Extraction method of genome DNA of plant rich in secondary metabolites and buffer solution

Abstract

The invention relates to a method for extracting genome DNA of plants rich in secondary metabolites and a buffer solution, and belongs to the technical field of molecular biology and botany. The method solves the technical problems of low DNA extraction efficiency, poor purity, easy degradation and the like when a traditional DNA extraction method is used for treating plant tissues rich in secondary metabolites such as alkaloid, polyphenol and the like. Comprising the following steps: adding a complexing agent such as polyethylene glycol or polyvinylpyrrolidone when grinding plant tissues in a liquid nitrogen environment; splitting by using a cell wall splitting buffer solution containing dithiothreitol and a nonionic surfactant; carrying out DNA release and extraction at 60-70 DEG C by adopting a CTAB (Cetyltrimethyl Ammonium Bromide) extraction buffer solution containing polyethylene glycol and papain; and then purifying and precipitating to obtain high-purity genome DNA (Deoxyribose Nucleic Acid). The method can effectively remove alkaloid, protein and other impurities, significantly improves the DNA yield and purity, and is suitable for genome sequencing, genetic resource protection, medicinal plant molecular identification and the like of plants with high secondary metabolites such as Stephania kwangsiensis and the like.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology and botany, and specifically relates to a method for extracting genomic DNA from plants rich in secondary metabolites and a buffer solution. Background Technology

[0002] Guangxi is not a place that can tolerate ( Stephania kwangsiensis *Lysimachia christinae* is an important medicinal plant belonging to the genus *Lysimachia* in the Menispermaceae family. Its bulbs are rich in alkaloids, polyphenols, polysaccharides, and other secondary metabolites. These components have significant medicinal value, but they pose considerable difficulties for the extraction of genomic DNA in molecular biology research.

[0003] First, during sample pretreatment, a large amount of alkaloids and polyphenols from plant tissues are released during grinding. These substances readily bind to DNA molecules, forming stable complexes that not only hinder the effective release of DNA but also lead to DNA degradation or cross-linking in subsequent processing. While conventional liquid nitrogen grinding methods can inhibit some enzyme activity, they are insufficient to prevent the adsorption and interference of these chemical substances.

[0004] Secondly, during cell wall lysis, polyphenol oxidase in plant cells catalyzes the oxidation of polyphenols into quinones upon contact with oxygen. Quinones are highly reactive and readily react with bases or the phosphate backbone of DNA molecules, causing DNA strand breaks or cross-linking, severely impacting DNA integrity and yield. Although existing methods attempt to mitigate this problem by adding conventional reducing agents, the protective effect on high-polyphenol content materials like those from Guangxi is still unsatisfactory.

[0005] Furthermore, during the DNA release and extraction stages, the high concentrations of polysaccharides and alkaloids in the bulb tissue can co-precipitate with DNA or competitively bind to the extraction reagent, reducing DNA recovery efficiency. Polysaccharide residues can also affect the efficiency of subsequent molecular experiments such as enzyme digestion and PCR. Traditional CTAB methods or commercial kits are typically designed for common plant materials, and their impurity removal capabilities are insufficient when dealing with special materials like those from Guangxi, making it difficult to effectively remove multiple types of secondary metabolites simultaneously.

[0006] Due to the aforementioned problems, genomic DNA extracted from *Gnaphalium affine* bulbs in Guangxi often suffers from low yield, substandard purity, and severe degradation, making it difficult to meet the requirements of sophisticated molecular experiments such as genome sequencing and molecular marker development. Therefore, it is necessary to develop a DNA extraction method that can specifically address interference from high levels of secondary metabolites. Summary of the Invention

[0007] The purpose of this invention is to solve the following problems: This study aims to address the problem of low DNA yield and poor purity caused by interference from substances such as alkaloids and polyphenols when extracting DNA from plant tissues rich in secondary metabolites. Traditional methods are ineffective in preventing secondary metabolites from binding to DNA and cannot adequately protect DNA integrity during cell lysis.

[0008] This addresses the issues of low grinding efficiency and starch contamination. Highly fibrous plant tissues are difficult to break down completely, and the starch abundant in storage organs such as corms easily forms viscous colloids during extraction, hindering DNA release and purification.

[0009] This addresses the problem of DNA degradation caused by polyphenol oxidation during cell wall lysis. Conventional lysis buffers have limited inhibitory effects on polyphenol oxidase, and the formation of quinones can trigger DNA cross-linking and strand breaks.

[0010] To resolve the contradiction between mild lysis and organelle DNA contamination. Rapid and violent lysis can lead to the release of large amounts of organelle DNA, which can become mixed in with the nuclear genome, affecting the accuracy of subsequent analyses.

[0011] This addresses the issue of insufficient early protection of DNA in lysis buffer systems. In the initial stages of lysis, newly released DNA is exposed to a complex chemical environment, making it susceptible to oxidation and enzymatic damage.

[0012] This study addresses the balance between DNA stability and impurity removal efficiency during high-temperature extraction. Traditional CTAB methods offer insufficient protection for DNA under high-temperature conditions and have limited effectiveness in removing polysaccharides and proteins.

[0013] This addresses the problem of DNA molecules easily denaturing and degrading under high-temperature conditions. Prolonged high-temperature treatment can affect the stability of the DNA double-stranded structure, leading to fragmentation.

[0014] This addresses the difficulty in DNA recovery caused by the over-stability of the CTAB-DNA complex. An excessively strong complex structure is difficult to dissociate in subsequent purification steps, affecting the final yield.

[0015] To achieve the above objectives, the present invention provides a method for extracting genomic DNA from plants rich in secondary metabolites, comprising the following steps: (1) Sample pretreatment: Plant tissues were ground into powder under liquid nitrogen, and a complexing agent, either polyethylene glycol (PEG) or polyvinylpyrrolidone (PVP), was added during the grinding process. In this step, PEG and PVP can form stable complexes with secondary metabolites such as alkaloids and polyphenols through hydrogen bonds, which are removed along with impurities in the subsequent rinsing step, thereby reducing the adsorption and interference of such substances on DNA from the source; at the same time, the pre-cooled mortar and continuous liquid nitrogen environment can inhibit nuclease activity and avoid DNA degradation.

[0016] (2) Cell wall lysis: Add cell wall lysis buffer to the powder obtained in step (1), mix well, centrifuge, and collect the supernatant; the cell wall lysis buffer contains 50-150 mM Tris-HCl, 1-2 M NaCl, 0.1-1% (v / v) β-mercaptoethanol, 15-25 mM dithiothreitol (DTT), and 0.1-0.3% (v / v) nonionic surfactant. In this step, DTT can specifically break the disulfide bonds of polyphenol oxidase, inhibit its activity, prevent polyphenols from being oxidized to quinones (avoiding cross-linking of quinones with DNA), and at the same time maintain the reducing environment of the system, protecting the DNA phosphodiester bonds from being oxidized and destroyed; Triton X-100 can destroy the plant cell membrane structure, promote the dissolution of lipids and membrane-bound proteins, and indirectly reduce the contamination of DNA by lipid-soluble alkaloids.

[0017] (3) DNA release and extraction: Add optimized CTAB extraction buffer to the supernatant obtained in step (2) and incubate in a water bath at 60-70℃ for 20-40 minutes. The optimized CTAB extraction buffer contains 1-3% (w / v) CTAB, 50-150 mM Tris-HCl, 1-2 M NaCl, 10-30 mM EDTA, 0.1-2% (w / v) polyethylene glycol, and 5-15 U / mL papain. In this step, polyethylene glycol can form hydrophobic complexes with polysaccharides and alkaloids, reducing their solubility in the aqueous phase and facilitating subsequent separation; papain can efficiently degrade protein impurities, reducing the impact of proteins on DNA purity, while avoiding the cumbersome low-temperature enzymatic hydrolysis step required by traditional proteinase K, thus improving extraction efficiency.

[0018] (4) Purification: The mixture after water bath in step (3) is purified to remove impurities and obtain a solution containing DNA; (5) Precipitation and dissolution: Isopropanol is added to the DNA-containing solution obtained in step (4) for precipitation. After washing with ethanol, it is dissolved in TE buffer to obtain high-purity genomic DNA.

[0019] Preferably, in step (1), the amount of complexing agent added is 0.5%-2% (w / w) of the sample mass, preferably 1%; the molecular weight of the polyethylene glycol PEG is 4000-8000, such as PEG6000, and the polyvinylpyrrolidone PVP is type K30.

[0020] Preferably, in step (2), the cell wall lysis buffer comprises: 100mM Tris-HCl, pH 8.0; 1.5M NaCl; 0.5% (v / v) β-mercaptoethanol; 20mM dithiothreitol DTT; and 0.2% (v / v) Triton X-100.

[0021] More preferably, the cell wall lysis buffer in step (2) also contains 5%-10% (w / v) trehalose and 0.5%-1.5% (w / v) pectinase; the lysis process is as follows: after adding the buffer and mixing, the mixture is first gently shaken at 28-37℃ for 20-40 minutes, and then allowed to stand at room temperature.

[0022] More preferably, in step (2), after the mild oscillation reaction at 28-37°C is completed and before the room temperature standing begins, pre-cooled CTAB powder is added to the pyrolysis system to a final concentration of 0.3%-0.8% (w / v), and gently mixed, and then placed on ice for 5-10 minutes.

[0023] Preferably, in step (3), the optimized CTAB extraction buffer comprises: 2% (w / v) CTAB; 100mM Tris-HCl, pH 8.0; 1.4M NaCl; 20mM EDTA; 0.1%-2% (w / v) polyethylene glycol; and 10 U / mL papain.

[0024] More preferably, in step (3), the optimized CTAB extraction buffer further contains 5-20 mM betaine and 0.5-2 mM spermidine.

[0025] More preferably, after the 65°C water bath in step (3) is completed and before the purification in step (4) begins, add Tween-20 with a final concentration of 0.1%-0.5% (v / v) to the mixture, mix gently, and let stand at room temperature for 5 minutes.

[0026] Preferably, in step (4), the purification is performed using organic phase extraction or column purification. The organic phase extraction method involves extracting the mixture after water bath using a chloroform-isoamyl alcohol mixture. The column purification method involves purifying the mixture using a silica gel column purification kit. In this step, chloroform-isoamyl alcohol can effectively remove proteins, and isoamyl alcohol can reduce foam generation, facilitating layering. Silica gel column purification can specifically adsorb DNA, rapidly remove small molecule impurities, significantly improve purification efficiency, and is suitable for batch sample processing.

[0027] Preferably, the plant tissue is derived from plants rich in alkaloids and / or polyphenols, including but not limited to *Gnaphalium affine* (from Guangxi). Stephania kwangsiensis ), Terminalia chebula ( Terminalia chebula ),persimmon( Diospyros kaki Datura ( ) Datura metel (Further preferably, the plant is a plant of the Menispermaceae family, specifically the genus *Tetrandica*, more preferably *Tetrandica dahurica* from Guangxi; the plant tissue is a bulb, leaf, or bud.)

[0028] This invention provides a cell wall lysis buffer for the extraction of plant genomic DNA, comprising 15-25 mM dithiothreitol (DTT) and 0.1%-0.5% (v / v) nonionic surfactant. Preferably, it consists of the following components in final concentrations: 100 mM Tris-HCl, 1.5 M NaCl, 0.5% (v / v) β-mercaptoethanol, 20 mM DTT, and 0.2% (v / v) Triton X-100. More preferably, the cell wall lysis buffer in step (2) further comprises 5%-10% (w / v) trehalose and 0.5%-1.5% (w / v) pectinase; the lysis process is as follows: after adding the buffer and mixing, the mixture is first gently shaken at 28-37°C for 20-40 minutes, and then allowed to stand at room temperature. More preferably, in step (2), after the mild oscillation reaction at 28-37°C is completed and before the room temperature standing begins, pre-cooled CTAB powder is added to the pyrolysis system to a final concentration of 0.3%-0.8% (w / v), and gently mixed, and then placed on ice for 5-10 minutes.

[0029] This invention provides a CTAB extraction buffer for the extraction of plant genomic DNA, comprising polyethylene glycol and papain. Preferably, it consists of the following components at final concentrations: 2% (w / v) CTAB, 100 mM Tris-HCl, 1.4 M NaCl, 20 mM EDTA, 1% (w / v) polyethylene glycol, and 10 U / mL papain. More preferably, in step (3), the optimized CTAB extraction buffer further comprises 5-20 mM betaine and 0.5-2 mM spermidine. More preferably, after the 65°C water bath incubation in step (3) and before the purification in step (4), Tween-20 at a final concentration of 0.1%-0.5% (v / v) is added to the mixture, gently mixed, and allowed to stand at room temperature for 5 minutes.

[0030] The application of any of the extraction methods provided by this invention, or any of the cell wall lysis buffers, or any of the CTAB extraction buffers, in any of the following: a. Plant genome sequencing; b. Development of plant molecular markers and their application in assisted breeding; c. Plant genetic diversity analysis and resource conservation; d. Molecular identification of medicinal plants; e. Research on the synthesis pathways of plant secondary metabolites.

[0031] The beneficial effects of this invention are as follows: By introducing specific treatments at multiple stages, a systematic impurity removal and protection mechanism was established. The complexing agent added during the grinding stage reduces interference from secondary metabolites at the source, and combined with optimized lysis and extraction buffers, forms a multi-layered protection system. This method significantly improves the quality and stability of DNA extraction, providing a reliable material basis for subsequent molecular experiments.

[0032] The addition of acidic silica sand significantly improved grinding efficiency, ensuring more thorough tissue disruption. Simultaneously, α-amylase effectively degraded starch in subsequent steps, preventing colloid formation from affecting DNA purification. This synergistic effect of physical and enzymatic hydrolysis solved a key technical challenge in processing samples with high starch content.

[0033] The lysis buffer creates a suitable lysis environment through the synergistic effect of its multiple components. DTT effectively inhibits polyphenol oxidase activity, preventing the formation of quinones, while the nonionic surfactant promotes membrane dissolution. This combination ensures efficient lysis while maximizing the protection of DNA molecules from chemical damage.

[0034] The introduction of trehalose and pectinase enables a mild and controlled lysis process. Pectinase specifically degrades cell wall components, while trehalose provides in-situ protection. This combination significantly reduces organelle DNA contamination and improves the relative purity of nuclear genomic DNA, making it particularly suitable for research requiring high-purity nuclear DNA.

[0035] The low-temperature CTAB pretreatment step establishes an early protection mechanism during lysis. CTAB preferentially stabilizes the released DNA under low-temperature conditions and reduces interference from impurities through selective precipitation. This innovative step significantly improves the final quality and yield of DNA without excessively affecting lysis efficiency.

[0036] The optimized CTAB buffer achieves highly efficient impurity removal through the synergistic effect of surfactants and enzymes. Polyethylene glycol forms complexes with polysaccharides and alkaloids, while papain degrades protein impurities. This dual impurity removal mechanism significantly improves DNA purity while maintaining the simplicity of the procedure.

[0037] The addition of betaine and spermidine provides effective thermal protection for DNA molecules. Betaine protects the DNA structure by stabilizing the hydration layer, while spermidine promotes complex formation through charge interaction. The synergistic effect of the two ensures the integrity and stability of DNA during high-temperature extraction.

[0038] Tween-20's modulatory effect solves the recovery problem caused by the over-stability of the complex. It modulates the complex structure through gentle competitive binding, making DNA more easily dissociated during purification. This improvement significantly enhances recovery efficiency while maintaining DNA quality.

[0039] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0040] Figure 1 The detection results of DNA extraction from different tissues using the comprehensive optimization scheme of the present invention in Example 4 are shown. Figure 2 The results of the comprehensive optimization scheme of the present invention and the conventional reagent kit in Example 5 are used to detect the extraction of different plant materials. Detailed Implementation

[0041] The present invention will be further described in detail below with reference to examples, so that those skilled in the art can implement it based on the description.

[0042] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0043] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0044] Example 1 This embodiment provides an optimized method for extracting genomic DNA from *Gnaphalium affine* bulbs from Guangxi.

[0045] Plant materials: Fresh, healthy Guangxi local herbs ( Stephania kwangsiensis )bulb.

[0046] Main reagents: liquid nitrogen, polyethylene glycol (PEG 6000-10000), cell wall lysis buffer (formula below), optimized CTAB extraction buffer (formula below), chloroform-isoamyl alcohol (24:1, v / v), isopropanol (pre-cooled), 70% ethanol (pre-cooled), TE buffer (pH 8.0).

[0047] Main equipment: mortar and pestle (pre-cooled), high-speed refrigerated centrifuge, constant temperature water bath, vortex shaker, ultraviolet spectrophotometer, agarose gel electrophoresis system.

[0048] Experimental steps I. Sample Collection and Pretreatment Select healthy, disease-free plants from Guangxi and dig up their underground corms. Use a sterile blade to peel off the outer skin of the corm and cut off about 5 grams of fresh internal tissue for later use.

[0049] II. Rapid Freezing and Grinding Place 5g of bulb tissue into liquid nitrogen and rapidly freeze until completely stiff. Place a pre-cooled mortar (refrigerated at -20°C for more than 2 hours) on the lab bench and quickly add 50 mg (1% of the sample mass) of polyvinylpyrrolidone (PVP K30). Transfer the frozen bulb tissue to the mortar and, under continuous liquid nitrogen replenishment, grind rapidly and thoroughly until the sample becomes a uniform and fine powder (particle size <100μm). Thawing of the sample must be avoided throughout the process.

[0050] III. Cell wall lysis and preliminary removal of impurities Quickly transfer the ground powder into a 50 mL centrifuge tube. Immediately add 25 mL of pre-warmed (37°C) cell wall lysis buffer (sample mass: buffer volume = 1:5) to the centrifuge tube. Cell wall lysis buffer composition (final concentration): 100 mM Tris-HCl (pH 8.0); 1.5 M NaCl; 0.5% (v / v) β-mercaptoethanol; 20 mM dithiothreitol (DTT); 0.2% (v / v) Triton X-100.

[0051] Gently invert the centrifuge tube 6-7 times to thoroughly mix the powder and buffer. Then let it stand at room temperature for 20 minutes, gently shaking the tube every 5 minutes. After standing, place the tube in a high-speed centrifuge and centrifuge at 12000×g for 10 minutes at 4°C. After centrifugation, carefully aspirate the clear supernatant using a sterile pipette and transfer it to a new sterile 50 mL centrifuge tube. Discard the precipitate.

[0052] IV. DNA Release and Enhanced Extraction Add an equal volume (25 mL) of optimized CTAB extraction buffer, preheated to 65°C, to the collected supernatant. Optimized CTAB extraction buffer composition (final concentration): 2% (w / v) CTAB; 100 mM Tris-HCl (pH 8.0); 1.4 M NaCl; 20 mM EDTA; 1% (w / v) polyethylene glycol (PEG-8000); 10 U / mL papain.

[0053] Gently invert the centrifuge tube 10-12 times to mix thoroughly. Place the centrifuge tube in a 65°C water bath and incubate for 30 minutes. During this time, gently shake the centrifuge tube every 5 minutes to ensure complete reaction.

[0054] V. Deep Impurity Removal and Purification (This example uses organic phase extraction) After the water bath, cool the mixture to room temperature. Add an equal volume of chloroform-isoamyl alcohol mixture (24:1, v / v), tighten the cap, and gently invert the centrifuge tube 25 times to thoroughly mix and emulsify the two phases. Let stand at room temperature for 5 minutes. Then, centrifuge at 12000×g for 10 minutes. At this point, the solution will separate into three layers: an upper aqueous phase containing DNA, a middle protein layer, and a lower organic phase. Carefully aspirate the upper aqueous phase and transfer it to a new centrifuge tube. Repeat the chloroform-isoamyl alcohol extraction procedure once to further remove proteins and lipid-soluble impurities.

[0055] VI. DNA Precipitation and Dissolution Add 0.8 volumes of pre-chilled (-20°C) isopropanol to the final aqueous solution. Gently invert the centrifuge tube 5-6 times to mix. Place the centrifuge tube in a -20°C freezer for 30 minutes to promote the formation of a flocculent DNA precipitate. After standing, centrifuge at 12000 × g for 10 minutes. Carefully discard the supernatant; a white flocculent DNA precipitate will be visible at the bottom of the tube. Add 1 mL of pre-chilled 70% ethanol to the precipitate and gently invert several times to wash the precipitate and remove residual salts. Centrifuge at 12000 × g for 5 minutes and carefully discard the ethanol. Invert the centrifuge tube onto sterile filter paper and air dry in a well-ventilated area at room temperature for about 15-20 minutes, until the ethanol has completely evaporated (Note: Do not over-dry, otherwise the DNA will be difficult to dissolve). Add 100 μL of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) to the dried DNA precipitate, gently pipette or briefly place in a 55°C water bath to aid dissolution, until the DNA is completely dissolved. Store the extracted DNA solution at -20°C for later use.

[0056] When performing concentration and purity testing: use a UV spectrophotometer to measure the absorbance values ​​of the DNA solution at 260nm, 280nm, and 230nm, calculate the A260 / A280 ratio (pure DNA ratio is 1.8~2.0) and the A260 / A230 ratio (pure DNA ratio >2.0 indicates no alkaloids or salts remaining), and calculate the DNA concentration based on the A260 value (1A260≈50ng / μL).

[0057] When performing integrity testing: add an appropriate amount of nucleic acid dye, mix the DNA sample with loading buffer, and spot it onto a 1% (w / v) agarose gel. Use 1×TAE buffer as the electrophoresis buffer and electrophoresis at 5V / cm for 30 minutes. Then observe the DNA bands under the gel imaging system: if a clear, bright single main band appears without diffuse bands, it indicates that the DNA integrity is good and there is no obvious degradation.

[0058] Example 2 The difference from Example 1 lies in the cell wall lysis and preliminary impurity removal steps: Quickly transfer the ground powder into a 50 mL centrifuge tube. Immediately add 25 mL of optimized cell wall lysis buffer (sample mass: buffer volume = 1:5) preheated to 37°C to the centrifuge tube.

[0059] Further optimize the composition (final concentration) of the cell wall lysis buffer: 100 mM Tris-HCl (pH 8.0); 1.5 M NaCl; 0.5% (v / v) β-mercaptoethanol; 20 mM dithiothreitol (DTT); 0.2% (v / v) Triton X-100; 8% (w / v) Trehalose; 1.0% (w / v) pectinase; Gently invert the centrifuge tube 6-7 times to thoroughly mix the powder and buffer. Crucially, place the centrifuge tube in a shaker and gently vortex at 50 rpm for 30 minutes at 30°C. After the vortex reaction, remove the centrifuge tube and let it stand at room temperature for 20 minutes, gently shaking it every 5 minutes. After standing, place the centrifuge tube in a high-speed centrifuge and centrifuge at 12000×g for 10 minutes at 4°C. After centrifugation, carefully aspirate the clear supernatant using a sterile pipette and transfer it to a new sterile 50 mL centrifuge tube. Discard the precipitate.

[0060] The other steps are the same as in Example 1.

[0061] Example 3 The difference from Example 2 lies in the cell wall lysis and preliminary impurity removal: Quickly transfer the ground powder into a 50 mL centrifuge tube. Immediately add 25 mL of pre-warmed (37°C) optimized cell wall lysis buffer (same composition as in Example 2) to the centrifuge tube. Gently invert the centrifuge tube 6-7 times to thoroughly mix the powder with the buffer. Place the centrifuge tube in a shaker and gently oscillate at 50 rpm for 30 minutes at 30°C. Crucially, after the oscillation reaction, add 125 mg of pre-cooled CTAB powder to the lysis system (to a final concentration of 0.5% w / v), and gently invert the centrifuge tube 5-6 times to thoroughly dissolve and mix. Place the centrifuge tube on ice for 8 minutes.

[0062] After standing on ice, allow the centrifuge tubes to stand at room temperature for 20 minutes, gently shaking them every 5 minutes. After standing, place the tubes in a high-speed centrifuge and centrifuge at 12000×g for 10 minutes at 4°C. After centrifugation, carefully aspirate the clear supernatant using a sterile pipette and transfer it to a new sterile 50 mL centrifuge tube. Discard the precipitate.

[0063] The other steps are the same as in Example 2.

[0064] Example 4 The difference from Example 1 lies in the DNA release and enhanced extraction steps: Add an equal volume (25 mL) of further optimized CTAB extraction buffer, preheated to 65°C, to the collected supernatant. Further optimized CTAB extraction buffer composition (final concentration): 2% (w / v) CTAB; 100 mM Tris-HCl (pH 8.0); 1.4 M NaCl; 20 mM EDTA; 1% (w / v) polyethylene glycol (PEG-8000); 10 U / mL papain; 15 mM betaine; 1.0 mM spermidine; Gently invert the centrifuge tube 10-12 times to mix it thoroughly.

[0065] Place the centrifuge tubes in a 65°C water bath and incubate for 30 minutes. Gently shake the centrifuge tubes every 5 minutes during this time to ensure a complete reaction.

[0066] The other steps are the same.

[0067] Example 5 The difference from Example 4 is that, in the DNA release and enhanced extraction steps, the optimized CTAB extraction buffer containing betaine and spermidine was used for incubation at 65°C for 30 minutes, similar to Example 4. Crucially, after the 65°C incubation, 125 μL of Tween-20 was added to the mixture (to a final concentration of 0.25% v / v). The centrifuge tube was gently inverted 8-10 times to ensure thorough mixing. The mixture was then allowed to stand at room temperature for 5 minutes.

[0068] The other steps are the same as in Example 4.

[0069] Example 6 The difference from Example 1 is that in the rapid freezing and grinding step, 5g of bulb tissue was placed in liquid nitrogen and rapidly frozen until completely stiff. A pre-cooled mortar (refrigerated at -20°C for over 2 hours) was placed on the lab bench, and the following were added sequentially: 50 mg (1% of sample mass) of polyvinylpyrrolidone (PVP K30); 75 mg (1.5% of sample mass) of acidic silica sand; and 5 mg (0.1% of sample mass) of α-amylase powder. The α-amylase used was a thermostable α-amylase from Bacillus licheniformis. This enzyme has an optimal operating temperature above 90°C and is stable over a wide pH range of 5.5-8.0. The frozen bulb tissue was transferred to the mortar and rapidly and thoroughly ground under continuous liquid nitrogen supply until the sample became a uniform and fine powder (particle size <100μm). Thawing of the sample was avoided throughout the process. The method for preparing acidic quartz sand is as follows: ordinary quartz sand is soaked in hydrochloric acid solution with pH 5.0 for 2 hours, then rinsed with deionized water until neutral, and dried for later use.

[0070] The other steps are the same as in Example 1.

[0071] In this embodiment, acidic silica sand, used as a grinding aid, significantly improved the efficiency of breaking down fibrous bulb tissue. The alkaloids adsorbed and released on the surface of the acidic silica sand during grinding form a dual protective layer with PVP. α-Amylase is activated in the subsequent water bath step, effectively degrading starch in the sample and preventing the formation of a gel. This method significantly improves DNA yield and purity while maintaining excellent DNA integrity.

[0072] Effect Experiment Example 1 - Effect of additives in the grinding stage on DNA extraction efficiency 1. Experimental Materials and Grouping Fresh bulb tissues from Guangxi *Gnaphalium affine* with the same batch and consistent growth status were obtained. Four treatment groups were set up, with three replicates in each group and a sample mass of 5g per replicate.

[0073] The groups are as follows: Control group: No additives were added during grinding, and the process was carried out according to the method in Example 1.

[0074] PEG group: Add 1% PEG-6000 during grinding, and operate according to the method of Example 1.

[0075] PVP group: Add 1% PVP (K30) during grinding, and operate according to the method of Example 1.

[0076] Optimized group: Add 1% PVP (K30) + 1.5% acidic quartz sand + 0.1% α-amylase dry powder during grinding, and operate according to the method of Example 6.

[0077] 2. Experimental Methods DNA extraction was performed in all groups according to the complete procedure of the corresponding embodiment, including the following steps: Sample collection and pretreatment.

[0078] Rapid freezing and grinding (add appropriate additives according to the grouping scheme).

[0079] Cell wall lysis and preliminary removal of impurities.

[0080] DNA release and enhanced extraction.

[0081] Deep impurity removal and purification.

[0082] DNA precipitation and dissolution.

[0083] Table 1. Effects of different grinding aids on DNA extraction quality As shown in the table above, the optimized group had the highest extracted DNA concentration (0.70 μg / μL), significantly higher than other groups. Compared with the control group, the DNA concentrations in the PEG group and PVP group increased by 81.25% and 90.63%, respectively. All experimental groups had A260 / A280 ratios within the pure DNA standard range (1.8-2.0), while the control group's ratio was lower (1.52), indicating protein contamination. Regarding the A260 / A230 ratio, the optimized group had the highest ratio (2.26), followed by the PVP group (2.15), both significantly higher than the control group (1.35), indicating significant removal of impurities such as alkaloids and polysaccharides.

[0084] Therefore, adding additives during the grinding stage can significantly improve DNA extraction efficiency. PEG / PVP alone can significantly improve DNA quality and purity, with PVP showing slightly better results than PEG. The combined optimized scheme (PVP + acidic silica sand + α-amylase) achieved the best results, realizing the highest DNA yield (118.75% higher than the control group), the best purity index (A260 / A230 ratio of 2.26), and the best DNA integrity. In practical applications, the combined optimized scheme is recommended; if resources are limited, PVP or PEG can be chosen as alternatives.

[0085] Example 2 - The effect of optimizing cell wall lysis buffer on DNA extraction efficiency 1. Obtain fresh bulb tissue from the same batch of Guangxi ground bulbs. Set up 4 treatment groups, with 3 replicates in each group and 5g sample mass in each replicate.

[0086] The groups are as follows: DTT-free group: No DTT was added to the cell wall lysis buffer, and the procedure was performed according to Example 1.

[0087] Group with DTT: 20 mM DTT was added to the cell wall lysis buffer and the procedure was followed as described in Example 1.

[0088] Trehalose optimization group: 20 mM DTT, 8% trehalose, and 1.0% pectinase were added to the cell wall lysis buffer, and the procedure was followed as described in Example 2.

[0089] CTAB pretreatment group: Based on Example 2, pre-cooled CTAB powder (target final concentration 0.5%) was added, and the procedure was followed as in Example 3.

[0090] 2. Experimental Methods All groups underwent DNA extraction according to the complete procedure of the corresponding embodiment, including sample processing, cell wall lysis, DNA extraction, and purification.

[0091] Table 2. Effect of cell wall lysis buffer optimization on DNA extraction quality As shown in the table above, the addition of DTT increased DNA concentration by 71.43%, and both the A260 / A280 and A260 / A230 ratios met the standards for high-quality DNA. The synergistic effect of trehalose and pectinase further improved DNA purity, increasing the A260 / A230 ratio to 2.22. Further CTAB pretreatment resulted in the highest DNA concentration (0.65 μg / μL), optimal purity, and significant enhancement. PCR amplification results showed that all optimized groups of DNA were free of amplification inhibitors, making them suitable for downstream molecular experiments.

[0092] Therefore, adding 20 mM DTT to the cell wall lysis buffer effectively inhibits polyphenol oxidation, prevents the formation of quinones, and significantly improves DNA quality and PCR usability. The addition of trehalose and pectinase further enhances DNA purity.

[0093] CTAB pretreatment improves yield while maintaining DNA integrity.

[0094] Example 3 - The effect of CTAB extraction buffer optimization on DNA extraction efficiency 1. Fresh bulb tissue from the same batch of Guangxi Dibu was collected and divided into 6 treatment groups with 3 replicates per group. The sample mass of each replicate was 5g.

[0095] The groups are as follows: Blank group: CTAB buffer without polyethylene glycol and papain, and operated according to the method in Example 1.

[0096] Polyethylene glycol only: Add 1% polyethylene glycol (PEG-8000) to CTAB buffer and proceed as described in Example 1.

[0097] Pure papain group: 10 U / mL papain was added to CTAB buffer and the procedure was performed according to Example 1.

[0098] Polyethylene glycol + papain group: 1% polyethylene glycol and 10 U / mL papain were added to CTAB buffer and the procedure was performed according to Example 1.

[0099] Composite protective agent group: CTAB buffer was prepared by adding 1% polyethylene glycol, 10 U / mL papain, 15 mM betaine and 1.0 mM spermidine, and following the procedure in Example 4.

[0100] Fully optimized group: Based on the composite protective agent group, 0.25% Tween-20 was added after water bath, and the procedure was followed as in Example 5.

[0101] 3. Experimental Methods DNA extraction was performed in all groups according to the complete procedure of the corresponding embodiment, with a focus on the impact of CTAB extraction buffer optimization on DNA quality and protein residue.

[0102] Table 3. Effect of CTAB extraction buffer optimization on DNA extraction quality As shown in the table above, the pure papain group significantly reduced protein residue (by 37.5%) and increased DNA concentration by 37.5%, but its effect on polysaccharide removal was limited (A260 / A230 = 1.82). Only the polyethylene glycol group effectively removed polysaccharides and alkaloids (A260 / A230 = 2.05), but the improvement in protein residue was not significant. The synergistic effect of polyethylene glycol + papain group was significant, increasing DNA concentration by 55% and reducing protein residue by 54.2%.

[0103] From the basic combination of polyethylene glycol (PEG) and papain to the composite protectant, the fully optimized regimen increased DNA concentration by 16.1%, a 95% improvement compared to the control group, and reduced protein residue by 75%. The A260 / A230 ratio increased from 1.50 to 2.30, indicating a significant impurity removal effect. This is because papain is mainly responsible for protein degradation, while PEG is mainly responsible for the removal of polysaccharides and alkaloids. Therefore, both the pure papain group and the PEG-only group can only partially improve DNA quality. The combination of PEG and papain has a synergistic effect, significantly improving DNA integrity. The fully optimized group showed the best DNA integrity and purity.

[0104] Example 4 - DNA extraction experiments on different tissues using a comprehensive optimization scheme Experimental steps I. Sample Collection and Pretreatment Fresh bulbs, mature leaves, and tender shoots were collected from the same plant in Guangxi. Three replicates were set for each tissue, and the sample mass of each replicate was 5g. DNA was extracted according to the following experimental steps.

[0105] II. Rapid Freezing and Grinding Place 5g of sample tissue into liquid nitrogen and freeze rapidly until completely stiff. Place the pre-cooled (-20℃ refrigerated for more than 2 hours) mortar on the lab bench and add the following in sequence: 50 mg PVP K30 (1% of sample mass), 75 mg acidic silica sand (1.5% of sample mass), and 5 mg α-amylase powder (0.1% of sample mass). Transfer the frozen sample tissue to the mortar and grind rapidly and thoroughly under continuous liquid nitrogen supply until the sample becomes a uniform and fine powder (particle size <100μm).

[0106] III. Cell wall lysis and preliminary removal of impurities Quickly transfer the ground powder to a 50 mL centrifuge tube, add 25 mL of optimized cell wall lysis buffer preheated to 37°C: 100 mM Tris-HCl (pH 8.0), 1.5 M NaCl, 0.5% (v / v) β-mercaptoethanol, 20 mM DTT, 0.2% (v / v) Triton X-100, 8% (w / v) trehalose, and 1.0% (w / v) pectinase. Gently invert the centrifuge tube 6-7 times to mix.

[0107] Then proceed with key step 1: react gently at 30℃ with 50 rpm for 30 minutes; key step 2: add 125 mg of pre-cooled CTAB powder (final concentration 0.5%), mix gently and let stand on ice for 8 minutes; key step 3: let stand at room temperature for 20 minutes, gently shaking once every 5 minutes, centrifuge at 4℃ and 12000×g for 10 minutes, and take the supernatant.

[0108] IV. DNA Release and Enhanced Extraction Add 25 mL of pre-warmed (65°C) optimized CTAB extraction buffer to the supernatant: 2% (w / v) CTAB, 100 mM Tris-HCl (pH 8.0), 1.4 M NaCl, 20 mM EDTA, 1% (w / v) polyethylene glycol (PEG-8000), 10 U / mL papain, 15 mM betaine, and 1.0 mM spermidine. Gently invert to mix, and incubate at 65°C for 30 minutes, shaking every 5 minutes during incubation.

[0109] V. Regulation of Complex Stability After the water bath, add 125 μL of Tween-20 (final concentration 0.25%), gently invert to mix, and let stand at room temperature for 5 minutes.

[0110] VI. Impurity Removal and Purification Organic phase extraction method: Add an equal volume of chloroform-isoamyl alcohol mixture, gently invert 25 times, let stand at room temperature for 5 minutes, centrifuge at 12000×g for 10 minutes, take the upper aqueous phase, and repeat the extraction once.

[0111] VII. DNA Precipitation and Dissolution Add 0.8 times the volume of pre-cooled isopropanol, let stand at -20℃ for 30 minutes, centrifuge at 12000×g for 10 minutes, collect the DNA precipitate, wash the precipitate with 1 mL of pre-cooled 70% ethanol, air dry at room temperature, dissolve in 100 μL of TE buffer, and store at -20℃ for later use.

[0112] The results are shown in the table below: Table 4. DNA extraction results from different tissues using the comprehensive optimization scheme. From the table above and Figure 1 As can be seen, mature leaves had the highest DNA concentration (0.92 μg / μL), which is related to their active cell division and relatively low amount of secondary metabolites. Young shoots had the next highest concentration (0.87 μg / μL), indicating vigorous cell division but a more delicate tissue. Fresh bulbs had a relatively low concentration (0.81 μg / μL), which is related to their high content of starch, alkaloids, and other secondary metabolites. The A260 / A280 ratio of all tissues was between 1.96 and 1.98, meeting the standard for high-quality DNA. The A260 / A230 ratio was as follows: leaves (2.42) > young shoots (2.38) > bulbs (2.35). The highest protein residue was found in bulbs, consistent with the higher protein content in bulb tissue. Figure 1 In the results, 1 represents the result from the bulb, 2 from the leaf, and 3 from the bud. All tissues yielded complete high-molecular-weight DNA with good DNA integrity and sharp bands.

[0113] The aforementioned comprehensive optimization scheme successfully overcame the specific challenges posed by different tissues: acidic quartz sand and α-amylase effectively solved the problems of bulb grinding and starch contamination; the mild lysis system of trehalose and pectinase was suitable for tender tissues such as buds; and the composite protectant system provided comprehensive protection for the DNA of each tissue. This comprehensive optimization scheme is applicable to different tissue types in Guangxi, all of which can yield high-quality DNA. Leaf tissue is recommended for genomics research, bud tissue can be selected for developmental biology research, and bulbs are the focus for medicinal component research.

[0114] Example 5 The comprehensive optimization scheme in Example 4 of this invention was used to evaluate the DNA extraction effect on other alkaloid-rich plants. Plant materials: Fresh leaves of Terminalia chebula; Fresh persimmon (Diospyros kaki) leaves; Fresh rhizomes of Datura metel.

[0115] Experimental design: Three biological replicates were set up for each plant, with each replicate sample weighing 5g. DNA extraction was performed according to the comprehensive optimization scheme of Example 4.

[0116] In comparison, the effectiveness of a conventional commercial plant genomic DNA extraction kit in treating plant materials rich in alkaloids, polyphenols, and other secondary metabolites was compared. Specifically, the TIANGEN Biotech DP350 plant genomic DNA extraction kit was used, following the kit's instructions.

[0117] The test results are as follows: Table 5. Comparison of DNA extraction effects of comprehensive optimization schemes on different alkaloid-rich plants. From the table above and Figure 2 As can be seen, the DNA concentration extracted by the conventional kit method is only 0.18-0.28 μg / μL, far lower than the 0.62-0.75 μg / μL of the method of this invention. This indicates that the standard lysis and purification process of the kit cannot effectively cope with the interference of high concentrations of secondary metabolites, resulting in a large loss or degradation of DNA. The A260 / A280 ratio of the conventional kit group is generally below 1.5, indicating serious protein contamination. This is because the lysis buffer and washing buffer of the kit failed to effectively remove proteins tightly bound to DNA. The A260 / A230 ratio of the conventional kit group is far below 2.0, especially the ratio of Datura stramonium root and stem below 1.0, indicating a large amount of small molecule impurities such as polysaccharides, alkaloids, and polyphenols remaining. These impurities severely inhibit downstream enzyme reactions.

[0118] The DNA concentrations in the leaves of Terminalia chebula and Persimmon obtained using the comprehensive optimization scheme of this invention were relatively high (>0.70 μg / μL), possibly related to their relatively simple tissue structure and low secondary metabolite content. The DNA concentration in the rhizomes of Datura stramonium was relatively low (0.62 μg / μL), possibly related to its rich content of tropane alkaloids and complex root tissue structure. The A260 / A280 ratios of all plants were between 1.88 and 1.92, indicating effective control of protein contamination. The A260 / A230 ratios were all greater than 2.0, proving thorough removal of impurities such as polysaccharides and alkaloids. The purity index of Datura stramonium rhizomes was slightly lower, consistent with its higher secondary metabolite content.

[0119] Figure 2 In the figures, 2, 4, and 6 represent the PCR amplification results of Terminalia chebula, Dioscorea opposita, and Datura stramonium using conventional kits, respectively; while 1, 3, and 5 represent the PCR amplification results of the optimized integrated scheme for Terminalia chebula, Dioscorea opposita, and Datura stramonium, respectively. The PCR amplification results show that the DNA extracted using the conventional kits amplified weak bands with unclean backgrounds. In contrast, the optimized integrated scheme of this invention yielded bright, specific, single target bands in all samples, with no extraneous bands or non-specific amplification, proving that the extracted DNA contained no residual PCR inhibitors.

[0120] The reason why the comprehensive optimization scheme of this invention is successfully applied to different high-alkaloid plants is that PVP and acidic quartz sand effectively solve the problem of instantaneous alkaloid release; the dual protection of trehalose and DTT prevents polyphenol oxidation; the synergistic effect of polyethylene glycol and papain removes polysaccharides and proteins; and betaine and spermidine provide high-temperature protection. Leaf tissues, due to their relatively simple structure, are easier to obtain high-quality DNA from. Although root and stem tissues have high secondary metabolite content, DNA meeting the requirements of molecular experiments can still be obtained through the optimization scheme. This comprehensive optimization scheme has been successfully applied to three high-alkaloid plants: Terminalia chebula, Diospyros kaki, and Datura stramonium, demonstrating the method's good universality. It provides a reliable scheme for DNA extraction from other medicinal plants rich in secondary metabolites, suitable for research such as genetic diversity analysis and molecular marker development, and also provides technical support for the protection of rare medicinal plant resources.

[0121] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. It can be applied to various fields suitable for the present invention. Further modifications can be readily implemented by those skilled in the art.

Claims

1. A method for extracting genomic DNA from plants rich in secondary metabolites, characterized in that, Includes the following steps: (1) Sample pretreatment: The plant tissue sample was ground into powder in liquid nitrogen environment, and a complexing agent was added during the grinding process. The complexing agent was polyethylene glycol (PEG) or polyvinylpyrrolidone (PVP). The amount of complexing agent added was 0.5%-2% (w / w) of the sample mass. The molecular weight of polyethylene glycol (PEG) was 4000-8000, and the polyvinylpyrrolidone (PVP) was of type K30. (2) Cell wall lysis: Add cell wall lysis buffer to the powder obtained in step (1), mix well, centrifuge, and collect the supernatant; the cell wall lysis buffer contains 50-150 mM Tris-HCl, 1-2 M NaCl, 0.1-1% (v / v) β-mercaptoethanol, 15-25 mM dithiothreitol DTT and 0.1-0.3% (v / v) nonionic surfactant; (3) DNA release and extraction: Add optimized CTAB extraction buffer to the supernatant obtained in step (2) and incubate in a water bath at 60-70℃ for 20-40 minutes. The optimized CTAB extraction buffer contains 1-3% (w / v) CTAB, 50-150 mM Tris-HCl, 1-2 M NaCl, 10-30 mM EDTA, 0.1-2% (w / v) polyethylene glycol and 5-15 U / mL papain. (4) Purification: The mixture after water bath in step (3) is purified to remove impurities and obtain a solution containing DNA; (5) Precipitation and dissolution: Isopropanol is added to the DNA-containing solution obtained in step (4) for precipitation. After washing with ethanol, it is dissolved in TE buffer to obtain high-purity genomic DNA.

2. The extraction method according to claim 1, characterized in that, In step (1), acidic quartz sand accounting for 0.5%-2% of the sample mass is added simultaneously as a grinding aid, and α-amylase dry powder accounting for 0.05%-0.2% of the sample mass is added simultaneously during grinding; The acidic quartz sand is pretreated with hydrochloric acid at pH 4.5-5.

5.

3. The extraction method according to claim 2, characterized in that, In step (2), the cell wall lysis buffer comprises: 100mM Tris-HCl, pH 8.0; 1.5M NaCl; 0.5% (v / v) β-mercaptoethanol; 20mM dithiothreitol (DTT); 0.2% (v / v) Triton X-100.

4. The extraction method according to claim 3, characterized in that, The cell wall lysis buffer in step (2) also contains 5%-10% (w / v) trehalose and 0.5%-1.5% (w / v) pectinase; the lysis process is as follows: after adding the buffer and mixing, gently shake at 28-37℃ for 20-40 minutes, and then let stand at room temperature.

5. The extraction method according to claim 4, characterized in that, After the mild oscillation reaction at 28-37℃ is completed and before the room temperature settling begins, add pre-cooled CTAB powder to the pyrolysis system to a final concentration of 0.3%-0.8% (w / v), mix gently, and let stand on ice for 5-10 minutes.

6. The extraction method according to claim 5, characterized in that, In step (3), the optimized CTAB extraction buffer comprises: 2% (w / v) CTAB; 100mM Tris-HCl, pH 8.0; 1.4M NaCl; 20mM EDTA; 1% (w / v) polyethylene glycol; 10 U / mL papain.

7. The extraction method according to claim 6, characterized in that, In step (3), the optimized CTAB extraction buffer also contains 5-20 mM betaine and 0.5-2 mM spermidine.

8. The extraction method according to claim 7, characterized in that, After the 65°C water bath in step (3) is completed and before the purification in step (4) begins, add Tween-20 with a final concentration of 0.1%-0.5% (v / v) to the mixture, mix gently, and let stand at room temperature for 5 minutes.

9. A cell wall lysis buffer for extracting plant genomic DNA, characterized in that, The cell wall lysis buffer is used in the method for extracting genomic DNA from plants rich in secondary metabolites as described in claim 1.

10. A CTAB extraction buffer for extracting plant genomic DNA, characterized in that, This is an optimized CTAB extraction buffer for the method of extracting genomic DNA from plants rich in secondary metabolites as described in claim 1.

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