Pubhlh35 gene and application and preparation method of biochar

Abstract

The application relates to the technical field of harmless treatment of hyperaccumulators, in particular to a PubHLH35 gene and application and a preparation method of biochar. The nucleotide sequence of the PubHLH35 gene is shown in SEQ ID No. 1. The application finds that the PubHLH35 gene improves the absorption capacity of Populus nigra for Cd stress, and the overexpression transgenic variety cultivated has important theoretical and practical significance in the preparation of biochar for remediation of soil Cd pollution.

Description

Technical Field

[0001] This invention relates to the field of harmless treatment technology for hyperaccumulating plants, and in particular to a PubHLH35 gene and its application, and a method for preparing biochar. Background Technology

[0002] Cadmium (Cd) pollution in soil is widespread worldwide. It is one of the most harmful elements, possessing toxicity. It can affect important physiological and metabolic processes such as photosynthesis and respiration through ion substitution, disrupting the balance of reactive oxygen species, damaging signal transduction pathways, and damaging biological lipid membrane structures, ultimately inhibiting plant growth and development and having a negative impact on plant growth.

[0003] Cadmium pollution has been a serious problem for decades, primarily due to increased pollution from industrial and agricultural practices such as wastewater irrigation and excessive use of fertilizers. Many approaches exist for remediating Cd-contaminated soils, including soil treatment, soil excavation, and chemical methods. In addition, phytoremediation has been proposed as a potentially more effective and cost-efficient method. Hyperaccumulating plants, particularly fast-growing woody species like poplar, can absorb and accumulate large amounts of the toxic metal Cd from contaminated soils. Generally, woody plants accumulate more Cd than herbaceous hyperaccumulators due to their deep root systems and large biomass. Therefore, this invention proposes a biochar recovery strategy as a safe and value-added method for processing Cd-rich poplar branches and leaves, providing a reference for the breeding of transgenic poplars to remediate Cd-contaminated soils. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a PubHLH35 gene and its applications, as well as a method for preparing biochar. The PubHLH35 gene provided by this invention has the effect of enhancing the accumulation of heavy metals in plants, and preparing biochar from plants overexpressing this gene can promote the accumulation of heavy metals.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] This invention provides a PubHLH35 gene, the nucleotide sequence of which is shown in SEQ ID No. 1.

[0007] This invention also provides the application of overexpressing the PubHLH35 gene described in the above technical solution in improving the accumulation of heavy metals in plants.

[0008] Preferably, the plant includes Populus tomentosa.

[0009] Preferably, the heavy metal includes cadmium.

[0010] This invention also provides a method for preparing biochar, comprising the following steps:

[0011] 1) Overexpress the PubHLH35 gene described in the above technical solution in plants to obtain overexpressed plants;

[0012] 2) The overexpressed plant obtained in step 1) is thermally decomposed to obtain biochar.

[0013] Preferably, the conditions for thermal decomposition in step 2) include: a heating rate of 20°C / min, a temperature of 500°C, and a time of 20min.

[0014] Preferably, in step 1), the PubHLH35 gene is ligated into the pBI121-GFP vector to obtain a recombinant vector;

[0015] The recombinant vector was transferred into Agrobacterium to obtain the transformant bacteria;

[0016] The transformed bacteria were used to infect plants to obtain overexpressed plants.

[0017] Preferably, the Agrobacterium includes Agrobacterium GV3101; the plant includes Populus tomentosa.

[0018] This invention also provides the application of cadmium-rich biochar prepared by the preparation method described above in the remediation of cadmium-contaminated soil.

[0019] Preferably, the application includes applying the biochar to the soil at an amount of 5-10% of the soil mass.

[0020] Beneficial effects:

[0021] The application of the Populus tomentosa PubHLH35 gene in this invention refers to enhancing the absorption and translocation capacity of Populus tomentosa under Cd stress and preparing biochar to remediate soil cadmium pollution.

[0022] This invention discloses the application of the PubHLH35 gene. Using Agrobacterium-mediated overexpression of this gene in *Populus tomentosa*, transgenic lines of *Populus tomentosa* were obtained, effectively enhancing the translocation and absorption capacity of *Populus tomentosa* for Cd. Under Cd stress, the transgenic lines showed significantly reduced plant height, diameter at root, and dry weight compared to wild-type plants, preliminarily indicating that under drought stress, the overexpressing PubHLH35 transgenic lines suffered relatively more damage and exhibited stronger Cd absorption capacity.

[0023] Under Cd stress, the stomatal conductance, transpiration rate, and net photosynthetic rate of transgenic lines were significantly lower than those of wild-type plants, while the intercellular CO2 concentration of transgenic plants was significantly higher than that of wild-type plants. Chlorophyll content analysis revealed that the chlorophyll content of overexpressing transgenic plants was significantly lower than that of wild-type plants. Furthermore, Cd... 2+ The determination of Cd content and comparison revealed that under Cd stress, the Cd content of transgenic plants was significantly lower. 2+ The content was significantly higher than that of the wild type, and these data also verified that the overexpression lines of the PubHLH35 gene are effective against Cd. 2+ The absorption capacity is significantly higher than that of the wild type. This invention reveals that PubHLH35 enhances the absorption capacity of Populus tomentosa under Cd stress, and the resulting overexpression transgenic variety has important theoretical and practical significance for the preparation of biochar to remediate Cd-contaminated soil. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0025] Figure 1 This is a gel electrophoresis image of the PCR product of the Populus alba PubHLH35 gene in this invention, where M is the DL2000 DNA Marker and 1-3 are the PCR products.

[0026] Figure 2 This is the nucleotide sequence of the Populus alba PubHLH35 gene in this invention;

[0027] Figure 3 This is a PCR diagram of the Populus alba PubHLH35 gene constructed into the pBI121-GFP overexpression vector in this invention, where M is the DL2000 DNA Marker and 1-9 are the PubHLH35 gene.

[0028] Figure 4 This invention relates to the detection of DNA levels in Populus tomentosa PubHLH35 gene overexpression lines.

[0029] Figure 5 This invention provides a quantitative analysis of RNA levels in Populus tomentosa PubHLH35 gene overexpression lines.

[0030] Figure 6 This study aims to observe the phenotypes of Populus tomentosa PubHLH35 gene transgenic and wild-type plants under Cd stress in this invention.

[0031] Figure 7 Stomatal conductance of Populus tomentosa PubHLH35 gene transgenic and wild-type plants in this invention;

[0032] Figure 8The transpiration rate of Populus tomentosa PubHLH35 gene transgenic and wild-type plants in this invention;

[0033] Figure 9 The CO2 concentration between transgenic Populus alba PubHLH35 and wild-type plants in this invention;

[0034] Figure 10 The net photosynthetic rate of the Populus tomentosa PubHLH35 gene transgenic and wild-type plants in this invention;

[0035] Figure 11 The present invention relates to the Cd gene transgenic Populus alba PubHLH35 and wild-type plants. 2+ Content determination;

[0036] Figure 12 The Cd content in biochar of Populus tomentosa PubHLH35 gene transgenic and wild-type plants in this invention. 2+ Content and Cd in eluted biochar 2+ content. Detailed Implementation

[0037] A PubHLH35 gene, the nucleotide sequence of which is shown in SEQ ID No. 1, as follows:

[0038] .

[0039] This invention also provides the application of overexpressing the PubHLH35 gene described in the above-described technical solution in enhancing the accumulation of heavy metals in plants. In this invention, the plant preferably includes *Populus tomentosa*. In this invention, the heavy metal preferably includes cadmium.

[0040] This invention provides a method for preparing biochar, comprising the following steps:

[0041] 1) Overexpress the PubHLH35 gene described in the above technical solution in plants to obtain overexpressed plants;

[0042] 2) The overexpressed plant obtained in step 1) is thermally decomposed to obtain biochar.

[0043] The PubHLH35 gene described in the above technical solution is overexpressed in plants to obtain overexpressing plants. In this invention, the PubHLH35 gene is ligated into the pBI121-GFP vector to obtain a recombinant vector. This invention does not have specific limitations on the method of ligating the PubHLH35 gene into the pBI121-GFP vector; those skilled in the art can follow conventional methods. This invention transforms the recombinant vector into Agrobacterium to obtain transformant bacteria. This invention does not have specific limitations on the method of transforming the recombinant vector into Agrobacterium; those skilled in the art can follow conventional methods. This invention infects plants with the transformant bacteria to obtain overexpressing plants. Those skilled in the art do not have specific limitations on the method used to infect plants with the transformant bacteria; those skilled in the art can use conventional methods. In this invention, the Agrobacterium preferably includes Agrobacterium GV3101. In this invention, the plant preferably includes Populus tomentosa.

[0044] This invention involves the thermal decomposition of the overexpressed plant material to obtain biochar. The preferred conditions for this thermal decomposition include: a heating rate of 20°C / min, a temperature of 500°C, and a time of 20 min.

[0045] This invention also provides the application of biochar prepared by the preparation method described above in the remediation of cadmium-contaminated soil. In this invention, the application preferably includes: applying the biochar to the soil at an amount of 5-10% of the soil mass.

[0046] To further illustrate the present invention, the following detailed description is provided in conjunction with embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0047] Unless otherwise specified, all methods described in the following examples are conventional. The materials, reagents, enzymes, competent cells, and plasmids used are commercially available unless otherwise specified.

[0048] Example 1

[0049] The PubHLH35 gene of Populus tomentosa was obtained as follows:

[0050] 1.1 Obtaining the target gene sequence

[0051] Primers were designed at both ends of the target gene sequence as shown in Table 1.

[0052] Table 1 Primer sequences for target gene cloning in this invention

[0053]

[0054] Total RNA was extracted from Populus alba PubHLH35 using the CTAB method and reverse transcribed into cDNA. Using this cDNA as a template, the target gene was cloned using a KOD enzyme system in a total reaction volume of 200 μL. The reaction conditions were: 95℃ pre-denaturation for 2 min, 98℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 1 min, for a total of 32 cycles; a final extension at 68℃ for 2 min. Detection was performed by 1.2% agarose gel electrophoresis. The PCR system is shown in Table 2.

[0055] Table 2. PCR reaction system for target gene cloning in this invention.

[0056] Element Dosage 10×KOD Buffer 10μL PubHLH35-F (10μM) 6μL PubHLH35-R (10μM) 6μL dNTP (25 μM) 40μL 2×PCR Buffer for KOD FX 100μL KOD enzyme 4μL <![CDATA[ddH2O]]> 34μL

[0057] 1.2 Target gene ligation into pClone EZ-Omni Vector and sequencing

[0058] The target fragment product was recovered via gel extraction and ligated into the pClone EZ-Omni Vector. After transformation with *E. coli* using the heat shock method, suitable single colonies were selected for amplification culture and PCR detection. Positive plasmids were extracted and sequenced. The nucleotide sequence was as follows: Figure 2 As shown, this gene fragment is named PubHLH35 and consists of 735 bases.

[0059] 1.3 Experimental Results

[0060] 1.3.1 Cloning of the PubHLH35 gene

[0061] Primers were designed using the *Populus alba* database, and PCR was performed on *Populus alba*. The PubHLH35 gene length was 735 bp, preliminarily indicating the correctness of the PCR result. The next step involved purifying the PCR product and ligating it into the pClone EZ-OmniVector vector. The recombinant plasmid was transformed into *E. coli* and plated on Kansas-treated selection plates. Several positive colonies were randomly selected and tested using culture PCR. If bands were present... Figure 1 .

[0062] 2. Obtaining PubHLH35 transgenic Populus tomentosa

[0063] 2.1 Construction of overexpression vectors

[0064] A pair of primers was designed based on the PubHLH35 sequence, primers for PubHLH35 overexpression, and plasmids were used as templates for PCR.

[0065] Table 3 Token Information

[0066]

[0067] (1) The PCR reaction system is shown in Table 4:

[0068] Table 4. PCR reaction system for target gene cloning in this invention.

[0069] Element Dosage cDNA 10μL PubHLH35-OE-F (10μM) 6μL PubHLH35-OE-R (10μM) 6μL dNTP (25 μM) 40μL 2×PCR Buffer for KOD FX 100μL KOD enzyme 4μL <![CDATA[ddH2O]]> 34μL

[0070] (2) Reaction Procedure

[0071] The bacterial culture was pre-denatured at 94℃ for 52 min, then denatured at 94℃ for 30 s, annealed at 56℃ for 30 s, and extended at 72℃ for 30 s, for a total of 35 cycles, with a final extension at 72℃ for 2 min. After double enzyme digestion, the culture was ligated into the pBI121-GFP vector and transformed into *E. coli*. Plasmids were extracted from the successfully constructed bacterial culture and transformed into *Agrobacterium* GV3101 using liquid nitrogen. The culture was screened using Kan and Rfp, and the bacterial culture was subjected to PCR detection, plasmid extraction, and sequencing. The successfully constructed bacterial culture was used for transgenic purposes.

[0072] 2.2 Obtaining Agrobacterium-mediated transgenic Populus tomentosa lines

[0073] (1) Take about 15 healthy and sterile wild-type Populus tomentosa tissue culture seedlings that have grown for three weeks. Use sterile scissors to cut off the 3rd to 5th leaves from the upper part of the stem of the seedling. Carefully place them into sterile 1 / 2MS liquid culture medium to avoid human damage (pay special attention to ensure the petiole is intact when taking the leaves). Take 40 to 50 leaves.

[0074] (2) Hold the blade (medical surgical type) and cut vertically from the petiole, and cut off about 1cm of the leaf blade along with the veins to form a flat wound, and try to avoid damage to the part being removed.

[0075] (3) Carefully transfer the cut leaves into the prepared engineered bacteria, gently shake at room temperature, and infect for 20 minutes to ensure that the bacterial solution is in full contact with the leaves;

[0076] (4) After infection, gently absorb the bacterial solution on the leaf surface with sterile paper, lay the leaf face down on a WPM co-culture plate, and incubate in the dark at a constant temperature for 40-48 hours.

[0077] (5) Sterilization: Wash the leaves with sterile water, using about 40 mL of sterile water each time, 4-6 times, 2 minutes each time. Then use 600 mL of sterile water containing Cef, wash 10-12 times, using about 50 mL of water each time, about 4 minutes each time. When washing, gently shake the vial for sterilization. The total washing time should not be too long to reduce damage to the leaves.

[0078] (6) After washing, use sterile tweezers to gently transfer the infected leaves to sterile paper, gently wipe away excess water, and then spread them on WPM differentiation selection medium containing 30 mg / L Kan, Cef, and Tim for dark incubation and screening.

[0079] (7) Observe regularly and remove leaves with bacteria (Cef is easily decomposed and ineffective). Depending on the condition of the culture medium, the culture medium should be changed every 5-8 days. When the wound swells and grows resistant buds, the leaves can be transferred to WPM differentiation and selection medium with a Kan concentration of 40 mg / L and cultured under light.

[0080] (8) After exposure to light, the resistant buds gradually turned green, grew taller and stronger. The buds and leaves were separated with sterile scissors and blades, and individual resistant seedlings were transferred to 50 mg / L Kan selection medium and cultured until rooting.

[0081] (9) After about 15-20 days of screening, the transgenic resistant seedlings did not die or weaken due to screening and were still able to grow healthily. That is, after preliminary screening, subsequent molecular testing can be carried out.

[0082] 2.3 Molecular detection of PubHLH35 transgenic plants

[0083] Genomic DNA was extracted from *Populus alba* using the CTAB method, using wild-type (WT) and overexpression transgenic materials respectively. PCR detection was performed on the selected overexpression plants, with pBI121-PubHLH35-GFP as a positive control and total DNA from WT as a negative control.

[0084] 2.4 Results and Analysis

[0085] 2.4.1 Vector construction of the PubHLH35 gene

[0086] Plasmids were extracted from colonies with correctly sequenced PubHLH35 genes. These colonies were ligated into the pBI121-GFP vector via homologous recombination and transformed into *E. coli* for PCR detection. After sequencing, if the sequence was correct, the plasmid was extracted and transformed into *Agrobacterium* GV3101. Single colonies were selected for PCR detection. Plasmids were extracted from bacterial cultures showing bands and sent for sequencing. Bacterial cultures with correct sequences were stored at -80°C for subsequent transgenic applications.

[0087] 2.4.2 Obtaining PubHLH35 gene transgenic plants

[0088] Activated Agrobacterium tumefaciens pBI121-PubHLH35-GFP was used to infect healthy leaves of Populus tomentosa. After co-culture, the bacteria were washed, followed by selection of transgenic leaves. PubHLH35 transgenic plants were obtained after selection.

[0089] 2.4.3 Molecular detection of PubHLH35 transgenic Populus tomentosa

[0090] DNA and RNA were extracted from PubHLH35 overexpressing transgenic plants, and the transgene was detected at the genomic and transcriptional levels, respectively. At the DNA level, PCR showed a fragment length of 835 bp, which is the sum of the PubHLH35 gene and the pBI121-GFP gene. Figure 4 .

[0091] RNA was extracted from PubHLH35-overexpressing transgenic plants and reverse transcribed into cDNA. PubHLH35 gene expression levels were analyzed, and the results are shown below. Figure 5 .

[0092] 3. Physiological analysis of transgenic Populus tomentosa under Cd stress

[0093] Poplar seedlings cultured for 21 days were transplanted into soil. Two months later, wild-type (WT) and transgenic poplars with similar growth performance were selected and treated with 200 mg / kg CdCl2 soil for two weeks. Seedlings not treated with CdCl2 served as controls. After harvest, the roots were carefully washed three times with sterile water to remove Cd from the root surface. 2+ Poplar plants were separated into root, stem, and leaf tissues, dried at 80℃ for 3 days, and used for Cd treatment. 2+ Content determination and biochar preparation.

[0094] 3.1 Measurement of photosynthetic indicators

[0095] Photosynthetic gas exchange parameters were measured using a Li-6400 portable photosynthetic fluorescence measurement system (Heinz Walz GmbH, Germany). Red and blue light sources with an intensity of 1600 μmol / m² were selected for the photosynthetic gas exchange parameters. 2 / s. The concentration was controlled at 400 μmol / s using a CO2 cylinder. Measurement parameters included stomatal conductance (Gs), net photosynthetic rate (Pn), transpiration rate (Et), and intercellular CO2 concentration (Ci). Three biological replicates were performed, with three technical replicates per replicate.

[0096] 3.2 Plant Cd under stress 2+ content

[0097] Roots, stems, and leaves of WT and PubHLH35-OE plants were collected, dried, ground into powder, and passed through a 100-mesh sieve. The powder was then immersed in a solution containing HNO3-HClO4. After microwave digestion, the cadmium concentration in the root, stem, and leaf tissues was determined using inductively coupled plasma mass spectrometry (ICP-MS) (ICP-OES 5110). The Cd concentrations in WT and transgenic poplar plants were calculated. 2+ content.

[0098] 3.3 Biochar preparation and cleaning

[0099] The entire plant sample was cleaned with deionized water, and any residual water adhering to the plant surface was then absorbed with gauze. Each plant sample treated with 0 or 200 mg / kg CdCl2 was pulverized and placed in a muffle furnace for thermal decomposition. An anaerobic environment was ensured during heating. The pyrolysis temperature was set to 500 °C, the heating rate to 20 °C / min, and the residence time to 20 min.

[0100] Weigh 0.1g of biochar sample into a digestion tube, fill the tube with 6mL of nitric acid, 2mL of hydrofluoric acid, and 2mL of hydrogen peroxide, shake, tighten the stopcock, and let stand for 1 hour. Then, place it in a microwave digester and set the program for digestion. After digestion, determine the Cd content of the sample using ICP-MS. 2+ concentration.

[0101] The biochar was pulverized and sieved, placed in an Erlenmeyer flask, and a 1 mol / L HCl solution was added. The flask was then placed in a constant-temperature shaker at 25°C and washed at 250 rpm for 24 hours. The supernatant was separated from the leached biochar by atmospheric pressure filtration. Deionized water was added repeatedly until the supernatant was neutral. The cleaned biochar was then baked at 80°C to obtain clean biochar. The Cd content of the washed biochar was determined using ICP-MS, as described above. 2+ concentration.

[0102] 3.7 Results and Analysis

[0103] 3.7.1 Growth of transgenic plants under Cd stress

[0104] There was no significant difference in growth between untreated wild-type and transgenic plants. After Cd treatment, the growth of transgenic plants was significantly inhibited compared with wild-type plants. In particular, the overexpressing plants showed a significant reduction in plant height, diameter at root, and dry weight compared with WT plants, which preliminarily indicates that under Cd stress, transgenic plants overexpressing PubHLH35 were subjected to more severe stress.

[0105] 3.7.2 Determination of photosynthetic indices in transgenic plants under Cd stress

[0106] Under Cd stress, transgenic plants exhibited higher photosynthetic efficiency and stomatal conductance. With increasing Cd treatment time, the levels of WT and overexpressed Gs, Et, and Pn all decreased. Furthermore, Pn, Gs, and Et in PubHLH35 were significantly lower than in WT, and the gas exchange parameter Ci in PubHLH35 overexpression increased more rapidly than in WT.

[0107] 3.7.3 Biochar preparation and cleaning

[0108] Biochar C-OE and C-WT were derived from PubHLH35 overexpression and WT, respectively. Results showed that C-OE contained Cd... 2+ The concentration was significantly higher than that of C-WT. These results indicate that, after Cd stress, PubHLH35 overexpressing transgenic plants exhibited higher Cd concentrations than WT. 2+ Concentration. This indicates that PubHLH35 overexpression converts to biochar for Cd extraction from contaminated soil. 2+ Their capabilities have been enhanced.

[0109] Acid washing effectively eluted Cd from biochar. 2+ This makes the treated biochar suitable for the remediation of Cd-contaminated soil.

[0110] Therefore, converting PubHLH35-overexpressing transgenic poplar biomass into biochar further confirms the feasibility of cadmium recovery. Utilizing the resulting biochar for phytoremediation in cadmium-contaminated soil provides a feasible method for ecological restoration while simultaneously achieving resource recycling.

[0111] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of overexpression of PubHLH35 gene in enhancing heavy metal accumulation in plants; The nucleotide sequence of the PubHLH35 gene is shown in SEQ ID No. 1; The plant in question is Populus alba; The heavy metal in question is cadmium.

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