A method for inactivating foreign DNA in agroforestry biomass and bio-breeding straw

Abstract

The application discloses a kind of forestry biomass and biological breeding straw-based foreign DNA inactivation method, comprising the following steps: step S1, providing a transgenic crop raw material waste, the waste is crushed, add hypochlorous acid solution, obtain waste slurry;Step S2, the waste slurry is placed in reaction kettle, stir at room temperature, acidification reaction occurs;Step S3, the reaction kettle is heated and pressurized, the hypochlorous acid solution and the waste slurry occur cleavage reaction, the lignin, cellulose, hemicellulose in the waste slurry are oxidized and degraded, obtain transgenic crop raw material residue;The temperature in the reaction kettle is 121 DEG C~140 DEG C, and the pressure is 0.3MPa~0.8MPa;Wherein, the pH of the hypochlorous acid solution is 5~6, the effective chlorine concentration is 100ppm~150ppm, and the oxidation-reduction potential is stably maintained in >=800mV working interval.The inactivation method can realize full-dimensional harmless to class II transgenic waste, and the residue liquid after treatment has higher organic content and structural integrity after adding appropriate proportions of nitrogen, phosphorus and potassium elements and the like through the cleavage process, and can be used for subsequent resource utilization.

Description

Technical Field

[0001] This invention relates to the field of waste disposal technology for bio-breeding crops, specifically to a method for inactivating exogenous DNA from agricultural and forestry biomass and bio-breeding straw. Background Technology

[0002] With the continuous expansion of commercial planting of genetically modified crops globally, the harmless treatment and resource utilization of their waste have become a crucial link in the risk management of the entire transgenic technology chain. In my country, the pilot area for commercialization of transgenic maize has exceeded ten million mu (approximately 6.67 million hectares), generating a massive amount of waste. Unlike conventional waste, transgenic crops carry artificially inserted exogenous genes (such as insect-resistant and herbicide-resistant genes). If not properly disposed of, recombinant DNA can be released into the soil environment through root exudates, pollen, and plant debris. Existing research indicates that exogenous recombinant DNA can be integrated into the genome of soil microorganisms through homologous recombination, directly or indirectly altering the structure and function of the microbial community. Field trials of transgenic sugar beets have shown that recombinant DNA can be detected in the soil for at least a year, and even after 25 days under laboratory soil culture conditions, it can still be detected, indicating that relying solely on natural degradation cannot ensure the complete inactivation of recombinant DNA.

[0003] According to the "Regulations on the Safety Management of Agricultural Genetically Modified Organisms" (State Council Decree No. 304), the state has established a safety assessment system for agricultural genetically modified organisms. Article 4 of the "Administrative Measures for the Safety Assessment of Agricultural Genetically Modified Organisms" (revised by the Ministry of Agriculture and Rural Affairs Decree No. 2 of 2022 on January 21, 2022) clarifies that safety assessment work is conducted according to three categories: plants, animals, and microorganisms, based on science and adhering to the principle of case-by-case review, and implemented in a graded and phased manner. Article 9 of these measures classifies agricultural genetically modified organisms into four safety levels, with safety level II being "low-risk." According to its safety management requirements, genetically modified organisms of safety level II and above must be reliably inactivated before waste disposal and discharge. However, currently, agricultural genetically modified organism processing enterprises uniformly adopt incineration to inactivate genetically modified waste, resulting in resource waste, environmental pollution, and carbon emissions. Summary of the Invention

[0004] The purpose of this invention is to provide a method that can both completely inactivate recombinant DNA in biological breeding crops and retain the resource value of waste matrix.

[0005] To achieve the above objectives, the present invention provides a method for inactivating exogenous DNA from agricultural and forestry biomass and straw-based materials used in biological breeding, comprising the following steps: Step S1: Provide waste from a genetically modified crop raw material, crush the waste, add hypochlorous acid solution, and obtain waste slurry; Step S2: Place the waste slurry in a reaction vessel and stir it with hypochlorous acid solution at room temperature to induce an acidification reaction; Step S3: The reactor is heated and pressurized, and the hypochlorous acid solution undergoes a pyrolysis reaction with the waste slurry to oxidize and degrade the lignin, cellulose, and hemicellulose in the waste slurry, thereby obtaining a genetically modified crop raw material residue liquid; the temperature inside the reactor is 121℃~140℃, and the pressure is 0.3MPa~0.8MPa; The hypochlorous acid solution has a pH of 5-6, an effective chlorine concentration of 100ppm-150ppm, and an oxidation-reduction potential of ≥800mV.

[0006] Optionally, the method for preparing the hypochlorous acid solution includes: using food-grade high-purity sodium chloride as raw material, preparing a dilute sodium chloride electrolyte of a specified mass concentration with deionized water; the electrolyte flows through a diaphragmless electrolytic cell and is electrochemically oxidized by a hypochlorous acid electrolysis generator to obtain a hypochlorous acid aqueous solution.

[0007] Optionally, in step S1, the solid-liquid ratio of the waste to the hypochlorous acid solution is 1:9.

[0008] Optionally, step S1 specifically includes: cutting the waste into sections, drying it at 65℃~85℃ to constant weight, and pulverizing it through a 100-mesh sieve.

[0009] Optionally, the stirring time at room temperature in step S2 is 30 min to 60 min, and the oxidation reaction time in step S3 is 30 min to 60 min.

[0010] Optionally, the total microbial inactivation efficiency of the residue liquid of the genetically modified crop raw materials is greater than or equal to 6log, the long fragments of exogenous genetic modification with a length of greater than or equal to 1000bp are completely degraded, the copy number of short fragments of exogenous genetic modification with a length of less than or equal to 100bp decreases by greater than or equal to 6log, and the total fragmentation rate of exogenous DNA is greater than or equal to 95%.

[0011] Optionally, the waste from the genetically modified crop raw materials is Class II genetically modified waste.

[0012] Optionally, the genetically modified crop raw material includes at least one of genetically modified corn, genetically modified soybeans, genetically modified cotton, and genetically modified sweet potatoes.

[0013] The present invention also provides an application of the method described above in the preparation of agricultural organic fertilizer.

[0014] Optionally, at least one element selected from nitrogen, phosphorus, and potassium can be added to the residue liquid of the genetically modified crop raw materials according to a predetermined formula ratio to prepare a water-soluble organic fertilizer rich in small molecule peptide components.

[0015] Compared to the prior art, the beneficial effects of the present invention include at least the following: (1) In this invention, when inactivating Class II genetically modified waste, hypochlorous acid with high α-HOCl activity and high redox potential is precisely electrolyzed and activated to oxidize and degrade the lignocellulose in the waste, achieving in-situ cell wall disruption and exposing recombinant DNA. Hypochlorous acid can react with the outer ring primary amino group (-NH2) and inner ring secondary amino group (inner ring NH) of the exposed recombinant DNA bases to generate a semi-stable N-chloramine intermediate. Chloramine further decomposes to generate nitrogen-centered free radicals, causing irreversible chemical modification and spatial configuration distortion of nucleoside bases, ultimately inducing the dissociation of hydrogen bonds in the DNA double strand and triggering the cleavage of phosphodiester bonds, thus achieving the fragmentation and inactivation of exogenous recombinant DNA double strands. Under high temperature and high pressure hydrothermal action, hypochlorous acid reacts with lignin to generate hydrochloric acid. The generation of an acidic environment further catalyzes the degradation of lignin, increases the oxidation rate of lignin by hypochlorous acid, and significantly improves the cell wall disruption efficiency. After inactivation, the triple harmlessness fully met the standards, plant cells were completely inactivated, no callus formation occurred, and the residual DNA showed no horizontal gene transfer activity in the E. coli transformation test and simulated soil co-culture. The inactivation effect met the control requirements for safety level II waste in the "Administrative Measures for Safety Evaluation of Agricultural Genetically Modified Organisms".

[0016] (2) The inactivation method of the present invention removes the lignin from the waste residue, but the damage to the fiber skeleton is relatively controllable, which helps to preserve the structural integrity of the solid residue, which can be further prepared into organic fertilizer or soil conditioner, etc., and retains the resource potential while treating the residue liquid. Attached Figure Description

[0017] Figure 1 This is a quantitative analysis chart of Van Soest in Example 2 of the present invention.

[0018] Figure 2 This is a scanning electron microscope image of the chemical cell wall disruption of straw lignocellulose by hypochlorous acid according to the present invention.

[0019] Figure 3 This is an agarose gel electrophoresis image of the present invention.

[0020] Figure 4 This is a diagram showing the TTC staining results of the loss of plant cell viability according to the present invention.

[0021] Figure 5 This is a target band diagram for PCR detection of exogenous gene residues in simulated soil co-culture according to the present invention. Detailed Implementation

[0022] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0023] I. Experimental Materials: The herbicide-tolerant genetically modified corn Jingke 938, which has obtained agricultural genetically modified organism safety certification, was selected, and its mature straw and leaves were collected.

[0024] II. Experimental Equipment: High-temperature and high-pressure hydrothermal reactor, biosafety cabinet (BSC-1300IIA2 type), autoclave (DSX-29L-I type), real-time PCR instrument (ABI 7500 type), droplet digital PCR (ddPCR) system (QX200 type), gel electrophoresis system (Bio-Rad PowerPac Basic), UV-Vis spectrophotometer (NanoDrop), high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS, Thermo Fisher TSQ Quantis), high-throughput sequencing platform (IlluminaNovaSeq).

[0025] III. Experimental Reagents: Hypochlorous acid stock solution (effective chlorine concentration calibrated), commercially available hypochlorous acid solution, nutrient agar, Bengal red medium, CTAB extraction buffer (containing 2% PVP and 0.2% β-mercaptoethanol), primers and TaqMan probes (targeting CaMV 35S, NOS, bar, and maize invertase genes), 5-cyano-2,3-dimethyltetrazolium (CTC) staining solution, Escherichia coli competent cells (DH5α), LB medium, DNA digestion reagents (DNase I, snake venom phosphodiesterase, alkaline phosphatase).

[0026] IV. Experimental Methods: (1) Characterization of lignocellulose degradation effect Chemical composition analysis: Fourier transform infrared spectroscopy (FTIR) analysis of lignin (1510, 1600 cm⁻¹) -1 ), cellulose (1160, 1060, 897 cm) -1 ) and hemicellulose (1735 cm) -1 Characteristic peak intensity variations; X-ray diffraction (XRD) was used to calculate the crystallinity index (CrI) using the Segal method.

[0027] Component quantification: Neutral detergent fiber (NDF), acid detergent fiber (ADF), and acid detergent lignin (ADL) were determined by the Van Soest method, and the lignin removal rate was calculated.

[0028] Microstructure: Scanning electron microscopy (SEM) was used to observe the surface of straw and the characteristics of cell wall damage.

[0029] (2) Detection of microbial inactivation effect Plate count method: Referring to GB 4789.2-2022, the dilution plating method was used to count bacteria (nutrient agar, 36°C, 48 h), molds and yeasts (Red Bengal, 28°C, 72 h), and heat-resistant spores (plating after heat treatment at 80°C for 10 min, 36°C, 72 h).

[0030] VBNC state microorganisms: The PMA-qPCR method was used. The sample was treated with PMA dye and then DNA was extracted. The DNA of viable cells was quantified by qPCR, which effectively distinguished between dead and live bacteria.

[0031] (3) Multiscale degradation analysis of transgenic DNA DNA extraction: A modified CTAB method was used, with 2% PVP and 0.2% β-mercaptoethanol added to the extraction buffer to remove polysaccharide and polyphenol interference. NanoDrop was used to detect concentration and A... 260 / A 280 The ratio (1.8–2.0 is acceptable).

[0032] Agarose gel electrophoresis: Equal amounts of DNA (200–500 ng) were electrophoresed on a 1.5% agarose gel (containing GelRed) at a constant voltage of 5V / cm for 40 min, and the band morphology was observed.

[0033] qPCR fragmentation fingerprinting: Amplicones of different lengths (100, 200, 500, 1000, 2000 bp) were designed, targeting exogenous genes (CaMV 35S promoter, NOS terminator, NPTII marker gene, cry1Ab), strain-specific sequences (MON8103′ flanking sequence), and internal reference genes (zein, 18S rRNA, actin). The Ct values ​​of each amplicon were determined by SYBR Green qPCR, and the fragmentation index (Ct value of each amplicon / Ct value of 100 bp amplicon) was calculated. The method detection limit was 0.05%–0.1% of transgenic components.

[0034] Absolute quantification of short fragments: Absolute quantification of 100 bp transgenic short fragments was performed using droplet digital PCR (ddPCR), and the logarithmic decrease in copy number was calculated.

[0035] (4) Detection of chloride base damage products DNA was digested with DNase I, snake venom phosphodiesterase, and alkaline phosphatase. 5-chlorouridine (5-ClUrA) and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dGuo) were quantitatively detected by HPLC-MS / MS (electrospray ionization mode, multiple reaction monitoring, MRM), at a concentration of 10⁻⁶ ppm. 6 The number of damaged bases is represented by the number of bases in the sample.

[0036] (5) High-throughput sequencing validation DNA from the experimental and control groups was selected, libraries were constructed, and Illumina high-throughput sequencing (target depth ≥10× coverage) was performed. The libraries were compared with the maize reference genome B73 RefGen_v4 and exogenous gene sequence databases to analyze the distribution of breakpoints and the proportion of fragmented sequences.

[0037] (6) Detection of plant cell viability TTC staining: Place the sample slices in 0.5% TTC staining solution and incubate at 30°C in the dark for 12 hours. Observe the red color development (active cells appear red).

[0038] Callus induction: After surface sterilization, sample segments were inoculated into MS medium (containing appropriate amounts of auxin and cytokinin) and cultured at 25°C±1°C for 12 h / d for 30 days. Callus formation was then observed.

[0039] (7) Validation of biological transformation activity of residual nucleic acids *E. coli* transformation assay: *E. coli* DH5α competent cells were prepared using the CaCl2 method. Equal amounts (100–200 ng) of DNA samples from each group were used to transform the competent cells and plated on LB agar plates containing kanamycin (50 μg / mL) (NPTII gene conferred resistance). Transformation efficiency (CFU / μg DNA) was calculated. The gold standard for safe inactivation was zero transformed colonies (or below the limit of detection). Transformants were picked for plasmid extraction and PCR validation (targeting 35S, NPTII, and cry1Ab).

[0040] Simulated soil co-culture: DNA before and after treatment was co-cultured with a mixed microbial community of soil (collected from the top layer of soil in the transgenic maize planting area and passed through a 2 mm sieve) for 30 days. Total DNA was extracted periodically and PCR amplification was performed using exogenous gene-specific primers to detect whether recombinant DNA fragments were horizontally transferred to soil bacteria.

[0041] (8) Data processing and statistical analysis All data are expressed as mean ± standard deviation (Mean ± SD), with three independent biological replicates per group. One-way ANOVA was used for comparisons among multiple groups, and Tukey HSD was used for post-hoc testing; p < 0.05 was considered statistically significant. The synergistic effect index (SI) was calculated using the formula SI = combined treatment effect / (sum of individual treatment effects – combined treatment effect), with SI > 1.0 indicating synergistic enhancement. SPSS 25.0 was used for statistical analysis.

[0042] Currently, there are significant shortcomings in the methods for inactivating and disposing of waste from genetically modified organisms: (1) Returning waste to the field: cannot inactivate exogenous genes and ice-borne microorganisms; (2) Incineration: wastes resources and causes air pollution, and has been strictly prohibited by policy; (3) Laboratory high-pressure sterilization: the laboratory usually sterilizes at 121℃ and 0.1MPa for 30 minutes, which is effective for microorganisms but has limited effect on DNA breakage. DNA may still exist after sterilization, thus bringing the risk of gene pollution.

[0043] To address the aforementioned technical problems, this invention provides a method for inactivating exogenous DNA from agricultural and forestry biomass and straw-based materials used in bio-breeding, comprising the following steps: Step S1: Provide waste from a genetically modified crop raw material, cut the waste into sections, dry it at 65℃~85℃ to constant weight, crush the waste, pass it through a 100-mesh sieve, add hypochlorous acid solution, with a solid-liquid ratio of 1:9, to obtain waste slurry.

[0044] Step S2: Place the waste slurry in a reaction vessel and stir at room temperature for 30-60 minutes to induce an acidification reaction.

[0045] The three-dimensional network structure formed by lignin and cellulose in crop cell walls is a major barrier preventing exogenous chemicals from entering the cells. The reaction of highly active α-HOCl with lignin is achieved through three tandem steps: chlorination, demethoxylation, and oxidative ring-opening, degrading macromolecular lignin into small-molecule organic acids (such as acetic acid and formic acid) and oligomers.

[0046] Step S3: The reactor is heated and pressurized, and the hypochlorous acid solution undergoes a pyrolysis reaction with the waste slurry to oxidize and degrade the lignin, cellulose, and hemicellulose in the waste slurry to obtain genetically modified crop raw material residue liquid; the temperature inside the reactor is 121℃~140℃ and the pressure is 0.3MPa~0.8MPa.

[0047] This invention utilizes a slightly acidic electrolytic water generator to prepare a precisely electrolyzed and activated hypochlorous acid solution. The preparation method includes: using food-grade high-purity sodium chloride as raw material, preparing a dilute sodium chloride electrolyte of a specified mass concentration with deionized water; the electrolyte flows through a diaphragm-free electrolytic cell, and is electrochemically oxidized by a hypochlorous acid electrolysis generator to prepare a hypochlorous acid aqueous solution. High-purity edible salt undergoes pretreatment such as dissolution and filtration before entering the electrolysis system, ensuring the purity and safety of the raw materials from the source. The hypochlorous acid solution exhibits a strictly pH-controlled dissociation equilibrium: α-HOCl⇌ H₂ + + OCl -Its apparent dissociation constant (pKa = 7.54). The main bactericidal and oxidizing agents are the undissociated α-HOCl molecules, whose electrophilic chlorination ability to lignin aromatic rings and the rate of chlorination damage to DNA bases are greater than those of the dissociated hypochlorite (OCl) ions. - The effective chlorine concentration is approximately 80-100 times higher. This invention employs precise constant current control via diaphragm electrolysis to maintain the pH of the hypochlorous acid solution at 5-6. Under these conditions, the effective chlorine concentration is 100-150 ppm, and the oxidation-reduction potential (ORP) is maintained within the range of ≥800 mV, ensuring efficient electrophilic attack on lignin, cellulose, and hemicellulose, and rapid generation of chloramine radicals. Precise electrolysis utilizes a high-purity titanium-based DSA anode (RuO2-IrO2 / Ti coating), which possesses extremely high chlorine evolution potential (η_Cl2) and extremely low oxygen evolution competition, ensuring the main reaction at the anode interface (2Cl2) is achieved. - - 2e - The current efficiency (η_current) of (→ Cl2(ad)→α-HOCl) reaches 85%~95%.

[0048] Conventional preparation of hypochlorous acid solution uses industrial-grade hydrochloric acid as the electrolytic medium. While industrial hydrochloric acid provides chloride ions, it often contains impurity ions such as iron, arsenic, and heavy metals. These impurities may participate in electrode side reactions during electrolysis, accelerating electrode passivation or corrosion, and posing a risk of introducing harmful residues. Impurity ions in the raw materials (carbonate, phosphate, etc.) interfere with the electrolytic field or cause uncontrolled oxygen evolution overpotential at the electrodes, leading to pH > 7.5. At this point, α-HOCl drops sharply to < 30%, and the system reacts with low-activity OCl. - Dominant. Conventional processes use inferior coatings or raw materials containing heavy metals (Fe). 3+ / Cu 2+ Excessive levels of [a certain substance] can induce a serious oxygen evolution side reaction (2H₂O - 4e⁻). - → O2↑ + 4H + This not only wastes electrical energy, but also causes dissolved oxygen generated at the anode interface to undergo free radical chain quenching with newly formed α-HOCl, producing chlorate ions (ClO3) with no bactericidal activity. - This results in a large amount of electrical energy being consumed for heat and oxygen production rather than chlorine production. Although conventional iodometric or DPD methods can still detect "available chlorine" readings, the system has actually lost its redox buffer capacity, falling into a false residual state of "apparently high concentration, actual oxidation capacity zero".

[0049] It is evident that the hypochlorous acid solution prepared in this invention aims to maintain a high protonated state (high α-HOCl) and high chemical stability. If the electrolysis process is out of control, even with a high nominal concentration of oxidant, the electron flow will be blocked by the high oxygen evolution side reaction and degradation pathway, failing to effectively convert into the chemical energy required for the opening of the lignin aromatic ring and the breaking of DNA phosphodiester bonds. It is precisely based on the above-mentioned precise electrochemical parameter preparation method that the prepared hypochlorous acid solution can release maximum oxidation potential in subsequent experiments.

[0050] The dense network of lignin and cellulose in the cell walls of crop straw is a core physical barrier restricting chemical oxidants from entering the cell and reaching recombinant DNA. Precisely electrolyzed and activated hypochlorous acid (high α-HOCl content, high ORP), as a strong oxidant, exhibits a clear oxidative degradation effect on lignocellulose. (1) Oxidative Degradation of Lignin: Lignin is a three-dimensional network aromatic polymer composed of phenylpropane units linked by carbon-carbon bonds and ether bonds, and is the most stubborn component of the cell wall. The reaction of highly active α-HOCl with lignin is achieved through three tandem steps: chlorination, demethoxylation, and oxidative ring-opening. Specifically, α-HOCl first electrophilically chlorinates the carbon-carbon double bonds and phenolic hydroxyl groups in the lignin structure to generate chlorinated lignin intermediates; then, it destroys the methoxy (-OCH3) structure through demethoxylation; finally, it initiates the ring-opening destruction of the aromatic ring structure, degrading the macromolecular lignin into small molecule organic acids (such as acetic acid, formic acid, etc.) and oligomers. In the early stage of the reaction (phenolic oxidation substitution stage), the UV spectrum of lignin remains unchanged, but as the reaction proceeds, the characteristic absorption peak near 280 nm disappears, indicating that the aromatic ring structure is completely destroyed and the lignin is degraded into low molecular weight components. It is worth emphasizing that the reaction between α-HOCl and lignin exhibits autocatalytic properties—the hydrochloric acid (HCl) generated during the reaction further strongly catalyzes the reaction, automatically accelerating the degradation efficiency as the reaction progresses. The core reaction equations involve three steps: chlorination, demethoxylation, and oxidative ring-opening. DNA base amino sites ↓ α-HOCl chloride Semi-stable chloramines are generated ↓ Homogeneous cracking Release nitrogen-centered free radicals → Base chemical modification → Dissociation of double-strand hydrogen bonds → Glycan backbone disruption → DNA strand breaks and fragmentation ↓ The genetically modified gene is completely inactivated, posing no risk of biodiversity spread. Specifically, this process generates α-HOCl (active molecular hypochlorous acid, structural formula HOCl, molecular formula HClO). This molecule can target the amino sites of DNA bases, undergoing a three-stage reaction of chlorination, demethoxylation, and oxidative ring-opening, thereby destroying the structure of recombinant exogenous DNA and achieving gene inactivation of more than 6 logs. The hypochlorite ions generated under alkaline conditions are not labeled α, and their oxidative inactivation ability is significantly reduced; therefore, they are not considered an effective component of this process.

[0051] 1. Chlorination (initial reaction) The active hypochlorous acid molecule targets the exposed amino site of the base, undergoing an electrophilic chlorination reaction. The chlorine atom covalently binds to the nitrogen atom of the amino group, generating an N-chloro base intermediate. This intermediate is unstable and easily undergoes homolytic cleavage to generate active free radicals, providing a chain reaction initiation site for subsequent second-order reactions.

[0052] R represents DNA base nucleus backbone residues.

[0053] 2. Demethoxylation The free radical active species generated in the first step attack the methoxy functional group on the side chain of the base, the CO ether bond breaks, the methoxy group detaches from the parent skeleton in the form of methanol, is replaced by a hydroxyl group at the original site, the original spatial configuration of the base is distorted, and the base pairing ability is initially destroyed.

[0054] 3. Oxidative ring-opening (final inactivation) The hydroxyl-modified base nucleus aromatic ring structure exhibits significantly enhanced reactivity. Under the synergistic oxidation of excess active hypochlorous acid and dissolved oxygen, the pyrimidine / purine aromatic ring backbone undergoes ring-opening cleavage, the intact base structure completely disintegrates, and the exogenous DNA loses its genetic replication activity, ultimately achieving a gene inactivation effect of ≥6 log.

[0055] Minimalist flowchart connection structure: DNA base → Chlorination to form chloramine → Free radical-induced demethoxylation → Oxidative ring opening → Permanent gene inactivation.

[0056] (2) Oxidative degradation of cellulose: Cellulose is a linear polysaccharide composed of D-glucose linked by β-1,4-glycosidic bonds. In neutral and acidic media, α-HOCl mainly oxidizes the hydroxyl groups at C2, C3, and C6 of cellulose to aldehyde and carboxyl groups, thereby initiating the cleavage of glucosidic bonds and leading to the depolymerization of cellulose molecules. Although α-HOCl can cause cellulose depolymerization, the damage to the strength of the fiber skeleton is relatively controllable, which helps to preserve the structural integrity of the solid residue and provides conditions for subsequent resource utilization.

[0057] (3) Degradation of hemicellulose: Hemicellulose is a key interfacial component connecting lignin and cellulose, and its branched structure is relatively sensitive to α-HOCl oxidation. α-HOCl can oxidize the acetyl and uronic acid groups on the side chains of hemicellulose, loosening the lignin-carbohydrate complex (LCC) structure and further promoting the dissolution of lignin.

[0058] Using a precisely electrolyzed activated hypochlorous acid solution to degrade lignin, cellulose, and hemicellulose in the waste of genetically modified crop raw materials can effectively break down the dense physical barrier of plant cell walls, exposing recombinant DNA inside the cells and providing contact conditions for subsequent DNA breakage.

[0059] After recombinant DNA exposure, hypochlorous acid solution first reacts with the outer ring -NH2 groups and inner ring NH groups of the DNA bases to generate semi-stable chloramines, which account for 50-95% of the added hypochlorous acid solution. The chloramines then decay through thermal and metal ion-catalyzed processes, generating nitrogen-centered free radicals. The tendency for free radical formation is: cytidine > adenosine = guanosine > uridine = thymidine. Pre-formed nucleoside chloramines can also induce plasmid breakage, especially when unstable chloramines derived from thymidine and uridine rapidly form free radicals. The reaction of α-HOCl with DNA leads to chemical modification of nucleoside bases and induces structural changes, thereby triggering the dissociation and breakage of double-stranded DNA. α-HOCl-induced DNA damage occurs at sequence-specific sites and can produce various stable chlorinated nucleoside base products, including 8-chloro-(2′-deoxy)-adenosine, 8-chloro-(2′-deoxy)-guanosine, 5-chloro-(2′-deoxy)-cytidine, and 5-chloro-uracil. Furthermore, the reactive nitrogen free radicals generated by the decay of chloramine can extract hydrogen atoms from the deoxyribose backbone of DNA, further exacerbating strand breaks.

[0060] Example 1 This embodiment 1 provides a method for inactivating exogenous DNA from agricultural and forestry biomass and straw-based bio-breeding materials, comprising the following steps: Step S1: Provide mature straw and leaves of transgenic insect-resistant corn Jingke 938. Cut the straw into 3-5 cm sections, dry at 65°C to constant weight, pulverize and pass through a 100-mesh sieve (particle size ≤0.18 mm), mix thoroughly, and then seal and dry for storage. Initial bacterial count was tested to be 10... 6 ~10 7 CFU / g, the detection rate of three types of transgenic elements (CaMV 35S promoter, NOS terminator, and bar gene) was 100%. The waste slurry was obtained by adding the hypochlorous acid solution prepared in this invention (effective chlorine concentration of 100 ppm, pH 5.8).

[0061] Step S2: Place the waste slurry in a reaction vessel and stir at room temperature for 30 minutes to induce an acidification reaction.

[0062] In step S3, the reactor is heated and pressurized, and the hypochlorous acid solution undergoes a pyrolysis reaction with the waste slurry to oxidize and degrade the lignin, cellulose, and hemicellulose in the waste slurry, thereby obtaining genetically modified crop raw material residue. The temperature inside the reactor is 130°C, the pressure is 0.5 MPa, the oxidation time is 60 min, and the total time for the entire step S3 is 120 min.

[0063] Example 2 This embodiment 2 provides a method for inactivating exogenous DNA from agricultural and forestry biomass and straw-based biological breeding materials, comprising the following steps: Step S1: Provide mature straw and leaves of transgenic insect-resistant corn Jingke 938. Cut the straw into 3-5 cm sections, dry at 65°C to constant weight, pulverize and pass through a 100-mesh sieve (particle size ≤0.18 mm), mix thoroughly, and then seal and dry for storage. Initial bacterial count was tested to be 10... 6 ~10 7 CFU / g, the detection rate of three types of transgenic elements (CaMV 35S promoter, NOS terminator, and bar gene) was 100%. The waste slurry was obtained by adding the hypochlorous acid solution prepared in this invention (effective chlorine concentration of 150 ppm, pH 5.25).

[0064] Step S2: Place the waste slurry in a reaction vessel and stir at room temperature for 60 minutes to induce an acidification reaction.

[0065] In step S3, the reactor is heated and pressurized, and the hypochlorous acid solution undergoes a pyrolysis reaction with the waste slurry to oxidize and degrade the lignin, cellulose, and hemicellulose in the waste slurry, thereby obtaining genetically modified crop raw material residue. The temperature inside the reactor is 130°C, the pressure is 0.5 MPa, the oxidation time is 60 min, and the total time for the entire step S3 is 120 min.

[0066] Comparative Example 1 Commercially available hypochlorous acid was added to waste materials from genetically modified insect-resistant corn variety Jingke 938 and stirred at room temperature for 60 minutes. The commercially available hypochlorous acid had an effective chlorine concentration of 100 ppm and a pH of 5.8.

[0067] Comparative Example 2 The hypochlorous acid solution prepared according to this invention was added to waste of genetically modified insect-resistant corn Jingke 938 and stirred at room temperature for 60 minutes. The effective chlorine concentration of the hypochlorous acid solution was 100 ppm, and the pH was 5.28.

[0068] Comparative Example 3 The hypochlorous acid solution prepared according to this invention was added to waste of genetically modified insect-resistant corn Jingke 938 and stirred at room temperature for 60 minutes. The effective chlorine concentration of the hypochlorous acid solution was 150 ppm, and the pH was 4.8.

[0069] Comparative Example 4 High-temperature hydrothermal treatment was carried out on the waste of genetically modified insect-resistant corn Jingke 938. The temperature was maintained in a reactor at 130℃ and 0.3MPa for 120 minutes.

[0070] Comparative Example 5 The waste from the genetically modified insect-resistant corn variety Jingke 938 is not treated in any way.

[0071] Experimental results: (1) The chemical cell-wall breaking effect of hypochlorous acid on straw lignocellulose After treatment with a hypochlorous acid solution with an effective chlorine concentration of 150 ppm as described in this invention at room temperature for 60 min, the characteristic peaks of lignin in the FTIR spectrum (1510, 1600 cm⁻¹) were observed. -1 The intensity decreased by 39.6%, confirming the disruption of the aromatic ring structure; the characteristic peak of hemicellulose (1735 cm⁻¹) -1 The concentration of cellulose in the solid residue decreases as lignin and hemicellulose preferentially degrade and dissolve, leading to a higher relative proportion of cellulose in the solid residue, corresponding to 1160 cm⁻¹. -1 1060 cm -1 897 cm -1 The absorption intensity of the characteristic peak of cellulose increased relatively, and XRD analysis showed that CrI decreased from 58.3% to 52.1%, indicating that the crystalline region was damaged to some extent.

[0072] like Figure 1 As shown, Van Soest quantitative analysis revealed that the lignin removal rate reached 42.7% (Example 2), the hemicellulose degradation was 38.3%, and the cellulose retention was 82.6% (the cellulose skeleton was retained, providing a basis for subsequent resource utilization).

[0073] like Figure 2 As shown, SEM observation revealed the peeling of the waxy layer on the straw surface, the appearance of cracks and pores in the thick-walled cell walls, and the loosening of the cell wall structure. These results indicate that precise activation of α-HOCl effectively oxidized and degraded the lignocellulose network, successfully breaking down the physical barrier.

[0074] In Comparative Example 3, the lignin removal rate was 28.5% with only hypochlorous acid treatment and no subsequent hydrothermal treatment; in Comparative Example 4, the lignin removal rate was 12.3% with only high-temperature hydrothermal treatment; and in Example 2, the lignin removal rate was 42.7% with a combination of hypochlorous acid and high-temperature hydrothermal treatment. This indicates that the precisely electrolytically activated hypochlorous acid solution of the present invention has a synergistic effect with temperature and pressure (SI=1.46). Although the effective chlorine concentrations of Comparative Examples 1 and 2 were both 100 ppm, the lignin removal rate of the commercially available hypochlorous acid in Comparative Example 1 was 16.3%, far lower than the 21.7% lignin removal rate of Comparative Example 2. This result indicates that the effective chlorine of ordinary commercially available products (Comparative Example 1) is reduced to a low-activity OCl due to pH drift to alkalinity (8.2) and catalytic degradation by impurities. - The nominal concentration cannot be equivalent to the actual oxidation capacity due to the existence of the form and the loss of the redox buffer capacity; while the food-grade hypochlorous acid (comparative example 2) with precise electrolytic activation maintains a high α-HOCl ratio (>95%) and a high ORP (≧800mV), thus providing sufficient thermodynamic driving force for lignin degradation.

[0075] (2) Microbial inactivation effect As shown in Table 1, the number of various microorganisms in the blank treatment group of Comparative Example 5 did not change significantly. The inactivation effect of commercially available hypochlorous acid in Comparative Example 1 was weaker than that of the precisely electrolyzed activated hypochlorous acid of the present invention at the same concentration, which is related to the difference in active species caused by the electrolysis process. The inactivation log values ​​of bacteria, molds, and spores in Comparative Examples 2 and 3 were ≤4.2 log, and VBNC-state microorganisms were still detected positively. The single high-temperature hydrothermal treatment in Comparative Example 4 had an inactivation log value of ≥4 log for bacteria and molds / yeasts, but only 3.2 log for heat-resistant spores, and VBNC could still be detected. The inactivation effects of Examples 1 and 2 were significantly better than the single treatment group (p<0.05), with Example 2 showing the best effect. The inactivation log values ​​of bacteria, molds / yeasts, and heat-resistant spores were all ≥6.0 log, no detection was found in plate counts, and no VBNC-state microorganisms were detected by PMA-qPCR.

[0076] Table 1. Microbial inactivation effect (log value of inactivation) of the examples and comparative examples Note: "≥6.0" indicates that the number of viable bacteria after treatment is below the detection limit; "positive / negative" indicates whether microorganisms in VBNC state are detected.

[0077] (3) Degradation effect of transgenic DNA like Figure 3As shown, agarose gel electrophoresis revealed that long fragment bands (≥1000 bp) of the three targets (35S promoter, NOS terminator, and bar gene) were clearly visible in the blank control group (Comparative Example 5); the long fragment bands of the single agent or single hydrothermal treatment groups (Comparative Examples 1-4) were less bright but still observable; some long fragment bands of the combined treatment group (Example 1) disappeared; and all long fragment bands of Example 2 did not appear.

[0078] As shown in Table 2, the ddPCR quantification results showed that the copy number of the 100 bp short fragment in Example 2 decreased by ≥6 log compared to before treatment, and the residual copy number was close to the detection limit (<10 copies / μL). In contrast, the single treatment groups (Comparative Examples 2-4) showed the highest decrease of only 3.5 log, significantly lower than the combined treatment groups (p < 0.05). The decrease in Comparative Example 1 (1.2 log) was significantly lower than that in Comparative Example 2 (1.8 log), further demonstrating the decisive influence of precise electrolytic activation on the DNA strand breaking activity of the oxidant.

[0079] Table 2. Logarithmic values ​​of copy number decrease for 100bp short fragments quantified by ddPCR qPCR fragmentation index analysis showed that in Example 2, neither the 2000 bp nor the 1000 bp amplicons showed amplification (Ct > 35). The Ct value of the 500 bp amplicon increased by 5.8 compared to the 100 bp amplicon, and the fragmentation index (500 bp / 100 bp Ct ratio) was 1.52, significantly higher than that of the single treatment group (approximately 1.1–1.2). HPLC-MS / MS detection showed that the content of the α-HOCl-specific damage marker 5-chlorouridine (5-ClUrA) in Example 2 was 32.7 ± 4.1 / 10 6 The base, 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dGuo), is 18.5 ± 2.3 / 10. 6 The base pairs were significantly higher than those in the single treatment group (p < 0.01). High-throughput sequencing showed that the proportion of reads from the transgenic sequence in Example 2 was <0.01%, and the breakpoints were evenly distributed in the coding region and flanking sequences of the exogenous gene, with no concentrated hotspots found.

[0080] (4) Effect of loss of plant cell activity like Figure 4As shown, TTC staining results indicated that the blank control group (Comparative Example 5) was distinctly red; the single 150 ppm precise activation α-HOCl treatment group (Comparative Example 3) and the single hydrothermal treatment group (Comparative Example 4) both showed varying degrees of light red, indicating that some cell activity remained; the two combined treatment groups (Example 1 and Example 2) did not show any red color, indicating complete inactivation. Callus induction experiments further verified that: after 7 days of culture, the blank control group (Comparative Example 5) showed visible callus tissue on the cut surface, and after 30 days, obvious callus formed; some segments of Comparative Example 3 and Comparative Example 4 produced a small amount of callus tissue after 30 days; while all segments of Example 1 and Example 2 did not produce any callus tissue, indicating that the combined effect of chemical cell disruption and oxidative chain scission completely eliminated the regeneration potential of plant cells.

[0081] (5) Verification of biological transformation activity of residual nucleic acids The results of the E. coli transformation assay showed that after DNA extracted from Comparative Examples 1-5 was transformed into competent cells, positive transformants (approximately 2.5 × 10⁻⁶) could grow on LB agar plates containing kanamycin (Comparative Example 5: approximately 2.5 × 10⁻⁶). 4 CFU / μg DNA, with ratios decreasing from 1-4 to 10. 2 ~10 3 (CFU / μg), PCR verification showed that the transformants contained the 35S, NPTII, and cry1Ab genes. However, the DNA samples from Examples 1 and 2 showed no colony growth on the selection plates (0 CFU / μg DNA), even when the DNA amount was increased to 1 μg, no transformants were found. Figure 5 As shown, after 30 days of co-culture in simulated soil, exogenous gene fragments could still be detected by PCR in Comparative Examples 1-5, while no exogenous gene signals were detected in Examples 1 and 2 at all sampling time points (days 0, 7, 14, and 30). The results indicate that the residual DNA after secondary co-treatment completely lost its biological transformation activity and horizontal gene transfer ability.

[0082] (6) Verification of synergistic effect and autocatalytic mechanism The Synergy Index (SI) was calculated. In Example 2, the SI was significantly greater than 1.0 in all four dimensions: lignin removal (SI=1.46), microbial inactivation (SI=1.52), DNA degradation (SI=1.48), and cell inactivation (SI=1.55), confirming the significant synergistic effect between precise activation of α-HOCl oxidation and high-temperature pressurized hydrothermal treatment.

[0083] Self-catalytic mechanism verification: Monitoring of pH changes during the reaction revealed that the pH of the final solution treated in Example 2 decreased from the initial 5.25 to 4.8-4.0, while the pH change in the comparative example (hydrothermal only, without α-HOCl) was <0.2. HPLC analysis confirmed the presence of small-molecule organic acids such as acetic acid (2.3 g / L) and formic acid (0.8 g / L) in the treated solution, originating from the oxidative ring-opening products of lignin aromatic rings. This spontaneously generated acidic environment further catalyzed the hydrolysis of nucleic acids, forming a positive feedback loop of "oxidative cell disruption → release of organic acids → pH decrease → accelerated nucleic acid cleavage," without the need for additional acid catalysts.

[0084] The treatment effect of this invention fully meets the control requirements for safety level II genetically modified waste under the "Administrative Measures for the Safety Evaluation of Agricultural Genetically Modified Organisms": no viable bacteria were detected after treatment, long genetically modified fragments were completely degraded (≥1000 bp), short fragment copy number decreased by ≥6 log, transformation test was negative, and no callus formation was observed. The technical parameters (150 ppm precise activation of α-HOCl, 130°C / 0.5 MPa) are all within the range that can be maturely achieved by existing industrial-grade hydrothermal equipment. Hypochlorous acid can be precisely prepared on-site through diaphragm electrolysis of saturated brine (to ensure high α-HOCl activity), and the single batch treatment capacity can be scaled up to tens of cubic meters. Because the treated straw has partially lost lignin and retained its cellulose intact, it is more conducive to the subsequent production of ethanol, organic fertilizer, or soil conditioner, achieving a seamless connection between "safe disposal + resource utilization".

[0085] The residue obtained by the inactivation method of the present invention can be made into small molecule peptide fertilizer, which can be reused in the planting base of the same crop to realize the ecological fertilizer efficiency cycle of core elements such as carbon, nitrogen, and phosphorus. Small molecule peptide fertilizer has the following advantages: (1) Direct absorption and signaling function: Small molecule peptides (especially oligopeptides of 2-10 amino acids) can be recognized and completely absorbed by plant root-specific transport proteins, with higher efficiency than amino acids. Some peptide segments act as signal molecules to regulate plant growth; (2) Improve the rhizosphere microecology: Peptides are high-quality nitrogen and carbon sources for beneficial microorganisms in the rhizosphere and can selectively stimulate the proliferation of probiotics (such as phosphate-solubilizing bacteria and nitrogen-fixing bacteria); (3) Integrate trace elements: Peptides and amino acids have chelating ability and can form stable organic complexes with metal ions such as Fe, Zn, and Mn.

[0086] In summary, the present invention provides a method for inactivating exogenous DNA based on agricultural and forestry biomass and straw used in biological breeding, comprising the following steps: Step S1, providing a waste product of a transgenic crop raw material, crushing the waste product, adding hypochlorous acid solution to obtain a waste slurry; Step S2, placing the waste slurry in a reaction vessel, stirring at room temperature to induce an acidification reaction; Step S3, heating and pressurizing the reaction vessel, wherein the hypochlorous acid solution reacts with the waste slurry in a pyrolysis reaction, oxidizing and degrading the lignin, cellulose, and hemicellulose in the waste slurry to obtain a transgenic crop raw material residue; the temperature inside the reaction vessel is 121℃~140℃, and the pressure is 0.3MPa~0.8MPa; wherein the pH of the hypochlorous acid solution is 5~6, the effective chlorine concentration is 100ppm~150ppm, and the oxidation-reduction potential (ORP) is ≥800mV. The inactivation method can achieve full-dimensional harmlessness of secondary genetically modified waste. The treated residue still has a high organic content and structural integrity, and can be used for subsequent resource utilization.

[0087] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A method for inactivating exogenous DNA from agricultural and forestry biomass and straw-based biological breeding, characterized in that, Includes the following steps: Step S1: Provide waste from a genetically modified crop raw material, crush the waste, add hypochlorous acid solution, and obtain waste slurry; Step S2: Place the waste slurry in a reaction vessel and stir it with hypochlorous acid solution at room temperature to induce an acidification reaction; Step S3: The reactor is heated and pressurized, and the hypochlorous acid solution undergoes a pyrolysis reaction with the waste slurry to oxidize and degrade the lignin, cellulose, and hemicellulose in the waste slurry, thereby obtaining a genetically modified crop raw material residue liquid; the temperature inside the reactor is 121℃~140℃, and the pressure is 0.3MPa~0.8MPa; The hypochlorous acid solution has a pH of 5-6, an effective chlorine concentration of 100ppm-150ppm, and an oxidation-reduction potential of ≥800mV.

2. The method as described in claim 1, characterized in that, The method for preparing the hypochlorous acid solution includes: using food-grade high-purity sodium chloride as raw material, preparing a dilute sodium chloride electrolyte of a specified mass concentration with deionized water; the electrolyte flows through a diaphragmless electrolytic cell and is electrochemically oxidized by a hypochlorous acid electrolysis generator to obtain a hypochlorous acid solution.

3. The method as described in claim 1, characterized in that, In step S1, the solid-liquid ratio of the waste to the hypochlorous acid solution is 1:

9.

4. The method as described in claim 1, characterized in that, Step S1 specifically includes: cutting the waste into sections, drying it at 65℃~85℃ to constant weight, and pulverizing it through a 100-mesh sieve.

5. The method as described in claim 1, characterized in that, The stirring time at room temperature in step S2 is 30 min to 60 min, and the oxidation reaction time in step S3 is 30 min to 60 min.

6. The method as described in claim 1, characterized in that, The total microbial inactivation efficiency of the residue liquid of the genetically modified crop raw materials is greater than or equal to 6log, the long fragments of exogenous genetic modification with a length of greater than or equal to 1000bp are completely degraded, the copy number of short fragments of exogenous genetic modification with a length of less than or equal to 100bp decreases by greater than or equal to 6log, and the total fragmentation rate of exogenous DNA is greater than or equal to 95%.

7. The method as described in claim 1, characterized in that, The waste from the genetically modified crop raw materials is classified as Class II genetically modified waste.

8. The method as described in claim 1, characterized in that, The genetically modified crop raw materials include at least one of genetically modified corn, genetically modified soybeans, genetically modified cotton, and genetically modified sweet potatoes.

9. The application of the method as described in any one of claims 1 to 8 in the preparation of agricultural organic fertilizer.

10. The application as described in claim 9, characterized in that, According to a predetermined formula ratio, at least one element selected from nitrogen, phosphorus, and potassium is added to the residue liquid of the genetically modified crop raw materials to prepare a water-soluble organic fertilizer rich in small molecule peptide components.

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