to a nutrient solution containing carbofuran (5.7 mg / L) and grown under the following conditions: a photoperiod of 16 h light / 8 h dark, a temperature of 25 / 20℃ (light / dark), a light intensity of 250 μmol / m² / s, and a relative humidity of 70%. After 3 days of exposure, the corn roots were cleaned and dried before being transferred to a nutrient solution without carbofuran. CTS was then applied using the following two methods: (1) hydroponic treatment, in which chitosan stock solution dissolved in pure water was added to the nutrient solution to concentrations of 1, 10, and 100 mg / L; (2) foliar treatment, in which 1, 10, and 100 mg / L chitosan aqueous solution was sprayed onto the leaves until the leaves were moist. A control group (CK) without chitosan was also set up. Plant samples were collected after 0, 1, 3, 5, and 7 days of cultivation to analyze the residues of carbofuran and its metabolites in the roots, stems, and leaves. Each treatment was replicated three times.

[0045] Example 2: The promoting effect of carboxylated chitosan on the metabolism of thiocarbamate in soil-grown maize.

[0046] The soil moisture content was adjusted to 40% of its maximum water holding capacity, mixed thoroughly, and passed through a 2 mm steel sieve. The equivalent of 300 g dry weight of soil was then divided into 300 g plastic flowerpots. Eight coated corn seeds (Funongyu 008) with carbofuran were placed in each flowerpot and cultured in a plant growth chamber (growth conditions: 16 h light / 8 h dark, temperature 25 / 20℃ (light / dark), light intensity set at 250 μmol / m²). 2 (Watering was carried out at 70% relative humidity, with watering twice daily, morning and evening). After all corn seedlings had sprouted, excess seedlings were removed. The remaining 5 seedlings continued to grow until they had two leaves and a central bud. Chitosan stock solution was then directly applied to the soil until the chitosan content in the soil reached 10 mg / kg (based on dry soil), and a 10 mg / L chitosan aqueous solution was sprayed onto the stems and leaves. After 7 and 14 days of continued growth, corn tissues were collected and separated. Butyric acid carbofuran residues in the corn roots, stems, and leaves were extracted and measured. A control group without chitosan application was also included. Each treatment was performed in triplicate.

[0047] Example 3: Possible mechanism by which chitosan promotes the metabolism of thiocarbofuran in maize plants

[0048] The following treatments were performed in a system containing 220 mL of nutrient solution and 3 corn seedlings:

[0049] (1) Nutrient solution blank group (CK): The corn seedlings were transferred to the nutrient solution and the growth conditions were the same as in Example 1.

[0050] (2) Butylcarbofuran treatment group (CAS): The corn seedlings were transferred to a nutrient solution containing butylcarbofuran (5.7 mg / L) to continue growing, with the same growth conditions and cycle as (1).

[0051] (3) Chitosan treatment group (CTS): Maize seedlings were transferred to a nutrient solution containing 10 mg / L chitosan to continue growing, with the same growth conditions as (1).

[0052] (4) Butylcarbofuran + chitosan treatment group (CAS+CTS): The corn seedlings were transferred to a nutrient solution containing butylcarbofuran (5.7 mg / L) and chitosan (10 mg / L) to continue growing, and the growth conditions were the same as (1).

[0053] Samples were collected and multiple indicators were measured 1, 3, 5 and 7 days after the corn grew. For each treatment, each indicator was replicated in three or five (transcriptome sequencing) replicates.

[0054] Specific measurement indicators include plant photosynthetic parameters, antioxidant enzyme activity, and transcriptome sequencing:

[0055] Residue extraction and determination:

[0056] Maize tissues were collected after each of the above experimental treatments. 1 g of root or 1.5 g of stem / leaf was weighed into a 50 mL centrifuge tube, 4 mL of ultrapure water and 5 mL of ethyl acetate were added, and the mixture was homogenized for 2 min using a disperser. After tightening the cap, the tube was placed on a shaker and shaken at the highest speed for 10 min. The mixture was then ultrasonically extracted at 100 Hz for 10 min. 2 g of NaCl and 3 g of anhydrous MgSO4 were added to the centrifuge tube, and the mixture was vortexed for 1 min to mix. The tube was then centrifuged at 8000 r / min for 3 min. 1.5 mL of the supernatant was transferred to a 2 mL centrifuge tube pre-filled with purification reagent (50 mg C18 + 50 mg GCB + 50 mg PSA + 150 mg MgSO4). The tube was vortexed for another 1 min and then centrifuged at 8000 r / min for 3 min. The supernatant was filtered through a 0.22 μm organic filter membrane and analyzed by GC-MS.

[0057] Measurement of photosynthesis in maize plants:

[0058] The photosynthetic parameters of maize leaves were measured using a portable LI-6400 photosynthesis system after 7 days of experimental treatment: net photosynthetic rate. P N (μmol / m 2 / s) and transpiration rate Tr (mmol / m 2 / s). The maximum light saturation intensity of maize was set at 1500 μmol / m. 2 / s, leaf temperature 25 ± 2℃, air humidity 80 ~ 90%, one leaf from each of the three seedlings in each treatment was selected for measurement, with the first fully unfolded leaf closest to the center of the leaf as the target. Three replicates were set up for each treatment.

[0059] GST, P450 and POD enzyme activity assays:

[0060] Following the method of Wan et al. (2017) with some modifications, the following steps were performed: Maize root tissues treated for 1, 3, 5, and 7 days were collected for crude enzyme extraction. Specifically, maize root tissues were mixed with pre-cooled PBS buffer at a ratio of 1:9 in centrifuge tubes, homogenized in an ice-water bath, centrifuged at 8000 r / min for 10 min, and the supernatant was collected. Enzyme activity was determined according to the operating instructions of various enzyme kits.

[0061] Transcriptome sequencing analysis:

[0062] The sequencing samples were maize roots treated for 3 days. Specific steps included:

[0063] (a) Library construction and sequencing

[0064] The sequencing was outsourced to Nanjing Paisennong Gene Technology Co., Ltd., and the main steps included: (1) total RNA extraction and detection; (2) mRNA isolation and fragmentation; (3) double-stranded cDNA synthesis; (4) PCR enrichment of the library; (5) library quality detection; and (6) sequencing.

[0065] (ii) Raw data filtering

[0066] Raw sequencing data (raw reads) were filtered using Cutadapt software to remove reads containing adapters, reads with indeterminate base information, and low-quality reads. The clean data Q20 and Q30 values, sequence error rate, and other metrics were calculated to assess the quality of the clean data.

[0067] (III) Data Analysis and Visualization

[0068] The transcriptome data analysis and visualization workflow is as follows:

[0069] (1) Alignment with the reference genome: Download the reference genome GCF_902167145.1_Zm-B73-REFERENCE-NAM-5.0_genomic.fna from the NCBI database, and use Tophat2 software to align the filtered Reads to the reference genome.

[0070] (2) Quantitative standardization analysis: The Read Count value of each gene was statistically compared using HTSeq and used as the original expression level of the gene. In order to make the gene expression levels comparable between different genes and different samples, FPKM was used to standardize the expression level. Genes with FPKM>1 were considered to be expressible.

[0071] (3) Differential gene expression analysis was performed using DESeq software. The results were based on the fold change |log2FoldChange|>1 and significance. P Differentially expressed genes were screened using a criterion of <0.05.

[0072] (4) Differential gene enrichment analysis: TopGO was used to perform GO enrichment analysis of differentially expressed genes to identify GO terms with significant differential gene enrichment. Clusterprofiler was used to perform KEGG enrichment analysis to identify KEGG pathways with significant differential gene enrichment.

[0073] (5) Data visualization was performed using IBM Spass Statistics 26, combined with analysis of variance and Duncan's test. P The significance of the difference was tested at the <0.05 level. Graphs were plotted using the Pasenuo Gene Cloud Platform (https: / / www.genescloud.cn / home) and Origin 8.5 software.

[0074] Result calculation method:

[0075] The concentrations of carbofuran and its metabolites in maize tissues were calculated using the external standard quantification method. Matrix effects were eliminated through a matrix-matched standard curve. The total concentration of carbofuran in each maize tissue was calculated as the sum of carbofuran and its metabolites, with concentration units of µmol / kg. The concentration conversion formula is as follows:

[0076]

[0077] in,

[0078] C µmol / kg : Compound concentration after unit conversion, unit µmol / kg;

[0079] C mg / kgThe concentration of the compound is obtained directly by quantitative detection, in mg / kg.

[0080] M analyte Molar mass of carbofuran (or its metabolites), in g / mol.

[0081] Subsequently, based on the fresh weight of the tissue, the concentration of carbofuran in maize tissue (µmol / kg, FW) was calculated as follows:

[0082]

[0083] C parent and C metabolite The values ​​represent the concentrations of thiocarbofuran and its metabolites in maize tissues, in µmol / kg, FW.

[0084] The total concentration (µmol / kg, FW) of carbofuran in the whole maize plant was calculated as follows:

[0085]

[0086] Where: m tissue m plant , representing the fresh weight of maize tissue and the whole maize plant, respectively, in g; C tissue and C plant The values ​​represent the concentrations of carbofuran in maize tissues and the whole plant, respectively, in µmol / kg, FW.

[0087] Test results:

[0088] (1) The promoting effect of chitosan on the metabolism of thiocarbamate in maize

[0089] 1) The promoting effect of adding chitosan to hydroponics on the metabolism of butyric acid carbofuran in corn

[0090] The dynamic changes of carbofuran residues in maize plants after CTS treatment in Example 1 are as follows: Figure 1 As shown in the figure, the addition of CTS to the culture medium accelerated the metabolism of carbofuran in maize plants and reduced its residues. After 1 day of treatment with 1, 10, and 100 mg / kg, the total concentration of carbofuran was 88.6%, 72.8%, and 59.1% of the control group, respectively, showing a concentration-dependent effect. After 5 days, the concentrations were 61.1%, 58.0%, and 57.6% of the control group, respectively, at which point there was no significant effect between different concentrations of CTS.

[0091] 2) The effect of chitosan spray treatment on the metabolism-promoting effect of carbofuran in corn.

[0092] In Example 1, similar to adding CTS directly to hydroponics, the total concentration of thiocarbofuran in the corn plants changed after spraying the aboveground parts of the corn plants with CTS equivalent to 1, 10, and 100 mg / kg of CTS, as shown in the figure. Figure 2 As shown, spray treatment with CTS (carbohydrate sulfadiazine) promoted the metabolism of carbohydrate. After 1 day of treatment with 1, 10, and 100 mg / kg, the total concentration of carbohydrate in the plants was 92.9%, 80.1%, and 72.0% of that in the control group, respectively. The promotion of carbohydrate metabolism by CTS increased with increasing concentration; after 5 days, the concentrations were 59.4%, 47.7%, and 47.3% of those in the control group. Treatments with 10 and 100 mg / kg of CTS showed significant effects, but the difference between the two treatments was not statistically significant.

[0093] The above results indicate that in the water-maize system, direct addition of CTS at concentrations of 1, 10, and 100 mg / kg to the nutrient solution and foliar spraying can promote the metabolism of carbofuran and reduce its residues. Considering the effects of adding CTS to the hydroponic solution and foliar spraying on carbofuran residues in maize plants, a soil cultivation experiment was conducted using 10 mg / kg as the CTS treatment concentration.

[0094] 3) The promoting effect of chitosan on the metabolism of butyric acid carbofuran in maize under soil cultivation system

[0095] Based on the fresh weight of the plant, the total concentration of thiocarbofuran and its metabolites in the whole maize plant (calculated as thiocarbofuran) was calculated. The results of Example 2 are as follows: Figure 3 As shown, at 7 days, the total concentrations of CTS in root-treated, sprayed, and control maize plants were 0.29 µmol / kg, 0.21 µmol / kg, and 0.39 µmol / kg, respectively. The residue levels of carbofuran in CTS-treated soil and sprayed plants were 74.4% and 53.9% of the control, respectively. At 14 days, the concentrations were 0.18 µmol / kg, 0.17 µmol / kg, and 0.23 µmol / kg, respectively. The carbofuran levels in CTS-treated root and sprayed plants were 78.3% and 73.9% of the control, respectively. These results indicate that both CTS root and spray treatments can effectively reduce carbofuran residues in maize plants after seed coating treatment.

[0096] (2) Effects of chitosan on maize photosynthesis

[0097] Net photosynthetic rates of different treatment groups in Example 3 P N And the transpiration rate Tr results are as follows Figure 4 As shown, where a is the net photosynthetic rate. P N The processing results, where b represents the transpiration rate Tr. Figure 4The results showed that, compared to the control group, the net photosynthetic rate of maize in the thiocarbamate treatment group, the chitosan treatment group, and the combination of both treatments was significantly higher. P N There was no difference in transpiration rate Tr, and no inhibitory effect was observed on fresh weight, plant height, and root length of maize plants during the experiment, indicating that the experimental concentrations of carbofuran and chitosan did not affect the growth of maize plants.

[0098] (3) Effects of chitosan on enzyme activity in maize plants

[0099] POD enzymes are key enzymes in the plant's antioxidant system. Their activity often increases under stress to scavenge reactive oxygen species damage and maintain homeostasis. Therefore, their activity can indicate the degree of external influence. The POD enzyme activity results of different treatment groups in Example 3 are as follows: Figure 5 As shown in Figure a. Figure 5 As shown in Figure a, compared with the blank control (CK), the addition of carbofuran to the nutrient solution slightly increased the POD enzyme activity in maize roots, indicating that although the tested concentration of carbofuran did not significantly affect maize plant growth, it did exert a certain stress effect on the plants, and the increased POD enzyme activity counteracted this stress effect. At 1 and 5 days after CAS+CTS treatment, POD enzyme activity significantly increased, suggesting that the combined treatment of carbofuran and chitosan may synergistically enhance their activation effect on the maize antioxidant system.

[0100] Cytochrome P450 enzymes are an important enzyme system for phase I pesticide metabolism in plants and play a crucial role in plant responses to pesticide stress. The P450 enzyme activity results for different treatment groups in Example 3 are as follows: Figure 5 As shown in Figure b, compared with the control (CK), P450 enzyme activity significantly increased 3-5 days after CAS addition; after CTS treatment, P450 enzyme activity also significantly increased relative to CK 3-5 days, but there was no significant difference compared with CAS treatment; however, after CAS+CTS combined treatment, P450 enzyme activity significantly increased 1-5 days, and was significantly higher than CAS single treatment, and significantly higher than CTS single treatment at 5 days. This indicates that maize promotes the metabolism of carbofuran by increasing P450 enzyme activity; CAS+CTS further enhances the promotion of carbofuran metabolism by increasing P450 enzyme activity.

[0101] Glutathione S-transferase (GST) exerts its detoxification effect by catalyzing the conjugated reaction between glutathione and pesticides. The glutathione S-transferase (GST) activity results of different treatment groups in Example 3 are as follows: Figure 5As shown in Figure c, compared with the control group, GST enzyme activity significantly increased 5-7 days after CAS treatment; GST enzyme activity significantly increased 3-7 days after CTS treatment, and was higher than that of CAS treatment at 7 days; the changes in GST enzyme activity after CAS+CTS combined treatment were similar to those after CTS treatment. These results indicate that both carbofuran and chitosan treatments significantly induced GST enzyme activity. This phenomenon may be because maize activates GST enzyme activity in order to alleviate the toxic stimulation of carbofuran and maintain cellular homeostasis, thereby accelerating phase II metabolism. CTS has a more significant activation effect on the plant's antioxidant system, which explains why carbofuran is metabolized faster in the presence of chitosan.

[0102] (4) Transcriptome analysis of the regulation of buthicocarbamate metabolism by the plant itself and by chitosan

[0103] 1) Quality control of transcriptome sequencing data and alignment with reference genome

[0104] As set up in Example 3, the groups were divided into CK group, CAS group (butyroxobin treatment), CTS group (chitosan treatment) and CAS+CTS group (butyroxobin and chitosan mixed treatment), with 5 biological replicates in each group to explore the molecular mechanisms that may be involved in butyroxobin metabolism itself and CTS-promoted butyroxobin metabolism. The results are shown in Tables 1 and 2.

[0105] Table 1. Transcriptome data statistics and quality assessment

[0106]

[0107] The percentages of bases with 99% and 99.9% accuracy in base identification in the raw sample data, Q20 and Q30 values, are greater than 97.8% and 94.0%, respectively, and the error rate Raw N% is not higher than 0.002%, indicating that the data quality is qualified and can proceed to the next step of analysis.

[0108] Table 2. Comparison information between sample and reference genomes

[0109] Sample Total Mapped Rate (%) Mapped to Gene Rate (%) CK-1 37494156 88.2 35493668 97.9 CK-2 39095318 86.6 36868381 97.5 CK-3 41820685 87.8 39571469 97.8 CK-4 38033687 86.2 35821355 97.5 CK-5 35260035 88.3 33419403 97.9 CAS-1 38800492 86.6 36604845 97.5 CAS-2 39606736 87.9 37410418 97.5 CAS-3 37182421 88.0 35216922 97.8 CAS-4 38644593 87.3 36199560 97.0 CAS-5 47010806 88.9 44422680 97.7 CAS+CTS-1 39623059 88.7 37255112 97.3 CAS+CTS-2 35357652 88.5 33181863 97.2 CAS+CTS-3 38936439 87.9 36472527 97.0 CAS+CTS-4 43586790 88.4 40975157 97.2 CAS+CTS-5 42058976 88.5 39793412 97.8 CTS-1 42247885 88.4 39892354 97.7 CTS-2 35710572 87.5 33733400 97.6 CTS-3 45208333 87.7 42549349 97.3 CTS-4 45487882 89.1 42927557 97.6 CTS-5 42216366 88.9 39871959 97.6

[0110] Total mapped represents the number of reads aligned to the reference genome, with a proportion >86.2%; Mapped to Gene represents the total number of reads that can be aligned to gene regions, with a proportion >97.2%. The results indicate that the tested species are closely related to the reference genome.

[0111] 2) Differentially expressed gene analysis

[0112] The results of differentially expressed gene analysis under different treatments in Example 3 are as follows: Figures 6-8As shown, by Figure 6 It can be seen that, when compared with the control group CK, each treatment yielded DEGs. The CAS treatment had a total of 1260 DEGs in maize roots, of which 593 were upregulated and 667 were downregulated (CK vs CAS); the CTS treatment had 455 DEGs, of which 197 were upregulated and 258 were downregulated (CK vs CTS); and the combined treatment group of the two produced 1220 DEGs, of which 668 were upregulated and 552 were downregulated (CK vs CAS+CTS).

[0113] Figure 7 The data shows the overlap of DEGs in the three treatment groups. The maize roots of the CAS, CTS, and CAS+CTS treatments contained 679, 287, and 644 DEGs, respectively, representing percentages of 53.9%, 63.1%, and 52.8%. CAS and CTS together contained 60 DEGs, CAS and CAS+CTS together contained 468 DEGs, and CTS and CAS+CTS together contained 55 DEGs. The three groups together contained 53 DEGs. This indicates that maize, in response to different treatments at the gene expression level, exhibits both common and unique DEGs.

[0114] POD is not only an antioxidant enzyme in plants but also a catalytic enzyme in phase I metabolism. Cytochrome P450, GST, and ABC transporters are common catalytic enzymes in the phases I, II, and III metabolism of pesticides in plants, respectively. Therefore, this study aims to identify DEGs containing cytochrome P450, POD, GST, and ABC transporters from the DEGs shared by pesticide treatments alone and those involving chitosan and pesticide mixtures, in order to explore key regulatory genes in the metabolism of carbofuran, such as... Figure 8 As shown.

[0115] like Figure 8 As shown in section a, the DEGs encoded by POD, cytochrome P450, GST, and ABC transporters shared by CAS and CAS+CTS are 10, 10, 4, and 3, respectively. Of the 10 DEGs encoding POD, the number upregulated (2) is less than the number downregulated (8), but POD enzymes are activated in maize roots from both CAS and CAS+CTS. This may be due to functional redundancy of multiple encoding genes. Secondly, cellular regulation of POD may occur at the protein level rather than the transcriptional level, thus causing the difference between protein level detection results and transcriptional level results. Therefore, in the presence of carbofuran, CTS may promote the translation of upregulated POD genes into proteins by enhancing cellular activity in maize roots, and upregulate POD activity at the protein level, participating in phase I metabolism of carbofuran.

[0116] Of the DEGs encoding P450 enzymes, 8 were upregulated and 2 were downregulated, with significant regulation observed in the coding genes. CYP72A5 and CYP81Q32 The upregulation was 4.02 and 4.09-fold in CAS, and 4.64 and 4.99-fold in CAS+CTS. This indicates that carbofuran induces the upregulation of the gene encoding cytochrome P450 in maize roots, leading to enhanced cytochrome P450 enzyme activity at the protein level, which is beneficial for carbofuran metabolism; the addition of CTS accelerates carbofuran metabolism by enhancing this upregulation effect.

[0117] All four common DEGs encoding GST enzymes were upregulated in CAS and CAS+CTS. GST8 The upregulation folds were as high as 23.30 and 39.91, indicating that maize roots may upregulate related coding genes, especially... GST8, It activates GST enzyme activity, catalyzing the conjugated reaction between glutathione and carbofuran, thereby promoting their metabolism.

[0118] All three common DEGs encoding ABC transporters were upregulated, with G family member 48 and B family member 21 being upregulated by 8.91 and 5.82, and 16.67 and 14.23-fold, respectively. In this study, ABC transporters ABCB21, ABCG39, and ABCG48 were significantly upregulated, indicating that maize roots activate ABC transporters by upregulating ABC-related encoding genes, accelerating the efflux of intracellular carbofuran, thereby alleviating the stress caused by the pesticide carbofuran. Furthermore, the upregulation folds of the aforementioned ABC transporter encoding genes in the CAS group were greater than those in the CAS+CTS group. This may be because the combined treatment of CTS and CAS exhibits a slight antagonistic effect on the activation of ABC transporters. Therefore, the process of accelerated carbofuran efflux and detoxification in maize roots is mainly induced by the pesticide itself.

[0119] In conclusion, chitosan may promote the metabolism of carbofuran by enhancing the upregulation of genes encoding POD, cytochrome P450, and GST enzymes in maize roots.

[0120] 3) GO enrichment analysis of differentially expressed genes

[0121] GO functional annotation and enrichment analysis of the selected DEGs revealed that 1260 DEGs in the CAS group were enriched into 868 GO entries, among which significant enrichment was found in (…). P <0.05 (the same below) 135; 455 DEGs processed by CTS were enriched in 421 GO items, with 48 significantly enriched; the expression of 1220 DEGs in the CAS+CTS group was enriched in 843 GO items, with 103 significantly enriched. The top 10 dominant items were selected for functional classification statistics, see Figure 9 .

[0122] The results of differentially expressed gene analysis under different treatments in Example 3 are as follows: Figure 9 As shown, Figure 9 As shown, the GO items significantly enriched in DEGs fall into three main categories: biological process (BP), molecular function (MF), and cellular component (CC). The GO item shared by all three treatments is redox processes within the BP category, indicating that both the exogenous substances thiocarbofuran and chitosan activated maize's own antioxidant system. The application of chitosan may promote thiocarbofuran metabolism by enhancing the induction of the antioxidant system. Furthermore, CAS and CAS+CTS also share GO items including metabolic processes (under the BP category, the same below), catalytic activity (MF category), and intrinsic membrane components (CC category), suggesting that the metabolism of thiocarbofuran and chitosan-promoted within the plant is achieved by enhancing catalytic enzyme activity.

[0123] Several processing groups' DEGs also have unique GO entries, such as Figure 9 In group a, the CAS group's DEGs significantly enriched GO entries included carbohydrate metabolism (18 DEGs), polysaccharide metabolism (13 DEGs), and drug catabolism (13 DEGs) under the BP category; and transporter activity (27 DEGs), oxidoreductase activity (20 DEGs), and transferase activity, as well as transfer of alkyl (excluding methyl) or aryl groups (16 DEGs) under the MF category. Figure 9 In the CTS group, DEGs-specific GO entries include methylation (5 DEGs), manganese ion transport (3 DEGs), manganese ion transmembrane transport (3 DEGs), and transition metal ion transport (3 DEGs) under the BP category; isomerase activity (5 DEGs), methyltransferase activity (5 DEGs), O-methyltransferase activity (5 DEGs), and single-carbon group transferase activity (5 DEGs) under the MF category; and extracellular regions (6 DEGs) under the CC category. Figure 9In the c group, the GO entries specific to the CAS+CTS group are catabolism processes (26 DEGs) and organic matter catabolism processes (25 DEGs) under the BP category; MF includes catalytic activity (151 DEGs) and hydrolytic enzyme activity (57 DEGs); the CC entries specifically involve membrane structure (149 DEGs), membrane components (117 DEGs), intrinsic membrane components (112 DEGs), and overall membrane components (109 DEGs). This indicates that different treatments produce differential stimuli to plants: CAS affects plant carbon metabolism and drug catabolism by regulating the activity of oxidoreductases and transferases; CTS mainly enhances root uptake of the micronutrient manganese by increasing transferase activity, thereby promoting growth and enhancing photosynthesis. Furthermore, hydrolytic enzymes in plants play a catalytic role in the phase I metabolism of pesticides. The hydrolytic enzyme activity in maize under the CAS+CTS group was regulated, with 20 upregulated DEGs and 37 downregulated DEGs enriched in this entry. While the number of upregulated DEGs was less than the number of downregulated DEGs, among the DEGs with significant regulation (>3.5-fold), 7 were upregulated and 5 were downregulated. Therefore, it is speculated that the hydrolytic enzymes may be activated by the combined treatment of carbofuran and chitosan. This indicates that chitosan may also accelerate the metabolism of carbofuran in maize by activating hydrolytic enzymes in the presence of carbofuran.

[0124] 4) KEGG enrichment analysis of differentially expressed genes

[0125] By comparing DEGs with the KEGG public database, pathway annotation and enrichment analysis were performed to clarify the metabolic pathways regulated by each treatment. DEGs in maize roots of the CAS group were annotated into 89 pathways, showing significant enrichment. P <0.05 (the same below) 13 pathways; DEGs in the CTS group were annotated into 36 pathways, with 3 significantly enriched; DEGs in the CAS+CTS group were annotated into 88 pathways, with 18 significantly enriched. An enrichment factor plot was created for the top 10 significantly enriched pathways in the CAS and CAS+CTS DEGs of Example 3, see [link to enrichment factor plot]. Figure 10 .

[0126] like Figure 10 In group a, based on the number of enriched DEGs, the main metabolic pathways in the CAS group are phenylpropane biosynthesis, plant hormone signal transduction, and cysteine ​​and methionine metabolism; considering the enrichment factors, the biosynthesis of various secondary metabolites, zeatin biosynthesis, and starch and sucrose metabolism are also major pathways.

[0127] The three metabolic pathways significantly enriched by CTS were the biosynthesis of sesquiterpenes and triterpenes, the biosynthesis of flavonoids, and the biosynthesis of zeatin, indicating that chitosan mainly activates the antioxidant system by regulating the secondary metabolites of maize plants and activating plant hormone signal transduction.

[0128] like Figure 10 In section b, in terms of the number of enriched DEGs, the most important pathways regulated by the CAS+CTS group are phenylpropane biosynthesis, photosynthesis, and glycolysis / glycogen synthesis. In terms of enrichment factors, in addition to the above three pathways, photosynthesis-antenna protein and diterpene biosynthesis are also important.

[0129] The CAS and CAS+CTS groups shared five significantly enriched metabolic pathways: phenylpropane biosynthesis, cysteine ​​and methionine metabolism, carbon fixation in photosynthetic organisms, pyruvate metabolism, and the MAPK signaling pathway in plants. Twelve common DEGs were involved in the phenylpropane biosynthesis pathway, with four upregulated and eight downregulated. For upregulated genes, the fold change in upregulation was greater in the CAS+CTS group than in the CAS treatment; for example, LOC103654125 was upregulated 8.48-fold in the CAS+CTS group but only 5.68-fold in the CAS group. The fold change in downregulation was less significant. Therefore, chitosan may promote thiocarbamate metabolism by enhancing the activation of the phenylpropane biosynthesis pathway. Three genes involved in cysteine ​​and methionine metabolism, LOC100274974, were upregulated in both the CAS and CAS+CTS groups. si603034f07 The presence of LOC103648465 (malate dehydrogenase, cytoplasm) and LOC103633793 (malate dehydrogenase, cytoplasm) suggests that the metabolism of carbofuran may be related to the accelerated phase II metabolism of the hormones ethylene and polyamines in the body. Pyruvate metabolism is related to respiratory metabolism; both treatment groups showed a total of 4 upregulated DEGs and 1 downregulated DEG, indicating that activation of energy metabolism also accelerates carbofuran metabolism. Three DEGs involved in the MAPK signaling pathway were found in both treatment groups, all of which were upregulated, specifically LOC100274400 (protein phosphatase). PP2C6 ) and LOC100279578 (protein phosphatase) PP2C68 The results showed that the pesticide thiocarbofuran and chitosan-regulated thiocarbofuran activated the MAPK signaling pathway to transmit stress signals.

[0130] In summary, the pesticide carbofuran (CTS) caused certain stress to maize plants. The stress resistance processes regulated by maize itself and by chitosan may involve promoting the synthesis of plant hormones ethylene and polyamines, thereby accelerating phase II metabolism. This is achieved by activating the MAPK pathway to transmit stress signals, enhancing carbon, nitrogen, and energy metabolism, inducing the phenylpropane pathway, and ultimately promoting the synthesis of secondary metabolites and accelerating carbofuran metabolism. The promoting effect of CTS on carbofuran metabolism is achieved by regulating photosynthesis and the biosynthesis of secondary metabolites, specifically by enhancing the photosynthesis-antenna protein pathway, carbon fixation by photosynthetic organisms, and the biosynthesis of secondary metabolites.

[0131] 5) Analysis of differentially expressed transcription factors in maize induced by chitosan

[0132] Transcription factors (TFs) are protein molecules that specifically bind to the upstream nucleotide sequences of target genes, regulating their transcription process. They are widely involved in plant growth and development and in responding to abiotic stresses. In the CKvs CAS study, 42 transcription factor families were involved, with six families containing more than 10 regulatory gene sets: bHLH, ERF, NAC, MYB-related, B3, and C2H2 families. Detailed information is shown in Table 3. Figure 11In the ERF, NAC, and MYB-related families, the number of upregulated DEG genes was 17, 12, and 11, respectively, which was more than the number of downregulated genes, which was 7, 10, and 9, respectively, indicating that upregulation was the main trend. In the bHLH, B3, and C2H2 families, the number of upregulated transcription factors was less than the number of downregulated transcription factors, with 12, 2, and 4 upregulated transcription factors and 21, 13, and 8 downregulated transcription factors, respectively, indicating that downregulation was the main trend. Among the bHLH family transcription factors, LOC103640286, LOC103639850, LOC542430, LOC100282060, LOC103647798, LOC100282518, and LOC100383548 showed the largest fold changes, ranging from 6.16 to 21.93, all of which were downregulated. Among the ERF family transcription factors, LOC103646120, LOC103654125, LOC100285530, LOC100272603, LOC103630419, LOC103633819, LOC118473014, and LOC103642141 showed the largest fold changes, with upregulation ranging from 4.21 to 21.93. The fold change in upregulation was 28.34, while the fold change in downregulation of transcription factors was less than 3.44, showing a relatively gradual increase, with upregulation being the dominant trend. The fold change in upregulation of NAC family transcription factors ranged from 2.09 to 10.90, showing significant variation; while the fold change in downregulation of transcription factors ranged from 2.05 to 4.96. In the MYB-related family, transcription factor LOC103655842 showed a fold change of 17.40, indicating significant upregulation. In the C2H2 family, LOC103637541 showed a fold change of 14.26, indicating significant downregulation.

[0133] In the CK vs CTS comparison (Table 4), CTS induced responses from 32 transcription factor families. The top five families in terms of the number of transcription factors included ERF, MYB-related, bHLH, NAC, and G2-like families, with a relatively small number of DEGs (determinants of genes involved) ranging from 3 to 5. Specifically, LOC100191813 of the ERF family was downregulated by 25.19-fold; LOC103632512 of the MYB-related family was upregulated by 21.17-fold; and LOC103650965 of the bHLH family was upregulated by 8.59-fold, all showing predominantly upregulation. Overall, in terms of the number of genes involved in regulating transcription factor families and the fold change, chitosan's induction effect was weaker than that of thiocarbofuran itself, consistent with the number of DEGs generated in both treatments.

[0134] In CK vs CAS+CTS (Table 5), a total of 44 transcription factor families were involved in regulation, of which 6 families had at least 10 transcription factors. Figure 11Specifically, the ERF, bHLH, MYB-related, MYB, NAC, and bZIP families were involved. Except for the bHLH family, where upregulated genes were fewer than downregulated genes, the other families were predominantly upregulated. Transcription factors with significant fold changes included: LOC103654125, LOC103636509, LOC103646120, LOC100274453, LOC542530, LOC100285530, and LOC103630419 of the ERF family, all of which were upregulated, with fold changes ranging from 4.65 to 14.41. In the bHLH family, LOC103650965 showed the largest fold change (13.75-fold), while LOC103641669 showed the largest fold change (4.54-fold). The MYB-related family showed relatively small changes, with the largest change observed in LOC103639349, which was downregulated by 4.75-fold. Within the MYB family, LOC103635916 was upregulated by 7.63-fold; within the NAC family, LOC100273182 was downregulated by 42.88-fold; and within the bZIP family, LOC103641119 was downregulated by 5.29-fold. These results indicate that, compared to CAS alone, chitosan application induced the involvement of more transcription factor families in regulating thiocarbofuran metabolism in response to stress.

[0135] The transcription factor families involved in both CAS and CAS+CTS treatments include bHLH, ERF, NAC, and MYB-related families. Compared with the CAS treatment, the upregulation effect of bHLH family transcription factors was significantly enhanced in the CAS+CTS group, specifically, the number of upregulated transcription factors increased, and the upregulation fold increased, ranging from 2.08 to 13.79. The number of upregulated ERF family transcription factors increased slightly from 17 to 18, with upregulation being the predominant factor in both groups. Both upregulation and upregulation of NAC and MYB-related family transcription factors were observed. It is speculated that in maize, CTS activates the signaling molecule ethylene to transmit the stress signal of thiocarbamate, bHLH promotes the synthesis of plant secondary metabolites such as flavonoids to promote thiocarbamate metabolism, and the NAC and MYB-related families may also be involved in the regulation of stress response.

[0136] Table 3. Transcription factor analysis of DEGs between the CK and CAS groups.

[0137] Gene ID Family Regulation Ratio of relative express level LOC100272601 bHLH Up 4.60 LOC100272761 bHLH Up 4.02 LOC542583 bHLH Up 2.61 LOC103641578 bHLH Down 3.98 LOC100282568 bHLH Down 2.41 LOC103650720 bHLH Down 2.38 LOC100275070 bHLH Up 2.50 LOC100501753 bHLH Up 2.92 LOC100282769 bHLH Up 2.06 LOC103628824 bHLH Down 4.80 LOC103640533 bHLH Down 2.71 LOC100272470 bHLH Down 4.09 LOC100286016 bHLH Up 2.35 LOC103651469 bHLH Up 2.37 LOC103643530 bHLH Down 2.27 LOC103640286 bHLH Down 19.62 LOC103639850 bHLH Down 19.71 LOC103652863 bHLH Down 5.79 LOC542430 bHLH Down 15.40 LOC103652574 bHLH Up 4.31 LOC103643529 bHLH Down 2.23 LOC103637976 bHLH Down 2.08 LOC100282060 bHLH Down 21.93 LOC103636104 bHLH Up 3.96 LOC103647798 bHLH Down 8.61 LOC103636714 bHLH Up 3.99 LOC100194106 bHLH Down 2.22 LOC100282518 bHLH Down 11.50 LOC103636726 bHLH Down 4.00 LOC103641360 bHLH Down 2.29 LOC100282766 bHLH Down - LOC100383548 bHLH Down 6.16 LOC103638616 bHLH Up 2.44 LOC103646120 ERF Up 28.34 LOC103654125 ERF Up 5.68 LOC100281688 ERF Up 2.16 LOC100285530 ERF Up 11.32 LOC103648032 ERF Down 2.52 LOC100383654 ERF Down 3.43 LOC100272603 ERF Up 4.21 LOC103644933 ERF Down 2.45 LOC109943658 ERF Up 2.21 LOC100286151 ERF Up 2.06 LOC100191919 ERF Up 3.29 LOC103627306 ERF Down 2.43 LOC103630419 ERF Up 6.21 LOC103654275 ERF Down 2.06 LOC103633819 ERF Up 4.71 LOC100281871 ERF Up 3.72 LOC100285811 ERF Down 3.44 LOC100193807 ERF Up 2.38 LOC100274079 ERF Up 2.29 LOC103639329 ERF Down 2.64 LOC118473014 ERF Up 17.98 LOC103642141 ERF Up 4.82 LOC100383492 ERF Up - LOC100280929 ERF Up 2.89 LOC103630472 NAC Up 6.51 LOC100274400 NAC Up 8.11 opr3 NAC Up 2.55 LOC103630400 NAC Up 7.01 LOC100280285 NAC Down 2.27 LOC103630022 NAC Down 3.39 LOC100275381 NAC Up 2.50 LOC103637346 NAC Up 2.39 LOC103625904 NAC Down 2.05 LOC100502408 NAC Up 2.09 LOC100273182 NAC Down 4.96 LOC100382216 NAC Down 2.48 opr4 NAC Up 3.22 LOC103633507 NAC Down 3.92 LOC100280927 NAC Down 2.22 LOC100277691 NAC Down 2.07 LOC100281942 NAC Down 2.35 LOC103637828 NAC Up 10.90 LOC100285546 NAC Down 2.28 LOC100283318 NAC Up 2.07 LOC103634518 NAC Up 2.44 LOC107275227 NAC Up 2.11 LOC103635032 MYB-related Up 8.91 LOC542191 MYB-related Down 2.42 LOC103638767 MYB-related Down 4.78 LOC103654816 MYB-related Up 3.85 LOC103626138 MYB-related Down 2.71 LOC103655842 MYB-related Up 17.40 LOC100276850 MYB-related Down 2.35 LOC100275329 MYB-related Up 2.29 LOC100283600 MYB-related Down 2.49 LOC103631028 MYB-related Up 2.48 LOC100285887 MYB-related Up 2.13 LOC100384044 MYB-related Up 2.57 LOC542233 MYB-related Up 3.16 LOC103633290 MYB-related Down 2.12 LOC103640852 MYB-related Down 3.37 LOC100282656 MYB-related Down 2.01 LOC103636453 MYB-related Up 2.04 LOC103635691 MYB-related Down 5.20 LOC100192937 MYB-related Up 4.33 LOC103647818 MYB-related Up 2.61 LOC542553 B3 Up 4.55 LOC100193148 B3 Down 3.21 LOC100280644 B3 Down 3.75 LOC542392 B3 Up 3.23 LOC100381520 B3 Down 6.78 LOC103629514 B3 Down 2.59 LOC100280053 B3 Down 2.21 LOC103653610 B3 Down 2.01 LOC100279348 B3 Down 2.06 LOC100382832 B3 Down 2.09 LOC100280719 B3 Down 2.18 LOC103627780 B3 Down 3.05 LOC103646471 B3 Down 2.71 LOC100192589 B3 Down 2.02 LOC103641629 B3 Down 2.12 LOC778439 C2H2 Down 2.37 LOC100281379 C2H2 Up 2.16 LOC100283770 C2H2 Down 2.21 LOC109943726 C2H2 Up 2.80 LOC100282331 C2H2 Down 4.50 LOC100280366 C2H2 Down 4.43 LOC103639076 C2H2 Up 4.32 LOC103633266 C2H2 Down 2.24 LOC103637541 C2H2 Down 14.26 LOC103643204 C2H2 Down 3.77 LOC100272665 C2H2 Down 4.61 LOC103636710 C2H2 Up 2.30

[0138] "-" indicates that the transcription factor is expressed only in the treatment group, so the fold difference with the CK group cannot be calculated, and the same applies below.

[0139] Table 4. Transcription factor analysis of DEGs between the CK and CTS groups.

[0140] Gene ID Family Regulation Ratio of relative express level LOC103651265 ERF Down 2.21 LOC103644933 ERF Down 2.12 LOC100191813 ERF Down 25.19 LOC100384725 ERF Down 4.05 LOC103637985 ERF Down 3.14 LOC100279070 MYB-related Down 3.81 LOC100276850 MYB-related Down 3.25 LOC100273616 MYB-related Down 2.09 LOC103632512 MYB-related Up 21.17 LOC103630399 MYB-related Down 3.71 LOC103639876 bHLH Up 3.23 LOC100381902 bHLH Up 2.98 LOC541904 bHLH Down 2.01 LOC103650965 bHLH Up 8.59 LOC100275381 NAC Up 2.20 LOC118473473 NAC Up 3.18 LOC107275227 NAC Up 2.47 LOC100382373 NAC Down 2.29 LOC103635802 G2-like Up 2.14 LOC100280967 G2-like Down 2.75 LOC109945291 G2-like Down 3.27

[0141] Table 5. Transcription factor analysis of DEGs between the CK group and the CAS+CTS group.

[0142] Gene ID TF Family Regulation Ratio of relative express level LOC103654125 ERF Up 8.48 LOC100281688 ERF Up 2.28 LOC100274079 ERF Up 2.90 LOC100280929 ERF Up 3.44 LOC103644933 ERF Down 3.58 LOC100285687 ERF Up 2.30 LOC103636509 ERF Up 6.66 LOC103646120 ERF Up 14.41 LOC542716 ERF Up 2.85 LOC103648032 ERF Down 2.02 LOC103650960 ERF Up 2.11 LOC100192969 ERF Down 2.03 LOC103649810 ERF Up 2.39 LOC100275504 ERF Up 2.68 LOC103627306 ERF Down 2.17 LOC100274453 ERF Up 7.30 LOC542530 ERF Up 5.40 LOC100272603 ERF Up 2.23 LOC100191919 ERF Up 2.68 LOC100285530 ERF Up 5.23 LOC100384381 ERF Down 2.58 LOC100273029 ERF Up 2.70 LOC103630419 ERF Up 4.65 LOC542583 bHLH Up 2.48 LOC100272761 bHLH Up 4.99 LOC100193144 bHLH Up 2.42 LOC103644325 bHLH Up 2.08 LOC100275070 bHLH Up 2.47 LOC103651368 bHLH Up 2.56 LOC100501753 bHLH Up 3.19 LOC103640533 bHLH Down 2.22 LOC100193902 bHLH Up 2.36 LOC103632970 bHLH Up 2.36 LOC541904 bHLH Down 2.30 LOC100381462 bHLH Up 3.06 LOC103630140 bHLH Up 2.69 LOC103633430 bHLH Up 2.49 LOC103633023 bHLH Up 4.16 LOC103635561 bHLH Down 3.16 LOC103650965 bHLH Up 13.75 LOC103651469 bHLH Up 2.73 LOC103652574 bHLH Up 2.40 LOC103641669 bHLH Down 4.54 LOC100276850 MYB-related Down 4.18 LOC103626138 MYB-related Down 3.52 LOC100384044 MYB-related Up 2.78 LOC100284598 MYB-related Up 2.95 LOC103631028 MYB-related Up 2.82 LOC103653395 MYB-related Down 2.30 LOC100282326 MYB-related Down 2.05 LOC542233 MYB-related Up 3.26 LOC100282656 MYB-related Down 2.05 LOC103633290 MYB-related Down 2.27 LOC103647750 MYB-related Down 2.16 LOC103640852 MYB-related Down 3.20 LOC103635032 MYB-related Up 5.82 LOC103655842 MYB-related Up 8.05 LOC103636453 MYB-related Up 2.52 LOC103654816 MYB-related Up 2.18 LOC103639349 MYB-related Down 4.75 LOC100194067 MYB-related Up 2.12 LOC100284932 MYB Down 2.09 LOC103629377 MYB Down 2.00 LOC100281063 MYB Up 5.70 LOC100274036 MYB Up 2.01 LOC100283222 MYB Down 2.05 LOC103637431 MYB Down 4.60 LOC100272381 MYB Up 2.10 LOC103635916 MYB Up 7.63 LOC100284755 MYB Down 2.09 LOC100285488 MYB Up 2.38 LOC109943068 MYB Down 2.20 LOC100284600 MYB Up 3.71 LOC100192609 MYB Up 2.68 LOC103634611 MYB Up 2.17 LOC100191533 MYB Up 2.30 LOC101027155 NAC Up 2.41 LOC100273092 NAC Up 2.05 LOC100273182 NAC Down 42.88 LOC100216881 NAC Down 2.08 opr3 NAC Up 3.31 LOC100275381 NAC Up 2.54 LOC100283318 NAC Up 2.42 LOC100274400 NAC Up 4.19 LOC100280285 NAC Down 2.11 LOC100281950 NAC Down 2.66 LOC103630472 NAC Up 4.77 LOC100285546 NAC Down 2.50 LOC103630022 NAC Down 2.21 LOC542154 NAC Up 2.44 LOC103630400 NAC Up 3.79 LOC100383828 bZIP Up 2.20 LOC100286113 bZIP Down 3.73 LOC103627791 bZIP Down 3.77 LOC100285656 bZIP Down 2.78 LOC103641119 bZIP Down 5.29 LOC100286138 bZIP Down 2.93 LOC100502540 bZIP Up 2.66 LOC100284301 bZIP Down 2.28 LOC100285149 bZIP Up 2.96 LOC100277204 bZIP Up 2.01

[0143] In summary, carbofuran exerts a certain stress on the surface of maize cells. This stress signal is amplified by chitosan, and the signaling molecule ethylene is activated and transduced to the cell through the MAPK pathway. Maize activates the phenylpropane pathway to synthesize secondary metabolites such as flavonoids by enhancing carbon metabolism, nitrogen metabolism and energy metabolism, and activates antioxidant enzyme POD and detoxification enzymes cytochrome P450 and GST, thereby promoting the metabolic response of carbofuran to stress.

[0144] summary:

[0145] This invention investigated the effects of chitosan at different concentrations on the metabolism-promoting effect of carbofuran in maize plants through root treatment and spraying. The possible molecular mechanisms by which chitosan promotes carbofuran metabolism in maize were explored through enzyme activity assays and transcriptome sequencing analysis. The conclusions are as follows:

[0146] (1) In both hydroponic and soil-based systems, the application of chitosan through root treatment and spraying effectively promoted the metabolism of carbofuran in maize plants of both varieties, thereby reducing residual levels. In the hydroponic system, after 5 days of root treatment with chitosan at doses of 1–100 mg / kg, the concentration of carbofuran in maize plants was 57.6%–61.1% of that in the control group; after 5 days of spraying with the same dose of chitosan, the concentration was 47.3%–59.4% of that in the control group; in the soil-based system, after 14 days of root treatment and spraying with chitosan at doses of 10 mg / kg, the concentration of carbofuran was 78.3% and 73.9% of that in the control group, respectively.

[0147] (2) The promoting effect of chitosan on the metabolism of thiocarbofuran in maize plants is due to the increase in the activity of peroxidase POD, cytochrome P450 and GST enzyme in plants after chitosan treatment.

[0148] (3) Transcriptome sequencing analysis showed that chitosan promotes the metabolism of carbofuran in maize plants by upregulating the expression of genes encoding peroxidase and activating the antioxidant system. Chitosan promotes the synthesis of secondary metabolites and activates the phenylpropane pathway by upregulating the expression of genes encoding peroxidase POD, cytochrome P450 and GST enzymes, effectively enhancing the carbon, nitrogen and energy metabolism processes of maize plant roots induced by carbofuran, and ultimately promoting the metabolism of carbofuran.

Claims

1. Use of carboxylated chitosan for reducing the residues of carbodoxacetin in crops, characterized in that, The carboxylated chitosan has a molecular weight of 1526.5 g / mol; a molecular formula of C 56 H 103 N9O 39 Carboxylated chitosan is applied exogenously to crops treated with thiabendazole.

2. Use according to claim 1, characterized in that, The crop is corn, wheat, rice or peanut.

3. A method of reducing the residue of buprofezin in a crop, characterized by, The application further relates to a method for improving the growth of a crop, comprising: Carboxylated chitosan having a molecular weight of 1526.5 g / mol; a molecular formula of C 56 H 103 N9O 39 ; The crop is a hydroponic or soil-cultivated crop; For a hydroponic crop, the method for exogenous application of carboxylated chitosan is: dissolving carboxylated chitosan into a mother liquor, and then directly adding the mother liquor into a nutrient solution to a set concentration or spraying the mother liquor diluted with water to a set concentration on the leaves of the crop; the set concentration is 1-100 mg / L; For a soil-cultivated crop, the method for exogenous application of carboxylated chitosan is: dissolving carboxylated chitosan into a mother liquor, directly adding the chitosan mother liquor into the soil to a content of 10 mg / Kg or spraying a carboxylated chitosan solution diluted to 10 mg / L on the stems and leaves of the crop.

4. The method of claim 3, wherein, The crop is corn, wheat, rice or peanut.

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