Application of PtNLP11 gene in improving the resistance of poplar to low nitrogen stress

Abstract

The present application relates to the field of plant genetic engineering technology, and specifically to the application of the PtNLP11 gene in improving the resistance of poplar to low nitrogen stress. The present application confirms through experiments that overexpressing the PtNLP11 gene in poplar plants under low nitrogen stress can promote nitrogen nutrition regulation of poplar plants, activate their adversity response, and regulate the root development and nutrient absorption of plants, thereby obtaining excellent poplar varieties that still have a faster growth and development rate and more biomass accumulation under nitrogen-poor conditions. The present application confirms that the PtNLP11 gene has potential application value in improving the resistance of poplar to low nitrogen stress, and provides theoretical support for the breeding of poplar varieties with high nitrogen utilization rate. At the same time, it has important theoretical and practical significance for the directional cultivation of artificial timber forests and the improvement of poplar wood yield and quality.

Description

Technical Field

[0001] The present application relates to the technical field of plant genetic engineering, and in particular to the application of the PtNLP11 gene in improving the resistance of poplar to low nitrogen stress. Background Art

[0002] Wood is one of nature's most important renewable resources and a primary raw material for pulp and papermaking, construction, and bioenergy. With rapid economic development, demand for wood continues to grow.

[0003] Poplar (Populus) is a model woody plant and the world's most widespread, adaptable, and fast-growing tree species. Characterized by rapid growth, strong wood, and excellent pulping and papermaking properties, poplars are widely used in construction, furniture, and papermaking, becoming a key raw material for plywood, fiberboard, paper matches, chopsticks, and packaging. They are of great economic and ecological significance. Among the environmental factors that constrain poplar growth, nitrogen plays a key role. In nature, nitrogen-poor conditions can slow wood formation in the aboveground parts of poplar trees. Nitrogen fertilizers are commonly applied to promote growth and increase wood yield.

[0004] However, nitrogen fertilizer application simply increases timber yields without improving timber properties. Furthermore, excessive nitrogen fertilizer application can inhibit plant growth and pollute the environment. Some of this nitrogen fertilizer is lost to the environment, causing serious environmental damage such as water pollution, global warming, and biodiversity loss. Therefore, developing a solution that improves timber properties and optimizes timber resource quality without harming the environment has become a key goal in current poplar breeding. Summary of the Invention

[0005] In view of this, the present application provides the application of the PtNLP11 gene in improving the resistance of poplars to low nitrogen stress. By overexpressing the PtNLP11 gene in poplar plants, the poplars can still maintain a faster growth rate under nitrogen-poor conditions.

[0006] The examples of the present application provide the use of the PtNLP11 gene in improving the low nitrogen stress resistance of poplar. The CDS nucleotide sequence of the PtNLP11 gene is shown in SEQ ID NO.1.

[0007] In some of the embodiments, the application of the PtNLP11 gene in improving the resistance of poplar to low nitrogen stress is specifically to construct a poplar plant overexpressing the PtNLP11 gene, including the following steps: (1) monoclonal amplification of the PtNLP11 gene; (2) construction of a plant overexpression vector; (3) transfer of the plant overexpression vector into a poplar plant using Agrobacterium-mediated method; and (4) functional verification of the PtNLP11 overexpressing transgenic plant.

[0008] In some embodiments, the above-mentioned construction of the PtNLP11 gene overexpressing poplar plant specifically includes the following steps:

[0009] S1, obtain mRNA from poplar roots, stems and leaves, mix and reverse transcribe to synthesize cDNA of PtNLP11 gene, use the synthesized cDNA as template and perform PCR amplification with specific primers to obtain the full-length CDS sequence of PtNLP11 gene;

[0010] S2, using KpnI restriction enzyme to digest the overexpression vector plasmid and purify and recover it, mixing the recovered digested vector fragment and the target gene fragment, ligating the target gene to the vector, and transforming the constructed ligation product into competent Escherichia coli. After colonies grow, positive verification is performed to confirm the correct overexpression vector is constructed;

[0011] S3, transforming the correctly constructed overexpression vector into competent Agrobacterium, and performing positive verification again after colonies grow to confirm that the correctly constructed overexpression vector has been transformed into Agrobacterium to obtain recombinant Agrobacterium;

[0012] S4, the recombinant Agrobacterium obtained in S3 was transfected into the poplar line, and through co-cultivation, differentiation culture, rooting culture, seedling hardening and transplanting, the PtNLP11 gene overexpressing poplar plants were obtained.

[0013] In some embodiments, the specific primers in S1 include an upstream primer and a downstream primer; the nucleotide sequence of the upstream primer is shown in SEQ ID NO.2, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.3.

[0014] In some embodiments, the PCR amplification in S1 is high-fidelity DNA polymerase amplification, and the amplification system of the high-fidelity DNA polymerase amplification includes: 5× Phusion HF Buffer, 10 μL; 10 mM dNTPs, 1 μL; high-fidelity DNA polymerase, 1 μL; template cDNA, 50 ng~100 ng; upstream primer, 20 μM; downstream primer, 20 μM; ddH2O, made up to 50 μL.

[0015] In some embodiments, the amplification program of PCR amplification in S1 is: (1) pre-denaturation at 98°C for 3 min; (2) denaturation at 98°C for 10 s, annealing at 60°C for 30 s, and extension at 72°C for 1 min, 35 cycles; (3) extension at 72°C for 10 min.

[0016] In some embodiments, under low nitrogen stress, the number of leaves of the overexpressing strain containing the PtNLP11 gene is greater than the number of leaves of the wild-type strain and the number of leaves of the strain in which the PtNLP11 gene is suppressed.

[0017] In some embodiments, under low nitrogen stress conditions, the plant height of the overexpression strain containing the PtNLP11 gene is higher than the plant height of the wild-type strain and the plant height of the PtNLP11 gene suppression expression strain.

[0018] In some embodiments, under low nitrogen stress, the fresh weight of plants of the overexpression lines containing the PtNLP11 gene is greater than the fresh weight of plants of the wild-type lines and the fresh weight of plants of the PtNLP11 gene suppression lines.

[0019] Based on the application of the PtNLP11 gene in the examples of this application to improve the low nitrogen stress resistance of poplar trees, the inventors discovered that overexpressing the PtNLP11 gene in poplar plants under low nitrogen stress can promote nitrogen nutrition regulation in poplar plants, activate their stress response, and regulate root development and nutrient absorption, thereby obtaining excellent poplar varieties that still have faster growth and development rates and higher biomass accumulation in nitrogen-poor environments. This provides theoretical support for the selection of improved forest varieties with high nitrogen utilization rates. At the same time, it has important theoretical and practical significance for the targeted cultivation of poplar artificial timber forests and improving the yield and quality of poplar wood. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 This is the process of constructing the poplar PtNLP11 overexpression vector in Example 1 of this application;

[0022] Figure 2 This is the process of constructing the poplar PtNLP11 inhibition expression vector in Example 1 of this application;

[0023] Figure 3 This is a photo of the poplar genetic transformation, differentiation, and budding stage in Example 2 of this application;

[0024] Figure 4 This is a photo of the adventitious bud elongation stage of poplar genetic transformation in Example 2 of this application;

[0025] Figure 5 This is the identification result of the poplar PtNLP11 transgenic strain in Example 2 of this application;

[0026] Figure 6 This is the growth and development of poplar trees under different nitrate concentrations in Example 2 of this application;

[0027] Figure 7 Example 2 of this application promotes the growth and biomass accumulation of poplars under different nitrate conditions;

[0028] Figure 8 This is Example 2 of the present application, which shows the effect of PtNLP11 on poplar photosynthesis under different nitrate conditions. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solutions and advantages of this application more clearly understood, the present application is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain this application and are not intended to limit this application.

[0030] Example

[0031] This example provides a method for constructing a poplar plant overexpressing the PtNLP11 gene, which specifically includes the following steps:

[0032] S1: mRNA was obtained from poplar roots, stems, and leaves, mixed, and reverse transcribed to synthesize cDNA of the PtNLP11 gene. Using the synthesized cDNA as a template, high-fidelity DNA polymerase amplification was performed using upstream and downstream primers to obtain the full-length CDS sequence of the PtNLP11 gene. The amplification system for high-fidelity DNA polymerase amplification included: 5× Phusion HF Buffer, 10 μL; 10 mM dNTPs, 1 μL; high-fidelity DNA polymerase, 1 μL; template cDNA, 100 ng; upstream primer, 20 μM; downstream primer, 20 μM; ddH2O, made up to 50 μL. The amplification program for high-fidelity DNA polymerase was as follows: (1) pre-denaturation at 98°C for 3 min; (2) denaturation at 98°C for 10 s, annealing at 60°C for 30 s, and extension at 72°C for 1 min, for 35 cycles; (3) extension at 72°C for 10 min.

[0033] S2: Use KpnI restriction enzyme to digest the overexpression vector plasmid 2301s and purify it. Mix the recovered digested vector fragment and the target gene fragment. Use a homologous recombination kit to ligate the target gene to the vector. Transform the ligation product into competent Escherichia coli. After colonies grow, perform positive verification to confirm the correct overexpression vector construction.

[0034] S3, transforming the correctly constructed overexpression vector into competent Agrobacterium, and performing positive verification again after colonies grow to confirm that the correctly constructed overexpression vector has been transformed into Agrobacterium to obtain recombinant Agrobacterium;

[0035] S4, the recombinant Agrobacterium obtained in S3 was transfected into the poplar line, and the PtNLP11 gene overexpressing poplar line was obtained through co-cultivation, differentiation culture, rooting culture, seedling hardening and transplanting.

[0036] Hereinafter, examples are given to more specifically illustrate the embodiments of the present application. The reagents and equipment used in the following examples can be purchased through commercial channels.

[0037] Example 1 Construction of overexpression vector and suppression expression vector of poplar PtNLP11 gene

[0038] 1. Amplification and Purification of the PtNLP11 Gene Fragment

[0039] 1.1 PCR amplification of target fragment

[0040] The PtNLP11 gene sequence was extracted from the genome of Populus tremula × P. alba (717 hybrid poplar). Specific primers containing the complete CDS sequence were designed at appropriate loci. The designed primers amplified a product of approximately 3000 bp. The primer sequences for the PtNLP11 gene are shown in Table 1. The primer sequences were submitted to Sangon Biotech (Shanghai) Co., Ltd. for synthesis. The primers were used for the first round of PCR amplification using Populus 717 cDNA as a template and a high-fidelity PCR system. The product was purified and recovered. Subsequently, primers containing homology arms were synthesized, and the recovered product was used as a template in a second round of PCR amplification using a high-fidelity PCR system. The amplified product was further purified and recovered.

[0041] To construct the RNAi vector, primers with amplification products of 150-300 bp were designed using the Primer3 online primer design tool. These primers were then infused with attB sequences and sent to Wuhan Qingke Biotechnology Co., Ltd. for primer synthesis (primer sequences are shown in Table 1). High-fidelity PCR amplification was performed using the 2301s-PtNLP11 plasmid containing the overexpression vector as a template. While amplifying the specific fragment, attB1 and attB2 sites were added to flank the target fragment to facilitate subsequent BP reactions. After agarose gel electrophoresis, the PCR amplification product was excised to the appropriate size for the target gene, placed in a 2 mL centrifuge tube, and stored at 4°C for DNA gel recovery experiments.

[0042] Table 1 Primers for expression vector construction

[0043]

[0044] Note: In the table above, linker sequences are distinguished by lowercase letters. The vector sequences in the primers below are all represented in this way.

[0045] 1.2 Product purification and recovery

[0046] The product was purified using the FastPure Gel DNA Extraction Mini Kit (Novagen, China) with the following steps:

[0047] (1) After electrophoresis, cut the PCR product into a gel and place it in a 1.5 mL centrifuge tube. Weigh the centrifuge tube first, then weigh the centrifuge tube containing the gel. Calculate the weight of the gel. 100 mg of gel is equivalent to 100 µL volume.

[0048] (2) Add an equal volume of Buffer GDP and completely dissolve the gel in a 55°C water bath for 10 min, inverting and mixing 2–3 times during the water bath.

[0049] (3) Place the FastPure DNA Mini Columns-G adsorption column in a 2 mL Collection Tube, transfer the sol to the adsorption column, and centrifuge at 12,000 rpm for 1 min.

[0050] (4) Discard the filtrate, place the adsorption column in a collection tube, add 300 μL of Buffer GDP to the adsorption column, let it stand for 1 minute, and centrifuge at 12,000 rpm for 1 minute;

[0051] (5) Discard the filtrate, place the adsorption column in a collection tube, and add 700 µL of Buffer GW to the adsorption column. Centrifuge at 12,000 rpm for 1 min.

[0052] (6) Repeat step 5;

[0053] (7) Discard the filtrate, place the adsorption column back into the collection tube, and centrifuge at 12,000 rpm for 2 min;

[0054] (8) Place the adsorption column in a 1.5 mL sterilized centrifuge tube, add 20 µL of ddH2O to the center of the adsorption column, let it sit for 2 minutes, and centrifuge at 12,000 rpm for 1 minute;

[0055] (9) Discard the adsorption column, detect the quality of the purified product by electrophoresis, and use a microplate to detect the product concentration. Then store the product at -20°C for future use.

[0056] 2. Construction of target gene expression vector

[0057] 2.1 Construction of PtNLP11 gene overexpression vector by homologous recombination

[0058] After activating the empty vector culture, shake the culture to extract the plasmid. After determining the DNA concentration, the vector plasmid was digested with the selected restriction endonuclease. The digestion reaction system is shown in Table 2-1. In this study, an overexpression vector was constructed through homologous recombination. The 2301s vector plasmid was used. The vector was linearized by single enzyme digestion using the KpnI restriction endonuclease. After the digestion, the digestion product was detected by electrophoresis and recovered and purified.

[0059] Table 2-1 Enzyme Digestion Reaction System

[0060]

[0061] After the linearized vector and target fragment are ready, the target gene is ligated into the vector using the ClonExpress® II One Step Cloning Kit. The specific steps are as follows:

[0062] (1) Determine the concentration of the target fragment and the vector and calculate the optimal dosage

[0063] The optimal amount of carrier used = [0.02 × carrier length] ng;

[0064] The optimal amount of fragment to be used = [0.04 × fragment length] ng;

[0065] When the target fragment length is greater than the vector length, the optimal usage calculation methods for the two are interchangeable. The usage amount of the linearized vector should be between 50-200 ng, and the usage amount of the target fragment should be between 10ng and 200ng. If the calculated amount is out of range, directly select the lowest or highest usage amount;

[0066] (2) Calculate the amount of DNA required for the recombination reaction according to the formula. To ensure the accuracy of the sample addition, the linearized vector and the inserted target gene fragment can be appropriately diluted before configuring the recombination reaction system. The sample volume of each component should be no less than 1 μL;

[0067] (3) Prepare the homologous reaction system on ice. The reaction system is shown in Table 2-2 below:

[0068] Table 2-2 Homologous recombination reaction system

[0069]

[0070] (4) Briefly centrifuge the reaction solution to collect it at the bottom of the tube, and use a pipette to gently pipette and mix (do not oscillate to mix);

[0071] (5) Incubate the reaction at 37°C for 30 min on a PCR instrument, then immediately cool on ice. After 5 min, the reaction can be used to transform E. coli. The ligation product can be stored at -20°C for one week and can be thawed when needed for transformation.

[0072] 2.2 Construction of PtNLP11 gene suppression expression vector using Gateway technology

[0073] Gateway technology consists of two reactions: BP and LR. In this example, the BP reaction was used to construct an RNAi expression vector. Two identical PtNLP11 gene-specific sequences were recombined into the vector via the BP reaction (see Table 2-3). This process forms hairpin RNA (hpRNA) in vivo, effectively reducing endogenous gene expression by leveraging the mechanism by which most eukaryotic double-stranded RNA (dsRNA) induces endogenous sequence-specific RNA degradation. This vector contains the ccdB gene. Therefore, only by replacing the ccdB gene with an exogenous gene via the BP reaction can the plasmid replicate normally in hosts other than DB3.1 (such as DH5α). This property was exploited to transform the recombined plasmid into the DH5α strain, and colonies were screened for successful clones.

[0074] Table 2-3 BP reaction system

[0075]

[0076] 2.3 E. coli transformation

[0077] (1) Remove the DH5α competent cells from -80°C and quickly place them on ice for 5-8 minutes. After thawing, aliquot 50 μL into a sterile 1.5 mL centrifuge tube.

[0078] (2) Add 5 μL of ligation product to 50 μL of competent medium, tap the tube wall a few times, and let it stand on ice for 25 min;

[0079] (3) Heat shock in a 42°C metal bath for 90 s, then quickly return to ice and let stand for 5 min;

[0080] (4) Add 700 μL of LB liquid medium to the centrifuge tube and incubate at 37°C on a shaker at 200 rpm for 1 h;

[0081] (5) Collect the cells by centrifugation at 5000 rpm for 1 min. Aspirate 650 μL of culture medium with a pipette and discard. Resuspend the cells with the remaining 100 μL of culture medium and spread them on a LB solid plate containing kanamycin (Kan, 2301S) or spectinomycin (Spec, pHEELSGATE4). After drying, invert the plate and culture in a 37°C incubator for 12-16 h.

[0082] (6) Pick a single colony from the plate and place it in LB liquid medium (containing Kan or Spec) and culture at 37°C with shaking at 200 rpm for 16 h. Perform PCR positive identification on the bacterial solution and send the selected positive bacterial solution to Wuhan Qingke Biological Company for sequencing.

[0083] 2.4 Recombinant plasmid extraction

[0084] Plasmid DNA was extracted using the FastPure Plasmid Mini Kit (Novagen, China) with the following steps:

[0085] (1) Add 25 mL of LB liquid medium to a 50 mL centrifuge tube, add 100 µL of bacterial solution and appropriate amount of Kan or Spec antibiotics, and culture at 37°C and 220 rpm for 12 h;

[0086] (2) Take 5 mL of bacterial solution and centrifuge at 13,000 rpm for 1 min at room temperature to collect the bacteria;

[0087] (3) Discard the supernatant, add 250 µL of Solution I / RNase A mixture, and vortex to completely suspend the cells;

[0088] (4) Add 250 µL of Solution II to the resuspended mixture, gently invert and mix 4-6 times, and let it stand at room temperature for 3 minutes to allow the cells to fully lyse;

[0089] (5) Add 350 μL of Solution III, gently invert several times until a white flocculent precipitate forms, and centrifuge at 13,000 rpm for 10 min;

[0090] (6) HiBind ® The Miniprep DNA binding column was placed in a 2 mL collection tube, and the supernatant was transferred to the collection tube and centrifuged at 13,000 rpm for 1 min.

[0091] (7) Discard the filtrate, reinstall the column into the collection tube, add 500 μL HBC Buffer, and centrifuge at 13,000 rpm for 1 min at room temperature;

[0092] (8) Discard the filtrate, return the column to the collection tube, add 700 μL DNA Wash Buffer, and centrifuge at 13,000 rpm for 1 min at room temperature;

[0093] (9) Repeat step (8) once;

[0094] (10) Discard the filtrate, reinstall the column into the collection tube, and centrifuge the empty column at 13,000 rpm for 1 min to dry the column;

[0095] (11) Place the column in a clean 1.5 mL centrifuge tube, add 50 µL of ddH2O to the column matrix, let it stand for 1 min, and centrifuge at 13,000 rpm for 1 min to elute the DNA;

[0096] (12) Detect DNA quality by electrophoresis and DNA concentration by microplate. Store plasmid DNA at -20℃.

[0097] 2.5 Agrobacterium tumefaciens transformation

[0098] (1) Take out a tube of Agrobacterium GV3101 competent cells and place it on ice. After it is completely thawed, pipette 50 μL into a 1.5 mL sterile centrifuge tube. Add 1 μL of pHEELSGATE4 recombinant plasmid to the centrifuge tube and gently tap the tube wall to mix.

[0099] (2) Place on ice for 5 min, freeze in liquid nitrogen for 5 min, heat shock at 37°C for 5 min, and place on ice again for 5 min;

[0100] (3) Add 700 μL of YEB liquid medium and shake at 220 rpm at 28°C for 2.5 h;

[0101] (4) Centrifuge at 5000 rpm for 1 min, aspirate 650 μL of supernatant with a 1 mL pipette and discard, mix thoroughly with a 100 μL pipette, and evenly spread the bacterial solution on YEB solid medium supplemented with Rif and the corresponding antibiotics of the vector. After air drying, culture in a constant temperature box at 28°C for two days.

[0102] (5) After colonies grow, pick a single colony and add it to YEB liquid culture medium containing Rif and the corresponding antibiotics of the vector. Place it in a shaker at 28°C and shake at 200 rpm in the dark overnight (12h~16h) for subsequent PCR identification.

[0103] 3. PCR Amplification

[0104] Experiments such as bacterial liquid PCR identification and positive seedling PCR verification used standard Taq amplification. The amplification system is shown in Table 3-1, and the reaction procedure is shown in Table 3-2. For higher accuracy, the full-length CDS of the target gene sequence was amplified using Thermo Scientific Phusion High-Fidelity DNA Polymerase. The reaction system and procedure are shown in Tables 3-3 and 3-4. In this study, Populus 717 cDNA was used as the template for CDS amplification.

[0105] Table 3-1 Common Taq enzyme PCR amplification system

[0106]

[0107] Note: If you need to expand the reaction system, just increase the reaction reagents in equal proportions.

[0108] Table 3-2 Common Taq enzyme PCR reaction procedure

[0109]

[0110] Table 3-3 High-fidelity enzyme PCR amplification system

[0111]

[0112] Table 3-4 High-fidelity enzyme PCR reaction procedure

[0113]

[0114] The experimental results of this embodiment are as follows Figure 1 、 Figure 2 shown. Figure 1 A is the full-length amplification of the PtNLP11 gene; Figure 1 B adds the KpnI homology arm to PtNLP11; Figure 1 C is DH5α colony PCR identification; Figure 1 D is the PCR identification of GV3101 colonies; Figure 1 E is a schematic diagram of the construction of the 2301s-PtNLP11 vector. The red arrows in the figure indicate the strains selected for the next experiment and the preserved strains. Figure 1 As shown in A, agarose gel electrophoresis detected a product size of about 3000 bp. According to the genomic information, the size of the PtNLP11 gene is 3009-3015 bp. The band sizes are similar, and it is preliminarily determined that the PtNLP11 gene was successfully amplified. The product was purified and recovered. A KpnI restriction site was added to the purified product ( Figure 1 B) At the same time, the 2301s vector was linearized using KpnI restriction enzyme. The product and the vector were homologously recombined and transformed into E. coli competent DH5α. After the colonies were grown by Kan screening, PCR positive verification was performed ( Figure 1 C), the band size was correct and sent for sequencing verification. The sequencing results were aligned with the genome sequence, indicating that the overexpression vector was successfully constructed. The plasmid of the 2301s-PtNLP11 expression vector was extracted and transformed into Agrobacterium competent GV3101. After the colonies grew after culturing at 28℃, PCR verification was performed again ( Figure 1 D) The band size is correct and the strain is saved for future use.

[0115] like Figure 2 As shown, Figure 2 A is the amplification of the PtNLP11 gene-specific fragment; Figure 2 B is the addition of the attLB site homology arm to PtNLP11; Figure 2 C is DH5α colony PCR identification, + represents the sense chain, and - represents the antisense chain; Figure 2 D is the PCR identification of GV3101 colonies, Figure 2 C. Figure 2 The red arrows in D indicate the strains selected for the next experiment and the preserved strains. Figure 2 E is a schematic diagram of the construction of RE-PtNLP11 vector. A small fragment specific to the gene PtNLP11 was cloned from the overexpression vector using primers containing site B ( Figure 2 A, Figure 2 B), the product was sequenced, and the sequencing results were highly matched only with the PtNLP11 gene in the two haplotypes (PtXaAlbH.03G114200, PtXaTreH.03G119300). Then, the amplified fragment was loaded into the pHELLSGATE4 vector by BP reaction and transformed into Escherichia coli competent DH5α ( Figure 2 C) After sequencing, the sequence alignment was correct and the suppression expression vector was constructed. After extracting the plasmid, it was transformed into Agrobacterium competent GV3101 and PCR verification was performed again ( Figure 2 D) The band size is correct and the strain is saved for future use.

[0116] Example 2 Effects of PtNLP11 on Poplars under Nitrogen Stress

[0117] 1. Acquisition and positive identification of transgenic poplar materials

[0118] To further determine the function of the PtNLP11 gene in poplar, the 230s-PtNLP11 overexpression vector and the pHELLSGATE4-PtNLP11 repression expression vector were genetically transformed into Populus 717. Poplar transformation requires four steps, including co-cultivation, bud differentiation, bud elongation, and rooting ( Figure 3 , Figure 4 A total of 8 poplar overexpression lines and 8 poplar suppression expression lines were obtained through genetic transformation.

[0119] In this embodiment, the transformation and differentiation budding stage is as follows Figure 3 As shown, Figure 3 A. Figure 3 B. Figure 3 C. Figure 3 D shows the growth status of plant materials after 10, 15, 20, and 30 days of antibiotic selection, respectively. The scale bar in the figure is 1 cm.

[0120] In this example, the adventitious bud elongation stage of genetic transformation is as follows Figure 4 As shown, Figure 4 A. Figure 4 B. Figure 4 C. Figure 4D shows the growth status of the cells after 3, 7, 20, and 30 days of culture on elongation medium. The scale bar is 1 cm.

[0121] The strains screened by antibiotics were positively identified. The DNA level of overexpression plants was identified using the primer position-specific primer PtNLP11-F and the vector universal primer 2301s-R. The DNA level of inhibition expression was identified using two pairs of primers for positive and antisense strand verification, including pHGRV35SF and PtNLP11-RE-R, and pHGRVOS and PtNLP11-RE-R. The results showed that the DNA quality of WT and transgenic strains was high and could be used for PCR identification. The identification results were as follows: Figure 5 A, Figure 5 As shown in B. Except for WT, 2301s-PtNLP11 plasmid and overexpression strains can amplify a band of about 3000 bp ( Figure 5 A). Similarly, the target bands were detected in both the positive and antisense chains of the pHELLSGATE4-PtNLP11 plasmid and the suppression expression strain ( Figure 5 B). To further verify the stability of the transgenic materials, they were verified at the RNA level. RT-qPCR results showed that the expression levels of the three overexpression lines OE-PtNLP11-2, OE-PtNLP11-4, and OE-PtNLP11-7 were the highest, upregulated by 2.53, 2.96, and 2.61 times, respectively, compared with the wild type ( Figure 5 C). The three expression-inhibiting strains RE-PtNLP11-1, RE-PtNLP11-14, and RE-PtNLP11-19 showed the most significant gene downregulation, with expression levels of 0.34, 0.28, and 0.34 times that of the wild type, respectively ( Figure 5 D). These results indicate that all transgenic lines are stable transformed positive lines and can be used for subsequent phenotypic verification experiments.

[0122] Figure 5 A. Figure 5 B is DNA level identification. Figure 5 A shows, from top to bottom, the DNA identification of the wild-type and overexpression strains, and the detection of internal reference genes. Figure 5 B shows, from top to bottom, the positive-sense DNA identification, antisense DNA identification, and internal reference gene detection of the wild-type and expression-inhibited strains. Figure 5 C. Figure 5 D is RNA level identification. Figure 5 C is the RNA level detection of wild type and overexpression strains. Figure 5D shows RNA level analysis for wild-type and repressed expression lines. pladmid1 represents the 2301s-PtNLP11 plasmid, and pladmid2 represents the pHELLSGATE4-PtNLP11 plasmid. OE-PtNLP11 indicates overexpression of the PtNLP11 gene in 717 Poplar, and RE-PtNLP11 indicates repressed expression of the PtNLP11 gene in 717 Poplar. Different numbers represent different lines.

[0123] 2. Effects of PtNLP11 on Poplar Growth and Development under Nitrogen Stress

[0124] To determine the role of the PtNLP11 gene in wood development under nitrogen stress, wild-type (WT) and transgenic poplars were exposed to different NO3 - The effect of the PtNLP11 gene on the growth and development of poplar trees was studied by monitoring the changes in leaf number and plant height at different time periods. The results of this example are shown in Figure 2. Figure 6 As shown in the figure, the growth and development of poplar trees under LN stress were significantly inhibited compared with HN conditions. Under LN conditions, plant height and leaf number were significantly reduced. - Under these conditions, the leaf number and plant height of the OE-PtNLP11 transgenic line were significantly higher than those of the WT at all developmental stages ( Figure 6 A- Figure 6 D). In contrast, the leaf number and plant height of the poplar RE-PtNLP11 transgenic line were significantly lower than those of the WT ( Figure 6 A- Figure 6 D). Under LN conditions, poplar leaves will show symptoms of nitrogen deficiency, with yellowing and stunted growth ( Figure 6 E). Compared with WT, the OE-PtNLP11 transgenic line showed better development and greener leaves, while the RE-PtNLP11 transgenic line showed more obvious growth inhibition ( Figure 6 E). Under HN conditions, there were small differences in plant height, leaf color, and leaf number between WT and transgenic lines ( Figure 6 F). These results indicate that the poplar PtNLP11 gene can promote plant leaf development and growth. Figure 6 A and Figure 6 B respectively shows the leaf development under LN and HN conditions; Figure 6 C and Figure 6 D respectively shows the changes in plant height under LN and HN conditions; Figure 6 E and Figure 6Figure F shows the growth of poplar trees after 40 days of nitrate treatment. Scale bar: 10 cm. LN represents 0.5 mM Ca(NO3)2, and HN represents 5 mM Ca(NO3)2.

[0125] 3. Effect of PtNLP11 on wood biomass accumulation in poplar under nitrogen stress

[0126] Depend on Figure 7 In the fresh weight analysis of WT and transgenic poplar lines, different NO3 - The concentration has a great influence on their growth and development. Regardless of LN or HN conditions, the fresh weight of roots, stems and leaves of OE-PtNLP11 transgenic lines were significantly higher than those of WT ( Figure 7 A- Figure 7 C). Under LN conditions, the fresh weights of roots, stems, and leaves of the RE-PtNLP11 transgenic line showed a downward trend compared to the WT ( Figure 7 A- Figure 7 C). Under HN conditions, the fresh weights of roots, stems, and leaves of almost all RE-PtNLP11 transgenic lines were significantly lower than those of WT ( Figure 7 A- Figure 7 C). In general, under both LN and HN conditions, overexpression of the PtNLP11 gene significantly increased plant fresh weight, while inhibition of PtNLP11 gene expression reduced plant fresh weight. Similarly, under LN conditions, the ground diameter of the OE-PtNLP11 transgenic line was larger than that of the WT, while the ground diameter of the RE-PtNLP11 transgenic line was smaller than that of the WT ( Figure 7 E). However, under HN conditions, there was almost no difference in ground diameter between the OE-PtNLP11 and RE-PtNLP11 transgenic lines ( Figure 7 E). Analysis of chlorophyll content revealed that the chlorophyll content in the OE-PtNP11 transgenic line was significantly higher than that in the WT, while the chlorophyll content in the RE-PtNLP11 transgenic line was lower than that in the WT ( Figure 7 F). These results indicate that the PtNLP11 gene can improve the absorption and utilization of nitrogen by poplars, especially under LN conditions, and can greatly promote the accumulation of biomass and chlorophyll in poplars. Figure 7 A. Figure 7 B. Figure 7 C. Figure 7 D represents the fresh weight of poplar roots, stems, leaves and total fresh weight, respectively; Figure 7 Figure E shows the ground diameter; Figure 7 Figure F shows the chlorophyll content of poplar leaves.

[0127] 4. Effects of PtNLP11 on photosynthesis in poplar trees under nitrogen stress

[0128] To investigate whether PtNLP11 regulates photosynthesis, photosynthetic indices of WT and transgenic poplar lines were measured. Compared with HN conditions, LN stress significantly affected the photosynthetic indices of poplars, significantly reducing Pn, Gs, Ci, and Tr ( Figure 8 ). Under LN conditions, the Pn value of the OE-PtNLP11 transgenic line was much higher than that of the WT, while the Pn value of the RE-PtNLP11 transgenic line was lower than that of the WT ( Figure 8 A). All transgenic lines showed no difference in Gs compared to WT ( Figure 8 B). In addition, the Ci and Tr of almost all transgenic lines were higher than those of WT ( Figure 8 C, Figure 8 D). Under HN conditions, the Pn value of the OE-PtNLP11 transgenic line was slightly higher than that of the WT, while the Pn value of the RE-PtNLP11 transgenic line was not significantly different from that of the WT ( Figure 8 A). Gs and Ci of all transgenic lines were higher than those of WT, but there were almost no differences among them ( Figure 8 B, Figure 8 C). Interestingly, the Tr of the OE-PtNLP11 transgenic line was lower than that of the WT, while there was no difference between the RE-PtNLP11 transgenic line and the WT ( Figure 8 D). These results indicate that PtNLP11 is involved in the photosynthesis pathway of poplar, and may adapt to the LN environment by enhancing photosynthesis-related indicators such as net photosynthetic rate, stomatal conductance, and transpiration rate through chlorophyll content.

[0129] From the above, it can be seen that overexpressing the PtNLP11 gene in poplar plants can effectively promote nitrogen nutrition regulation in poplar plants under low-nitrogen stress, activate their adversity response, and regulate the root development and nutrient absorption of the plants, thereby obtaining excellent poplar varieties that still have faster growth and development rates and more biomass accumulation under nitrogen-poor conditions. This application confirms that the PtNLP11 gene has potential application value in improving poplar resistance to low-nitrogen stress, provides theoretical support for the selection of poplar varieties with high nitrogen utilization efficiency, and has important theoretical and practical significance for the targeted cultivation of artificial timber forests and improving the yield and quality of poplar wood.

[0130] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present application should be included in the scope of protection of the present application.

Claims

1. Application of the PtNLP11 gene in improving resistance to low nitrogen stress in poplars, characterized in that: The CDS nucleotide sequence of the PtNLP11 gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that The application is to construct a poplar plant with overexpression of the PtNLP11 gene, comprising the following steps: (1) Monoclonal amplification of the PtNLP11 gene; (2) Construction of plant overexpression vectors; (3) Using Agrobacterium-mediated method to transfer plant overexpression vector into poplar plants; (4) Functional verification of transgenic plants overexpressing PtNLP11.

3. The use according to claim 2, characterized in that The specific steps include: S1, obtain mRNA from poplar roots, stems and leaves, mix and reverse transcribe to synthesize cDNA of PtNLP11 gene, use the synthesized cDNA as template and perform PCR amplification with specific primers to obtain the full-length CDS sequence of PtNLP11 gene; S2, using KpnI restriction enzyme to digest the overexpression vector plasmid and purify and recover it, mixing the recovered digested vector fragment and the target gene fragment, ligating the target gene to the vector, and transforming the constructed ligation product into competent Escherichia coli. After colonies grow, positive verification is performed to confirm the correct overexpression vector is constructed; S3, transforming the correctly constructed overexpression vector into competent Agrobacterium, and performing positive verification again after colonies grow to confirm that the correctly constructed overexpression vector has been transformed into Agrobacterium to obtain recombinant Agrobacterium; S4, the recombinant Agrobacterium obtained in S3 was transfected into the poplar line, and through co-cultivation, differentiation culture, rooting culture, seedling hardening and transplanting, the PtNLP11 gene overexpressing poplar plants were obtained.

4. The use according to claim 3, characterized in that In S1, the specific primers include an upstream primer and a downstream primer; The nucleotide sequence of the upstream primer is shown in SEQ ID NO.2, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.

3.

5. The use according to claim 3, characterized in that In S1, the PCR amplification is high-fidelity DNA polymerase amplification, and the amplification system of the high-fidelity DNA polymerase amplification includes: 5×Phusion HF Buffer, 10 μL; 10 mM dNTPs, 1 μL; High-fidelity DNA polymerase, 1 μL; Template cDNA, 50 ng–100 ng; upstream primer, 20 μM; downstream primer, 20 μM; Add ddH2O to 50 μL.

6. The use according to claim 3, characterized in that In S1, the amplification procedure of the PCR amplification is: (1) Pre-denaturation at 98°C for 3 min; (2) denaturation at 98°C for 10 s, annealing at 60°C for 30 s, and extension at 72°C for 1 min, for 35 cycles; (3) Extension at 72°C for 10 min.

7. The use according to claim 1, characterized in that Under low nitrogen stress, the number of leaves of the overexpression strain containing the PtNLP11 gene is greater than the number of leaves of the wild-type strain and the number of leaves of the PtNLP11 gene suppression expression strain.

8. The use according to claim 1, characterized in that Under low nitrogen stress conditions, the plant height of the overexpression strain containing the PtNLP11 gene is higher than that of the wild-type strain and the plant height of the PtNLP11 gene inhibition expression strain.

9. The use according to claim 1, characterized in that Under low nitrogen stress, the fresh weight of plants in the overexpression line containing the PtNLP11 gene is greater than that of the wild-type line and the PtNLP11 gene suppression expression line.

Images