PubHLH35 gene and application thereof, and preparation method of biochar

Abstract

The invention relates to the technical field of hyperaccumulator harmlessness, in particular to a PubHLH35 gene and application thereof and a preparation method of biochar. The nucleotide sequence of the PubHLH35 gene is as shown in SEQ ID No. 1 (sequence identifier number 1). According to the invention, the PubHLH35 gene is found to improve the Cd stress absorption capability of populus japonica, and the cultivated overexpression transgenic variety has important theoretical and practical significance in the aspect of preparing biochar for repairing soil Cd pollution.

Description

Technical Field

[0001] The invention relates to the technical field of harmless super-enriched plants, and in particular to a PubHLH35 gene and its application and a method for preparing biochar. Background Art

[0002] Cadmium (Cd) pollution in soil is widely distributed around the world. It is one of the most harmful elements and is toxic. It can affect important physiological metabolic processes such as photosynthesis and respiration through ion substitution, breaking the balance of active oxygen, destroying signal transduction pathways, and damaging the structure of biological lipid membranes, ultimately inhibiting plant growth and development and having a negative impact on plant growth.

[0003] Cadmium pollution has been severe over the past few decades, mainly due to increased pollution caused by industrial and agricultural practices such as wastewater irrigation and excessive use of fertilizers. Many options can be applied to remediate Cd contamination of soil, such as soil treatment, soil excavation, and chemical methods. In addition to this, phytoremediation has been proposed as a more potentially effective and less costly approach. Hyperaccumulators that can absorb and accumulate large amounts of the toxic metal Cd from contaminated soils are used, especially for fast-growing woody species such as poplars. Generally, woody plants accumulate a higher total amount of Cd than herbaceous hyperaccumulators because of their deep root systems and large biomass. Therefore, the present invention proposes a biochar recovery strategy as a safe and value-added method for treating 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] In order to solve the above problems, the present invention provides a PubHLH35 gene and its application and a method for preparing biochar. The PubHLH35 gene provided by the present invention has the function of increasing the heavy metal enrichment of plants, and preparing biochar from plants overexpressing the gene can promote the enrichment of heavy metals.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides a PubHLH35 gene, and the nucleotide sequence of the PubHLH35 gene is shown as SEQ ID No.1.

[0007] The present invention also provides the use of over-expressing the PubHLH35 gene described in the above technical solution in improving the heavy metal enrichment of plants.

[0008] Preferably, the plant includes Populus ussuriensis.

[0009] Preferably, the heavy metal includes cadmium.

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

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

[0012] 2) Thermally decomposing the overexpressed plant obtained in step 1) 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 20 min.

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

[0015] Transforming the recombinant vector into Agrobacterium to obtain transformed bacteria;

[0016] The transformed bacteria are used to infect plants to obtain overexpression plants.

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

[0018] The present invention also provides the use of cadmium-rich biochar prepared by the preparation method described in the above technical scheme in repairing soil cadmium pollution.

[0019] Preferably, the application comprises: applying the biochar to the soil in an amount of 5 to 10% of the soil mass.

[0020] Beneficial effects:

[0021] The application of the PubHLH35 gene of Populus ussuriensis in the present invention refers to improving the absorption and transport capacity of Populus ussuriensis for Cd under Cd stress, and preparing biochar to repair the cadmium pollution in soil.

[0022] The present invention discloses the application of PubHLH35 gene. The present invention uses Agrobacterium-mediated method to overexpress the gene in Populus ussuriensis, and the obtained Populus ussuriensis transgenic strain effectively improves the characteristics of Populus ussuriensis's ability to transport and absorb Cd. Under Cd stress, the height, ground diameter and dry weight of the transgenic strain are significantly reduced compared with the wild-type plants, which preliminarily shows that under drought stress, the damage of the transgenic strain overexpressing PubHLH35 is relatively serious, and the absorption capacity of Cd is stronger.

[0023] Under Cd stress, the stomatal conductance, transpiration rate and net photosynthetic rate of the transgenic lines were significantly lower than those of the wild type, and the intercellular CO2 concentration of the transgenic plants was significantly higher than that of the wild type. The chlorophyll content of the overexpressing transgenic plants was significantly lower than that of the wild type. In addition, 2+ The content of Cd in transgenic plants was determined by comparison. 2+ The content was significantly higher than that of the wild type. These data also verified that the overexpression strain of PubHLH35 gene was sensitive to Cd 2+ The absorption capacity is significantly higher than that of the wild type. The present invention finds that PubHLH35 improves the absorption capacity of Populus ussuriensis for Cd stress, and the cultivated overexpression transgenic varieties have important theoretical and practical significance in preparing biochar to repair soil Cd pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0025] Figure 1 The figure is the gel recovery electrophoresis diagram of the PCR product of the PubHLH35 gene of Populus ussuriensis in the present invention, wherein M is DL2000 DNA Marker, and 1-3 are PCR products;

[0026] Figure 2 is the nucleotide sequence of the PubHLH35 gene of Populus ussuriensis in the present invention;

[0027] Figure 3 The PCR diagram of the construction of the PubHLH35 gene of Populus ussuriensis into the pBI121-GFP overexpression vector in the present invention, wherein M is the DL2000 DNA Marker, and 1-9 are the PubHLH35 gene;

[0028] Figure 4 It is the detection of the PubHLH35 gene overexpression strain of Populus ussuriensis at the DNA level in the present invention;

[0029] Figure 5 It is a quantitative analysis of RNA levels in the strains overexpressing PubHLH35 gene of Populus ussuriensis in the present invention;

[0030] Figure 6 The present invention is to observe the phenotypes of the PubHLH35 gene transgenic and wild-type plants of Populus ussuriensis under Cd stress;

[0031] Figure 7 is the stomatal conductance of the transgenic and wild-type plants of Populus ussuriensis PubHLH35 gene in the present invention;

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

[0033] Fig. 9 is the intercellular CO2 concentration of the PubHLH35 gene transgenic and wild-type plants of Populus ussuriensis in the present invention;

[0034] Fig.10 The net photosynthetic rate of the PubHLH35 gene transgenic and wild-type plants of Populus ussuriensis in the present invention;

[0035] Fig.11 The Cd 2+ Content determination;

[0036] Fig.12 The Cd content in the biochar of the PubHLH35 gene transgenic and wild-type plants of Populus ussuriensis in the present invention is 2+ Content and Cd in biochar after elution 2+ content. DETAILED DESCRIPTION

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

[0038] .

[0039] The present invention also provides the use of overexpressing the PubHLH35 gene described in the above technical solution in improving the heavy metal enrichment of plants. In the present invention, the plant preferably includes Populus ussuriensis. In the present invention, the heavy metal preferably includes cadmium.

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

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

[0042] 2) Thermally decomposing the overexpressed plant obtained in step 1) to obtain biochar.

[0043] The PubHLH35 gene described in the above technical scheme is overexpressed in plants to obtain overexpressed plants. In the present invention, the PubHLH35 gene is connected to the pBI121-GFP vector to obtain a recombinant vector. The present invention does not specifically limit the method for connecting the PubHLH35 gene to the pBI121-GFP vector, and a person skilled in the art can follow the routine. The present invention transfers the recombinant vector into Agrobacterium to obtain a transformed bacterium. The present invention does not specifically limit the method for transferring the recombinant vector into Agrobacterium, and a person skilled in the art can follow the routine. The present invention infects plants with the transformed bacteria to obtain an overexpressed plant. A person skilled in the art does not specifically limit the method used by the transformed bacteria to infect plants, and a person skilled in the art can follow the routine. In the present invention, the Agrobacterium preferably includes Agrobacterium GV3101. In the present invention, the plant preferably includes Populus ussuriensis.

[0044] The present invention thermally decomposes the obtained overexpressed plant to obtain biochar. In the present invention, the thermal decomposition conditions preferably include: a heating rate of 20°C / min, a temperature of 500°C, and a time of 20 min.

[0045] The present invention also provides the use of the biochar prepared by the preparation method described in the above technical solution in remediating soil cadmium pollution. In the present invention, the use preferably includes: applying the biochar to the soil, and the application amount is 5-10% of the soil mass.

[0046] In order to further illustrate the present invention, the present invention is described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0047] The following examples are all conventional methods unless otherwise specified. The materials, reagents, enzymes, competent cells and plasmids used can be obtained from commercial sources unless otherwise specified.

[0048] Example 1

[0049] 1 The PubHLH35 gene of Populus ussuriensis 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 of the present invention

[0053]

[0054] The total RNA of Populus ussuriensis PubHLH35 was extracted by CTAB method and reverse transcribed into cDNA. The cDNA was used as template and the KOD enzyme system was used to clone the target gene according to the total reaction system of 200 μL. The reaction conditions were: 95℃ pre-denaturation for 2min, 98℃ denaturation for 30s, 56℃ annealing for 30s, 72℃ extension for 1min, and the number of cycles was 32; finally, 68℃ extension for 2min and 1.2% agarose gel electrophoresis detection. The PCR system is shown in Table 2.

[0055] Table 2 PCR reaction system for target gene cloning of the present invention

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

[0057] 1.2 Target gene connected to pClone EZ-Omni Vector vector for sequencing

[0058] The target fragment product was recovered by gel recovery, and the recovered product was connected to the pClone EZ-Omni Vector vector. After heat shock E. coli transformation, a suitable single colony was selected for expansion culture PCR detection, and the positive plasmid was extracted and sequenced. After testing, the nucleotide sequence was as follows Figure 2 As shown, the gene fragment was named PubHLH35 and consisted of 735 bases.

[0059] 1.3 Experimental Results

[0060] 1.3.1 Cloning of PubHLH35 gene

[0061] Using the Populus ussuriensis database, primers were designed and PCR was performed in Populus ussuriensis. The length of PubHLH35 gene was 735bp, which preliminarily indicated that the PCR result was correct. The next step was to purify the PCR product and connect it to the pClone EZ-OmniVector vector. The recombinant plasmid was transformed into Escherichia coli and spread on the Kan screening plate. Several positive colonies were randomly selected and tested by bacterial solution PCR. If there were any bands, Figure 1 .

[0062] 2. Obtaining PubHLH35 transgenic Populus ussuriensis

[0063] 2.1 Construction of overexpression vector

[0064] A pair of primers were designed based on the sequence of PubHLH35, primers for overexpression of PubHLH35, and PCR was performed using the plasmid as a template.

[0065] Table 3 Token information

[0066]

[0067] (1) PCR reaction system, as shown in Table 4:

[0068] Table 4 PCR reaction system for target gene cloning in the present invention

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

[0070] (2) Reaction procedure

[0071] Pre-denaturation at 94℃ for 52min, denaturation at 94℃ for 30s, annealing at 56℃ for 30s, extension at 72℃ for 30s, for a total of 35 cycles, and finally extension at 72℃ for 2min. After double restriction digestion, connect to pBI121-GFP vector and transform E. coli. The plasmids extracted from the bacterial solution successfully sequenced were transformed into Agrobacterium GV3101 by liquid nitrogen method, and screened with Kan and Rfp. The bacterial solution was tested by PCR, the plasmids were extracted and sequenced, and the successfully constructed bacterial solution was used for transgenic standby.

[0072] 2.2 Obtaining transgenic lines of Populus ussuriensis mediated by Agrobacterium

[0073] (1) Take about 15 three-week-old, strong and sterile wild-type Populus ussuriensis tissue culture seedlings, cut off the 3rd to 5th leaves at the top of the stem with sterile scissors, and carefully place them in sterile 1 / 2MS liquid culture medium to avoid human damage (pay special attention to ensure that the petiole is intact when taking leaves), and take 40 to 50 leaves;

[0074] (2) Use a surgical blade to cut vertically from the petiole and cut about 1 cm of the leaf perpendicular to the veins to form a smooth wound, and try to avoid damaging the removed part;

[0075] (3) Carefully transfer the cut leaves to the prepared engineered bacteria, gently shake at room temperature, and infect for 20 minutes to allow the bacterial solution to fully contact the leaves;

[0076] (4) After infection, use sterile paper to gently absorb the bacterial liquid on the leaf surface, spread the leaf surface face down on the WPM co-culture plate, and culture in the dark at a constant temperature for 40-48 hours;

[0077] (5) Degerming: Wash the leaves with sterile water, about 40 mL each time, 4-6 times, 2 min each time, then use 600 mL of sterile water containing Cef, wash 10-12 times, about 50 mL each time, about 4 min each time. Shake the vial gently during sterilization, and the total washing time should not be too long to reduce damage to the leaves;

[0078] (6) After washing, use sterile tweezers to gently move the infected leaves onto sterile paper, gently wipe to remove excess water, and then spread them onto WPM differentiation screening medium containing 30 mg / L Kan, Cef, and Tim, and culture them in the dark for 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 replaced every 5-8 days. When the wound swells and grows resistant buds, the leaves can be transferred to WPM differentiation screening medium with a Kan concentration of 40 mg / L and cultured under light.

[0080] (8) After light culture, the resistant buds gradually turn green, grow taller and become stronger. Use sterile scissors and blades to separate the buds and leaves, take out a single resistant seedling and transfer it to 50 mg / L Kan screening medium and culture until rooting;

[0081] (9) After about 15-20 days of screening, the transgenic resistant seedlings did not die or become weak due to the screening and were still able to grow healthily. This means that they have passed the preliminary screening and can be used for subsequent molecular testing.

[0082] 2.3 Molecular detection of PubHLH35 transgenic plants

[0083] The wild type (WT) and the overexpressed transgenic plants were used as materials, and the CTAB method was used to extract the genomic DNA of Populus ussuriensis. The overexpressed plants were tested by PCR using pBI121-PubHLH35-GFP as the positive control and the total DNA of WT as the negative control.

[0084] 2.4 Results and Analysis

[0085] 2.4.1 Construction of PubHLH35 gene vector

[0086] Extract plasmid from the colony with correct PubHLH35 gene sequencing. Connect to pBI121-GFP vector through homologous recombination and transfer into Escherichia coli for PCR detection. Send for sequencing. After the sequence is correct, extract the plasmid and transfer into Agrobacterium GV3101. Pick a single clone for PCR detection. Extract plasmid from bacterial solution with bands and send for sequencing. Store the bacterial solution with correct sequence at -80℃ for subsequent transgenic.

[0087] 2.4.2 Obtaining PubHLH35 gene transgenic plants

[0088] The activated pBI121-PubHLH35-GFP Agrobacterium was used to infect the leaves of Populus ussuriensis with good growth status. After co-cultivation, the bacteria were washed and then the transgenic leaves were screened. After the screening was completed, PubHLH35 transgenic plants were obtained.

[0089] 2.4.3 Molecular Detection of PubHLH35 Transgenic Populus ussuriensis

[0090] From the PubHLH35 overexpressing transgenic plants, plants were selected to extract DNA and RNA, and the transgenics were tested at the genomic and transcriptional levels. At the DNA level, the PCR fragment length was 835 bp, which was the length of PubHLH35 gene plus pBI121-GFP gene. Figure 4 .

[0091] The RNA of the PubHLH35 overexpressing transgenic plants was extracted and reverse transcribed into cDNA, and the expression level of the PubHLH35 gene was analyzed. The results are shown in Figure 5 .

[0092] 3. Physiological analysis of transgenic Populus ussuriensis 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 were used as controls. After harvesting, the roots were carefully washed three times with sterile water to remove Cd on the root surface. 2+ Poplar plants were separated into root, stem and leaf tissues and dried at 80 °C for 3 days for Cd 2+ Content determination and biochar preparation.

[0094] 3.1 Determination of photosynthetic indexes

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

[0096] 3.2 Plant Cd after stress 2+ content

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

[0098] 3.3 Biochar preparation and cleaning

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

[0100] Weigh 0.1g of biochar sample into a digestion tube, fill the tube with 6mL nitric acid, 2mL hydrofluoric acid and 2mL hydrogen peroxide, shake, tighten the piston and leave for 1h. Then, put it into a microwave digestion instrument and set the program for digestion. After digestion, use ICP-MS to determine the Cd content of the sample. 2+ concentration.

[0101] The biochar was crushed and sieved, placed in a conical flask, and 1 mol / L HCl solution was added. It was placed in a 25°C constant temperature shaker and washed at 250 r / min for 24 h. The supernatant was separated from the leached biochar by atmospheric filtration, and then deionized water was added several times until the supernatant was neutral. The cleaned biochar was baked at 80°C to obtain clean biochar. As mentioned above, the Cd content of the cleaned biochar was determined using ICP-MS. 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 the growth conditions of untreated wild-type and transgenic plants; after Cd treatment, the growth of transgenic plants was significantly inhibited compared with the wild-type, especially the overexpression plants, compared with WT, the plant height, ground diameter and dry weight were significantly reduced, preliminarily indicating that under Cd stress, the transgenic plants overexpressing PubHLH35 were more severely stressed.

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

[0106] Under Cd stress, transgenic plants showed higher photosynthetic efficiency and stomatal conductance. With the increase of Cd treatment time, Gs, Et and Pn of WT and overexpression decreased. In addition, Pn, Gs and Et of PubHLH35 were significantly lower than those of WT, and the gas exchange parameter Ci in PubHLH35 overexpression increased faster than that in WT.

[0107] 3.7.3 Biochar preparation and cleaning

[0108] Biochars C-OE and C-WT were derived from PubHLH35 overexpression and WT, respectively. 2+ These results indicate that after Cd stress, PubHLH35 overexpressing transgenic plants showed higher Cd 2+ This indicates that PubHLH35 overexpression converted into biochar can extract Cd from contaminated soil 2+ capacity enhancement.

[0109] Acid washing effectively eluted Cd from biochar 2+ , making the treated biochar suitable for soil Cd pollution remediation.

[0110] Therefore, the conversion of PubHLH35-overexpressing transgenic poplar biomass into biochar further confirmed the feasibility of Cd recovery. The use of the resulting biochar for phytoremediation in Cd-contaminated soils provides a feasible approach for ecological restoration while achieving resource recycling.

[0111] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A PubHLH35 gene, characterized in that The nucleotide sequence of the PubHLH35 gene is shown in SEQ ID No.

1.

2. Use of overexpressing the PubHLH35 gene described in claim 1 in improving heavy metal accumulation in plants.

3. The use according to claim 2, characterized in that: The plants include Populus ussuriensis.

4. The use according to claim 2, characterized in that: The heavy metals include cadmium.

5. A method for preparing biochar, characterized in that: The following steps are involved: 1) overexpressing the PubHLH35 gene according to claim 1 in a plant to obtain an overexpression plant; 2) Thermally decomposing the overexpressed plant obtained in step 1) to obtain biochar.

6. The preparation method according to claim 5, characterized in that: 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 20 min.

7. The preparation method according to claim 5, characterized in that: The step 1) connects the PubHLH35 gene to the pBI121-GFP vector to obtain a recombinant vector; Transforming the recombinant vector into Agrobacterium to obtain transformed bacteria; The transformed bacteria are used to infect plants to obtain overexpression plants.

8. The preparation method according to claim 7, characterized in that: The Agrobacterium includes Agrobacterium GV3101; and the plant includes Populus ussuriensis.

9. Use of the biochar prepared by the preparation method according to any one of claims 5 to 8 in remediating soil cadmium pollution.

10. The use according to claim 9, characterized in that: The application comprises: applying the biochar to soil, with the application amount being 5-10% of the soil mass.

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