Poplar water use efficiency related ptoerf54 gene and application thereof

Abstract

This invention discloses a PtoERF54 gene related to water use efficiency in poplar trees and its applications. The gene sequence is shown in SEQ ID NO.1. By constructing an overexpression recombinant vector of this gene and introducing it into poplar trees, PtoERF54 overexpressing transgenic plants are obtained, which can significantly improve the water use efficiency of poplar trees, enhance their drought resistance, and effectively mitigate the impact of drought stress on tree growth. This invention also discloses a breeding method for enhancing the drought resistance of poplar trees by improving their water use efficiency. This is beneficial for selecting new tree germplasm with stronger drought resistance, improving the environmental adaptability of poplar trees, and laying a theoretical foundation for molecular breeding of drought-resistant poplar trees.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to genes related to the drought resistance of poplar trees, and particularly to a PtoERF54 gene related to the water use efficiency of poplar trees and its application. Background Technology

[0002] Intensifying global climate change has led to increasingly severe drought stress, which has become a major abiotic stressor limiting tree growth, timber yield, and quality. The continued expansion of arid regions significantly constrains the sustainable development of forestry. Poplar (Populus) occupies an important position in global forestry due to its significant ecological value (such as in plantation construction and ecological restoration) and economic value (such as as a raw material for the paper industry). However, drought stress severely restricts the normal growth and geographical distribution range of poplar.

[0003] Utilizing molecular breeding techniques and genetic engineering to enhance the drought resistance of trees is an effective way to tap the potential of forestry production in arid areas and improve the environmental adaptability of poplar. There is an urgent need to breed new tree germplasm with stronger drought resistance. The key to achieving this goal lies in deeply analyzing the molecular mechanisms by which poplar copes with drought stress and identifying the key genes that play a core regulatory role.

[0004] Although poplars have developed their own drought-resistance mechanisms through long-term evolution, water use efficiency remains an inherent pathway for different poplar species to adapt to drought stress. Therefore, developing drought-resistant poplar varieties from the perspective of water use efficiency is an effective approach. Current research on genes related to poplar drought resistance, especially water use efficiency, is limited. It is necessary to develop key genes that enhance poplar drought resistance by improving water use efficiency, in order to deeply elucidate the molecular mechanisms by which poplars cope with drought stress, shorten the breeding cycle, and lay a theoretical and genetic resource foundation for the creation of new stress-resistant poplar germplasm. Summary of the Invention

[0005] To overcome the aforementioned problems, the inventors identified a PtoERF54 gene significantly associated with poplar water use efficiency. By constructing an overexpression recombinant vector of the PtoERF54 gene and introducing it into poplar trees, PtoERF54-overexpressing transgenic plants were obtained. Analysis of the drought stress phenotype of these plants revealed that, compared to wild-type plants, the overexpressing plants exhibited stronger resistance to drought stress and better growth. This indicates that the PtoERF54 gene plays a crucial regulatory role in controlling poplar water use efficiency. Overexpression of this gene can effectively improve poplar water use efficiency, thereby enhancing its drought resistance. This lays a theoretical foundation for further elucidating the molecular mechanisms by which poplar responds to drought stress and provides new insights for the creation of new stress-resistant poplar germplasm, thus completing this invention.

[0006] Specifically, the object of the present invention is to provide the following aspects:

[0007] In one aspect, a PtoERF54 gene related to water use efficiency of poplar trees is provided, the sequence of which is shown in SEQ ID NO.1.

[0008] Secondly, a protein encoded by the PtoERF54 gene, which is associated with water use efficiency in poplar trees, is provided, the encoded protein having the amino acid sequence shown in SEQ ID NO.3.

[0009] Thirdly, an overexpression recombinant vector for the PtoERF54 gene related to water use efficiency of poplar, as described in the first aspect, is provided, wherein the base vector of the overexpression recombinant vector is the 35S-PZP211-3Flag vector.

[0010] Fourthly, a breeding method is provided to enhance the drought resistance of poplar trees by improving their water use efficiency. This method is achieved by overexpressing the PtoERF54 gene described in the first aspect or increasing the content of the encoded protein described in the second aspect in the poplar plant.

[0011] Fifthly, the application of the gene described in the first aspect or the protein encoded in the second aspect in enhancing the drought resistance of poplar trees is provided, wherein the enhancement of the drought resistance of poplar trees is achieved by improving the water use efficiency of poplar trees.

[0012] The sixth aspect provides the application of the overexpression recombinant vector described in the third aspect in improving the water use efficiency of poplar trees.

[0013] The beneficial effects of this invention include:

[0014] (1) The PtoERF54 gene related to water use efficiency of poplar provided by the present invention can significantly improve the water use efficiency of poplar by overexpressing the gene in poplar plants, thereby enhancing the drought resistance of poplar and effectively mitigating the impact of drought stress on forest growth.

[0015] (2) The breeding method provided by the present invention, which enhances the drought resistance of poplar by improving the water use efficiency of poplar, is conducive to the selection of new forest tree germplasm with stronger drought resistance, improves the environmental adaptability of poplar, and lays a theoretical foundation for the molecular breeding of drought-resistant poplar.

[0016] (3) The application of the PtoERF54 gene or its encoded protein related to water use efficiency of poplar provided by the present invention in enhancing the drought resistance of poplar can increase the water use efficiency of PtoERF54-OE plants by 48.76% under light saturation conditions and by 56.68% under CO2 saturation conditions. At the same time, under drought stress, the cell membrane loss of overexpressing plants is significantly reduced, photosynthetic performance and photosynthetic capacity are significantly improved, and the reactive oxygen species scavenging capacity is significantly enhanced. Attached Figure Description

[0017] Figure 1 A structural diagram of the basic carrier described in Example 2 is shown;

[0018] Figure 2 The results of transgenic line identification in Example 4 are shown, where (A) represents the electrophoresis diagram of PCR identification; (B) represents the expression level of the PtoERF54 gene in the RT-qPCR results.

[0019] Figure 3 Figures (A) and (B) show the water use efficiency index analysis of poplar in Example 5. In this figure, (A) represents the light response curve of water use efficiency (WUE); (B) represents the CO2 response curve of water use efficiency. All data are the average of 8 biological replicates, and ± represents the standard error (n=8).

[0020] Figure 4 (A) shows a morphological comparison of wild-type (WT) and PtoERF54-overexpressing poplar lines (OE3, OE5) before and after drought treatment in Example 6, scale bar = 5 cm;

[0021] Figure 4 (B) shows a morphological comparison of leaves of wild-type (WT) and PtoERF54-overexpressing poplar lines (OE3, OE5) after drought treatment in Example 6;

[0022] Figure 4 (C) in Example 6 shows the comparison results of the relative water content (RWC) of leaves under normal and drought conditions;

[0023] Figure 4 (D) in Example 6 shows the comparison results of the relative electrical conductivity (REC) of the leaves under normal and drought conditions;

[0024] Figure 5(A) shows the maximum quantum efficiency (Fv / Fm) and non-photochemical quenching (NPQ) of photosystem II in Example 6 under normal and drought conditions.

[0025] Comparison results;

[0026] Figure 5 (B) shows the comparison results of peroxidase (POD) and superoxide dismutase (SOD) activities of plants in Example 6 under normal and drought conditions. Detailed Implementation

[0027] The present invention will be further described in detail below through preferred embodiments and examples. Through these descriptions, the features and advantages of the present invention will become clearer and more apparent.

[0028] The term “exemplary” as used herein means “serving as an example, embodiment, or illustration.” Any embodiment illustrated herein as “exemplary” is not necessarily to be construed as superior to or better than other embodiments.

[0029] In a first aspect, the present invention provides a PtoERF54 gene associated with water use efficiency of poplar trees, the gene sequence of which is shown in SEQ ID NO.1.

[0030] The inventors combined drought stress transcriptome data from five different poplar species—P. tomentosa, P. trichocarpa, P. alba × P. davidiana, P. davidiana, and P. deltoides—and conducted weighted co-expression network analysis (WGCNA) to identify the PtoERF54 gene, which is associated with drought tolerance in poplars. Specifically, it is significantly correlated with water use efficiency, a key indicator of drought tolerance in poplars.

[0031] The PtoERF54 gene is a member of the ethylene responsive factor (ERF) gene family. This family typically contains an AP2 / ERF domain and is a core regulator of responses to biotic and abiotic stresses and hormonal signals (especially ethylene). It plays a central role in plant growth and development (root, leaf, flower, fruit, and seed development) and stress adaptation (high temperature, high salinity, drought, and viral infection). ERF family proteins recognize and bind to the cis-acting element GCC-box (AGCCGCC) through their conserved AP2 / ERF domain, activating or inhibiting the transcription of downstream target genes, thereby achieving their regulatory functions.

[0032] Preferably, the CDS sequence of the PtoERF54 gene associated with poplar water use efficiency is shown in SEQ ID NO.2.

[0033] In this invention, the poplar is white poplar, preferably hairy white poplar (Populus tomentosa) or 84k poplar.

[0034] Among them, the 84k poplar is an asexual variety produced by the hybridization of silver poplar and glandular poplar (silver poplar × glandular poplar, Populus alba × Populus glandulosa). It belongs to the preferred tree species of the poplar school. Due to its easy rooting, rapid growth, excellent wood quality and wide adaptability, and because its transformation conditions are easier to standardize, it is often selected as an ideal model for genetic transformation.

[0035] In a second aspect, the present invention provides a protein encoded by the PtoERF54 gene, which is associated with water use efficiency in poplar trees, the encoded protein having an amino acid sequence as shown in SEQ ID NO. 3.

[0036] In a third aspect, the present invention provides an overexpression recombinant vector for the PtoERF54 gene, which is related to water use efficiency of poplar trees as described in the first aspect, wherein the base vector of the overexpression recombinant vector is the 35S-PZP211-3Flag vector.

[0037] Preferably, the overexpression recombinant vector of the PtoERF54 gene, which is related to the water use efficiency of poplar trees, is obtained by constructing the CDS sequence of the PtoERF54 gene onto a base vector.

[0038] More preferably, the CDS sequence of the PtoERF54 gene is shown in SEQ ID NO.2.

[0039] In a fourth aspect, the present invention provides a breeding method for enhancing the drought resistance of poplar trees by improving their water use efficiency, the method being achieved by overexpressing the PtoERF54 gene described in the first aspect or increasing the content of the encoded protein described in the second aspect in poplar plants.

[0040] Preferably, the method includes the following steps:

[0041] Step 1: Construct a recombinant vector for overexpression of the PtoERF54 gene.

[0042] Preferably, the overexpression recombinant vector is obtained by constructing the CDS sequence of the PtoERF54 gene into a base vector, wherein the base vector is the 35S-PZP211-3Flag vector.

[0043] More preferably, step 1 includes the following sub-steps:

[0044] Step 1-1: Obtain the CDS sequence of the PtoERF54 gene.

[0045] In a preferred embodiment, the CDS sequence of the PtoERF54 gene was obtained by amplification using overexpression primers P1 and P2.

[0046] The nucleotide sequence of the overexpression primer P1 is: 5'-ctcacgggtacccgaggatccATGGATGCCTCTATCTTTCACTCTG-3';

[0047] The nucleotide sequence of the overexpression primer P2 is: 5'-agtgagagatctttggtcgacCCAAGGACTCGCAGCCTTT-3'.

[0048] In this invention, the primers are gene primers with added homologous arms (lowercase letter portions in the primer sequences described above).

[0049] Preferably, using Populus tomentosa cDNA as a template, the target fragment is cloned using overexpression primers P1 and P2, and the cloned target fragment has homologous arms.

[0050] Steps 1-2 involve ligating the CDS fragment of PtoERF54 to the digested base vector.

[0051] In a preferred embodiment, the base vector is digested with the restriction enzymes BamHI and SalI.

[0052] Furthermore, the digested base vector was mixed with the CDS fragment and ligated at room temperature for 10 min using T4 ligase.

[0053] Preferably, the system for the above ligation reaction is as follows: 10 μL CDS fragment; 7 μL of enzyme-digested basic vector; 2 μL 10× buffer; 1 μL T4 ligase.

[0054] Steps 1-3: Identify the recombinant vector after ligation.

[0055] In a preferred embodiment, the ligation product is transferred into competent E. coli cells, and positive clones are identified by bacterial PCR.

[0056] Furthermore, plasmids from positive clones were extracted, ultimately yielding a successfully constructed recombinant vector for overexpressing the PtoERF54 gene.

[0057] Step 2: Transform the constructed overexpression recombinant vector into Agrobacterium.

[0058] Preferably, the Agrobacterium is GV3101.

[0059] Step 3: Poplar materials were infected, proliferated, differentiated, and rooted using Agrobacterium-mediated transformation to obtain overexpressing transgenic plants.

[0060] Preferably, the poplar material is 84k poplar.

[0061] According to a preferred embodiment of the present invention, the breeding method for enhancing the drought resistance of poplar trees further includes:

[0062] Step 4: Identify the obtained overexpressing transgenic plants.

[0063] Preferably, the obtained overexpressing transgenic plants are identified using PCR detection and RT-PCR (real-time quantitative PCR).

[0064] According to a preferred embodiment of the present invention, the PCR detection is performed using primers P3 and P4, wherein the sequence of primer P3 is 5'-ATGGATGCCTCTATCTTTCACTCTG-3' and the sequence of primer P4 is 5'-TGTGCTGCAAGGCGATTAAG-3'.

[0065] Preferably, the RT-PCR detection is performed using primers P5 and P6, with primer P5 having the sequence: 5'-TGACTCCGACGAAATGCTCC-3'; and primer P4 having the sequence: 5'-GGAATCCCTTATCTCCGCTG-3'.

[0066] More preferably, the transgenic lines that exhibit specific bands in PCR detection and show a significant increase in PtoERF54 gene transcription level in RT-qPCR results are overexpressing transgenic lines.

[0067] According to a preferred embodiment of the present invention, photosynthetic indicators and drought resistance were measured and evaluated in plants overexpressing the PtoERF54 gene that were identified as positive. Compared with wild-type plants, the water use efficiency of plants overexpressing the PtoERF54 gene was significantly improved and the drought resistance was significantly enhanced.

[0068] Preferably, compared with the wild type, the PtoERF54-OE plant has a 48.76% higher water use efficiency under light saturation conditions and a 56.68% higher water use efficiency under CO2 saturation conditions.

[0069] More preferably, drought resistance evaluation showed that after 6 days of drought treatment, the leaves of the overexpressing plants only showed slight wilting, and could be restored to normal morphology after rehydration;

[0070] Under drought stress, the cell membrane damage of the overexpressing plants was low, and their ion permeability was only 37.31%-38.54%.

[0071] Under drought stress, the overexpressing plants maintained a higher maximum quantum efficiency (Fv / Fm) in photosystem II and a lower non-photochemical quenching (NPQ).

[0072] Under drought stress, the activities of peroxidase (POD) and superoxide dismutase (SOD) in overexpressing plants were significantly increased.

[0073] As can be seen from the above, poplar plants overexpressing the PtoERF54 gene obtained by the method including the above steps have significantly improved water use efficiency, thereby significantly enhancing drought resistance.

[0074] A fifth aspect of the invention provides the application of the gene described in the first aspect or the protein encoded in the second aspect in enhancing the drought resistance of poplar trees.

[0075] Preferably, the enhancement of poplar drought resistance is achieved by improving the poplar's water use efficiency.

[0076] In a sixth aspect, the invention provides the application of the overexpression recombinant vector described in the third aspect in improving the water use efficiency of poplar trees.

[0077] Example

[0078] The present invention is further described below through specific examples; however, these examples are merely exemplary and do not constitute any limitation on the scope of protection of the present invention.

[0079] Unless otherwise specified, the reagents involved in the following examples are all commercially available conventional reagents, and the methods used are all methods commonly used in this technical field.

[0080] Example 1: Obtaining the target fragment of the PtoERF54 gene

[0081] Based on the genome sequence of *Populus tomentosa* (website: https: / / db.cngb.org / search / sample / CNS0752799 / ; reference paper: https: / / onlinelibrary.wiley.com / doi / full / 10.1111 / pbi.14108), the CDS sequence of the drought-resistant PtoERF54 gene of *Populus tomentosa* (as shown in SEQ ID NO.2) was obtained. Taking into account various principles of primer design, overexpression primers P1 and P2 for amplifying the target fragment were designed using the Primer BLAST tool (NCBI, https: / / blast.ncbi.nlm.nih.gov). The sequence of primer P1 (forward primer) is as follows:

[0082] 5'-

[0083] ctcacgggtacccgaggatccATGGATGCCTCTATCTTTCACTCTG-3';

[0084] The sequence of primer P2 (reverse primer) is: 5'-

[0085] agtgagagatctttggtcgacCCAAGGACTCGCAGCCTTT-3'.

[0086] Among them, primers P1 and P2 are gene primers with added homologous arms (lowercase letters in the primer sequences above), which are used to amplify the target fragment (the CDS fragment of the PtoERF54 gene) using Populus tomentosa cDNA as a template.

[0087] The cDNA of Populus tomentosa was obtained according to the following steps:

[0088] ① First, use the Novizan RNA Extraction Kit (product name: FastPure Plant TotalRNAIsolation Kit; catalog number: RC401-01) to extract total RNA from fresh leaf tissue of Populus tomentosa. Refer to the instructions for the RNA extraction kit (https: / / bio.vazyme.com / viewfilebizce / 1767349705707180032 / FastPure%20Plant%20Total%20RNA%20Isolation%20Kit-V22.1.pdf) for the procedure.

[0089] ②Then, using the Novozymes reverse transcription kit (product name: HiScriptIII 1st Strand cDNASynthesis Kit; catalog number: R312-01), following the instructions (https: / / bio.vazyme.com / viewfilebizce / 1767349619996704768 / R312%E8%AF%B4%E6%98%8E%E4%B9%A6-V22.1.pdf), the total RNA from Populus tomentosa obtained in step ① was reverse transcribed into cDNA, thereby obtaining the Populus tomentosa cDNA template.

[0090] The amplification reaction system (25 μL) is shown in Table 1, and the reaction procedure is shown in Table 2.

[0091] Table 1

[0092]

[0093] Table 2

[0094]

[0095]

[0096] Example 2: Construction of a recombinant vector for overexpression of the PtoERF54 gene

[0097] (1) Enzyme digestion vector

[0098] The selected overexpression vector was the 35S-PZP211-3Flag vector, such as... Figure 1 As shown.

[0099] The 35S-PZP211-3Flag overexpression vector was digested with BamHI and SalHI to obtain double-stranded fragments. The digestion system is shown in Table 3, and the reaction conditions were: digestion at 37℃ for 30 min.

[0100] Table 3

[0101]

[0102] The enzyme digestion products were subjected to gel extraction and purification using the Novizan Gel Extraction Kit (FastPure Gel DNA Extraction MiniKit; catalog number: DC301-01), following the instructions (instructions link: https: / / bio.vazyme.com / viewfilebizce / 1767349165158465536 / DC301%E8%AF%B4%E6%98%8E%E4%B9%A6-V22.1.pdf). The purified products were used as the vector backbone and stored at -20℃.

[0103] (2) Connection transformation

[0104] (2.1) Ligation of expression vector

[0105] The CDS fragment of the PtoERF54 gene obtained in Example 1 was mixed with the linear vector digested from the base vector and ligated using T4 ligase at room temperature for 10 minutes. The reaction mixture consisted of: 10 μL of CDS fragment; 7 μL of digested base vector; 2 μL of 10× buffer; and 1 μL of T4 ligase. The ligation product was used to transform Escherichia coli.

[0106] (2.2) Transformation of Escherichia coli DH5α competent cells

[0107] The main process for transforming E. coli is as follows:

[0108] ① Remove the competent E. coli cells from -80℃ and place them on ice to thaw for 10 minutes beforehand.

[0109] ② Add 8 μL of ligation product to the competent cells, gently mix with a pipette, and let stand on ice for 20 minutes.

[0110] ③ Heat shock the competent states containing the ligation product at 42°C for 60-90 seconds.

[0111] ④ After the heat shock ends, immediately take an ice bath for 2 minutes.

[0112] ⑤ Add 950 μL of antibiotic-free LB liquid medium to the tube and incubate at 37°C for 1 hour using a shaker.

[0113] ⑥ Centrifuge at 8000 rpm for 1 minute at room temperature.

[0114] ⑦ Discard the supernatant in a clean bench, leaving a small amount (about 50 μL). Mix the precipitate thoroughly and spread it onto LB solid medium containing rifampicin antibiotic.

[0115] ⑧Incubate overnight in a 37℃ incubator, inverted.

[0116] (2.3) The PCR identification system for positive clones is shown in Table 4.

[0117] Table 4

[0118]

[0119]

[0120] Remove the overnight culture plates, select normally growing bacterial spots, and place them into the mixed screening bacterial system for PCR reaction (see Table 5 for the reaction procedure).

[0121] Table 5

[0122]

[0123] (2.4) Extraction of positive clone plasmids

[0124] Plasmid extraction was performed using the Novizum Plasmid Mini Extraction Kit (name: 8min).

[0125] The procedure is performed using the FastPure Plasmid Mini Kit (item number: DC221-01). For detailed instructions, please refer to the manual: https: / / bio.vazyme.com / viewfilebizce / 1859125199465156608 /

[0126] DC221%20%E5%8D%95%E9%A1%B5%E7%89%88%E8%AF

[0127] The following is a PDF file (%B4%E6%98%8E%E4%B9%A6%20V24.1).

[0128] ① Take 1 ml of overnight culture, centrifuge at 12,000 rpm (13,400 × g) for 1 minute, and discard the supernatant completely.

[0129] ② Add 150 μl of Buffer P1 containing RNase A, and vortex or pipette until the cells are completely dispersed (without any clumps).

[0130] ③ Add 150 μl of Buffer P2, gently invert 15 times until the bacterial solution turns into a clear, viscous blue color (the operation should not exceed 3 minutes).

[0131] ④ Add 350 μl of Buffer NP3, quickly invert 15 times until the blue color disappears (a white flocculent precipitate appears), and centrifuge at 12,000 rpm for 2 minutes.

[0132] ⑤ Transfer the supernatant to the adsorption column (place in the collection tube), centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate.

[0133] ⑥ Add 700 μl of Buffer PW containing anhydrous ethanol, centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate.

[0134] ⑦ Return the adsorption column to the collection tube and centrifuge at 12,000 rpm for 1 minute (to remove residual liquid).

[0135] ⑧ Transfer the adsorption column to a 1.5 ml centrifuge tube, add 30 μl of preheated (65 °C) Elution Buffer, centrifuge at 12,000 rpm for 1 minute, and discard the adsorption column.

[0136] ⑨ Store the extracted plasmids at -20℃.

[0137] The recombinant plasmid obtained in this embodiment is: 35S::PtoERF54-PZP211-3Flag.

[0138] Example 3: Genetic transformation of the PtoERF54 gene

[0139] (1) Transformation of Agrobacterium tumefaciens with recombinant plasmid

[0140] ① Configure the conversion system (as shown in Table 6). YEP stands for Yeast.

[0141] Extract Peptone Medium (Yeast Extract Peptone Medium)

[0142] Table 6

[0143]

[0144] ②Ice bath for 5 minutes, then liquid nitrogen freezing for 8 minutes, and finally water bath at 37°C for 5 minutes.

[0145] ③ After adding YEP, shake in a 28°C incubator for 1 hour.

[0146] ④ Centrifuge at 8000 rpm for 1 minute and collect the bacterial cells.

[0147] ⑤ Spread the Agrobacterium tumefaciens transformed with the expression plasmid onto a solid culture medium containing rifampicin antibiotic; invert the medium and incubate overnight at 37°C.

[0148] (2) Infection, proliferation, differentiation and rooting culture of poplar materials

[0149] (2.1) Preparation of explants.

[0150] Select wild-type 84K poplar seedlings that are 4-6 weeks old, take the 3rd to 5th flat and tender green leaves, cut off the leaf tips in a clean bench, cut the leaves in half along the main vein, and then make 2-3 cuts perpendicular to the main vein.

[0151] (2.2) Preparation of Agrobacterium infection solution

[0152] ①Activate the target Agrobacterium once by streaking it onto YEP solid medium containing rifampicin.

[0153] ② Pick a single colony and inoculate it into 20 mL of liquid YEP medium. Place it in a shaker at 28°C and shake at 200 rpm for 12 hours.

[0154] ③ Take 100 μL of bacterial culture and transfer it to 100 mL of YEP liquid medium containing rifampicin antibiotic. Incubate overnight (10 hours) at 28°C with shaking at 200 rpm.

[0155] ④ Centrifuge at 8000 rpm for 10 minutes at room temperature, discard the supernatant, resuspend the bacterial cells in sterile suspension to prepare the infection solution (as shown in Table 7).

[0156] Table 7

[0157]

[0158] (2.3) Infection

[0159] Immerse the cut leaves in the infection solution and gently shake for 10 minutes. Blot off excess bacterial solution with sterile filter paper. Place the leaves face down on a differentiation medium containing acetylsuccinone (AS) at pH 5.2 and incubate in the dark at 25°C for 48 hours. The composition of the differentiation medium is shown in Table 8.

[0160] Table 8

[0161]

[0162] (2.4) Sterilization and screening

[0163] ① After dark culture, the leaves were washed three times with sterile water containing 100 mg / L termethin, then washed twice with sterile water and dried.

[0164] ② Transfer the leaves to a differentiation medium containing termethin at pH 5.8, and change the medium every 8 days.

[0165] (2.5) Rooting culture

[0166] When the resistant shoots grow to 3-5 cm, they are cut off and transferred to a rooting medium containing termethin. The rooting medium is shown in Table 9.

[0167] Table 9

[0168]

[0169]

[0170] In this embodiment, reagents such as yeast extract and Typtone, as well as plant hormones such as NAA and IBA, were all obtained from Sigma-

[0171] Purchased by China Limited Company.

[0172] Example 4: Identification of plants overexpressing the PtoERF54 gene

[0173] (1) Genomic DNA extraction

[0174] Using the Novizan Plant Genomic DNA Extraction Kit (name: FastPure)

[0175] Plant DNA Isolation MiniKit (catalog number: DC104-01), follow the standard procedure for this kit (website: https: / / bio.vazyme.com / viewfilebizce / 1767350474141548544 / F).

[0176] (astPure%20Plant%20DNA%20Isolation%20Mini%20Kit-V20.1.pdf) was used to extract genomic DNA from control and overexpression lines. The main procedure is as follows:

[0177] ① Take ≤100mg of fresh plant tissue or ≤20mg of dried tissue, grind it into powder with liquid nitrogen, and transfer it to a 1.5ml centrifuge tube.

[0178] ② Add 400 μl Buffer A1 and 4 μl RNase A, vortex to mix, and incubate at 65°C.

[0179] Water bath for 10 minutes.

[0180] ③ Add 130 μl of Buffer A2, mix well, incubate on ice for 5 minutes, centrifuge at 14,000 rpm for 5 minutes, and collect the supernatant.

[0181] ④ Add 1.5 times the volume of Buffer A3 (containing anhydrous ethanol) according to the volume of the supernatant, and immediately mix by pipetting.

[0182] ⑤ Transfer the mixture to the adsorption column in several portions, centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate.

[0183] Add 600 μl of Buffer AW (containing anhydrous ethanol) and centrifuge for 30 seconds. Repeat once.

[0184] ⑥ Centrifuge the empty column at 12,000 rpm for 2 minutes; then add 50-100 μl of preheated (65-70℃) Elution Buffer to the center of the membrane, let it stand at room temperature for 3-5 minutes, centrifuge at 12,000 rpm for 1 minute, and collect the DNA.

[0185] ⑦ Store DNA at -20°C.

[0186] (2) PCR identification

[0187] ①The PCR system is shown in Table 10:

[0188] Table 10

[0189]

[0190] The primer P3 sequence (gene primer) is as follows:

[0191] 5'-ATGGATGCCTCTATCTTTCACTCTG-3';

[0192] The primer P4 sequence (vector primer) is as follows:

[0193] 5'-TGTGCTGCAAGGCGATTAAG-3'.

[0194] ②Reaction program: 95℃ pre-denaturation for 5 min; (95℃ denaturation for 15 s, 58℃ annealing for 2 min, 72℃ extension for 30 s), 35 cycles; 72℃ final extension for 5 min, store at 4℃.

[0195] ③ Electrophoresis detection: PCR products are subjected to 1% agarose gel electrophoresis and compared with the positive control (target gene PCR product). Plants showing specific bands are positive overexpression lines, such as... Figure 2As shown in (A), it can be seen that the PCR detection results of the overexpressing plants numbered OE1 to OE12 are positive, where P represents the PCR identification result of the 35S::PtoERF54-PZP211-3Flag vector, which is used as a comparison for the identification of transgenic lines.

[0196] (3) Quantitative expression

[0197] ①cDNA synthesis: Total RNA was extracted from overexpressing plants using the Novizan RNA Extraction Kit (Product Name: FastPure PlantTotalRNAIsolation Kit; Catalog No.: RC401-01); then cDNA templates were obtained using the Novizan Reverse Transcription Kit (Product Name: HiScriptIII 1st Strand cDNA Synthesis Kit; Catalog No.: R312-01).

[0198] ②The RT-qPCR system is shown in Table 11:

[0199] Table 11

[0200]

[0201] The quantitative primer P5 sequence is: 5'-TGACTCCGACGAAATGCTCC-3';

[0202] The quantitative primer P6 sequence is: 5'-GGAATCCCTTATCTCCGCTG-3'.

[0203] ③ Reaction program: 95℃ pre-denaturation for 30s; (95℃ denaturation for 10s, 60℃ annealing for 30s), 40 cycles; dissolution curve analysis.

[0204] ④ Data Calculation: Using the Actin gene as an internal reference, 2 -ΔΔCt The relative expression levels of the target gene were calculated using the method described in the reference "Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method" (link: https: / / www.sciencedirect.com / science / article / pii / S1046202301912629?via%3Dihub). The expression differences between the transgenic lines and the WT lines were compared. The results are as follows: Figure 2 As shown in (B) in the diagram.

[0205] After the above PCR and RT-PCR identification, the overexpressing plants numbered OE3 and OE5 were found to have specific bands, and the expression levels were 9.2 and 8.5 times that of the control, respectively. Therefore, the two lines OE3 and OE5 were designated as PtoERF54 overexpressing transgenic plants.

[0206] Example 5: Analysis of photosynthetic indicators in plants overexpressing the PtoERF54 gene

[0207] To further elucidate the physiological basis behind the enhanced drought response in the overexpression lines, this embodiment used a LI-6400 portable photosynthesis system to measure water use efficiency parameters under controlled intercellular CO2 concentration (Ci) and photosynthetically active radiation (PAR) conditions.

[0208] Measurement results as follows Figure 3 Figures (A) and (B) show the light response curve of intrinsic water use efficiency (WUE) and the CO2 response curve of WUE. The data are the average of 8 biological replicates, with ± indicating the standard error (n=8).

[0209] Figure 3 The CO2 response curves and light response curves of water use efficiency both showed that the water use efficiency of PtoERF54 overexpressing plants (OE3 and OE5) was significantly higher than that of wild-type plants (WT).

[0210] Example 6: Assessment of drought resistance in plants overexpressing the PtoERF54 gene

[0211] To evaluate the effect of PtoERF54 gene overexpression on the drought resistance of poplar, PtoERF54 overexpressing plants and wild-type (WT) poplar were cultured under the same conditions and subjected to drought stress treatment. After the stress treatment, watering was resumed for 2 days.

[0212] The specific treatment method is as follows: transplant the tissue culture seedlings into a mixed soil (vermiculite: peat moss: potting soil = 1:1:3) and place them in an artificial climate chamber for growth (artificial climate chamber light cycle: 16 hours of light, 8.0 hours of darkness; relative humidity: 40%~45%).

[0213] (Temperature 24℃ during light, temperature 20℃ during darkness). Short-term drought treatment was applied to PtoERF54 overexpressing plants and wild-type plants that had grown normally for 7 weeks after transplanting. Before treatment, the plants were thoroughly watered, and the short-term drought stress treatment began when the relative soil moisture content dropped to 70%.

[0214] The morphological comparison results between overexpressing plants and wild-type plants are as follows: Figure 4As shown in (A) and (B), it can be seen that on the sixth day of drought stress, the leaves of wild-type plants wilted severely, while the leaves of PtoERF54 overexpressing lines wilted less. After rehydration, the recovery ability of WT plants was limited, while the PtoERF54 overexpressing lines could recover to normal morphology.

[0215] Furthermore, the results of the relative water content test of the plant leaves are as follows: Figure 4 As shown in (C), the relative water content of the leaves of the PtoERF54 overexpressing line is 1.42 times that of the wild-type plant, which also supports the result that the leaves of the PtoERF54 overexpressing line are less wilted than those of the wild-type under drought stress.

[0216] Electrolyte leakage in plants was assessed using relative conductivity (REC), and the results were as follows: Figure 4 As shown in (D), both overexpressing and wild-type plants showed increased cell membrane damage under drought stress, but the PtoERF54-overexpressing plants exhibited significantly lower membrane damage, with an ion leakage rate of 37.31%-38.54%, compared to 53.80% for the wild-type. This result indicates that the improved water use efficiency of the PtoERF54-overexpressing lines contributes to their greater tolerance to drought stress. Therefore, compared to the wild-type, the PtoERF54-overexpressing lines showed lower levels of wilting and cell membrane damage.

[0217] The relative conductivity was measured according to the method described in the literature “PeCHYR1,a ubiquitin E3 ligase from Populus euphratica, enhances drought tolerance via ABA-induced stomatal closure by ROS production in Populus” (link: https: / / onlinelibrary.wiley.com / doi / full / 10.1111 / pbi.12893).

[0218] Furthermore, this embodiment also compared the photosynthetic performance and photoprotective capacity of overexpressing plants and wild-type plants before and after drought stress, and the results are as follows: Figure 5 As shown in (A), it can be seen that under drought conditions, the PtoERF54-OE line maintained a higher maximum quantum efficiency (Fv / Fm) of photosystem II and a lower non-photochemical quenching (NPQ), indicating that its photosynthetic performance and photoprotection ability were improved.

[0219] The maximum quantum efficiency of the optical system II and the non-photochemical quenching were determined according to the method described in the literature "Chlorophyllfluorescence—a practical guide" (link: https: / / academic.oup.com / jxb / article / 51 / 345 / 659 / 652534?login=true).

[0220] To further evaluate the oxidative stress response, the activities of key antioxidant enzymes were quantitatively analyzed, and the results are as follows: Figure 5 As shown in (B) in the figure, it can be seen that compared with WT, the activities of peroxidase (POD) and superoxide dismutase (SOD) of the PtoERF54 overexpression line were significantly increased under drought stress, indicating that its reactive oxygen species scavenging ability was enhanced.

[0221] POD and SOD activities were determined according to the method described in the literature “PeCHYR1,aubiquitin E3 ligase from Populus euphratica, enhances drought tolerance via ABA-induced stomatal closure by ROS production in Populus” (https: / / onlinelibrary.wiley.com / doi / full / 10.1111 / pbi.12893).

[0222] The above results indicate that PtoERF54 overexpression plants exhibit significantly enhanced resistance and drought tolerance under drought stress.

[0223] The present invention has been described in detail above with reference to specific embodiments and exemplary examples. However, these descriptions should not be construed as limiting the present invention. Those skilled in the art will understand that various equivalent substitutions, modifications, or improvements can be made to the technical solutions and embodiments of the present invention without departing from the spirit and scope of the present invention, and all such modifications and improvements fall within the scope of the present invention.

Claims

1. A breeding method for enhancing drought tolerance of poplar by improving water use efficiency of poplar, characterized by, The method is achieved by overexpressing in a poplar plant a gene associated with water use efficiency of poplar or increasing the content of a protein encoded by a gene associated with water use efficiency of poplar PtoERF54 . The method is achieved by overexpressing in a poplar plant a gene associated with water use efficiency of poplar or increasing the content of a protein encoded by a gene associated with water use efficiency of poplar PtoERF54 . The sequence of the gene is shown as SEQ ID NO. 1, and the amino acid sequence of the encoded protein is shown as SEQ ID NO.

3.

2. The breeding method according to claim 1, characterized by, The method comprises the following steps: Step 1, Construction PtoERF54 Recombinant vectors for overexpression of genes; Step 2, the constructed overexpression recombinant vector is transformed into Agrobacterium; Step 3, the poplar material is infected, proliferated, differentiated and rooted cultured by the Agrobacterium-mediated method to obtain the overexpression transgenic plant.

3. The breeding method according to claim 2, characterized in that, Step 1 comprises the following sub-steps: Step 1-1, obtaining PtoERF54 CDS sequence of the gene; Steps 1-2, ligation of CDS fragments of PtoERF54 with the digested base vector; Step 1-3, the connected recombinant vector is identified.

4. A gene associated with water use efficiency in poplar or a protein encoded by a gene associated with water use efficiency in poplar for use in enhancing drought tolerance in poplar, characterized in that, The enhanced drought resistance of the poplar is realized by improving the water use efficiency of the poplar; The sequence of the gene is shown as SEQ ID NO. 1, and the amino acid sequence of the encoded protein is shown as SEQ ID NO.

3.

5. Overexpression of a gene related to water use efficiency of poplar PtoERF54 application of the recombinant vector in improving water use efficiency of poplar, characterized in that, The basic vector of the overexpression recombinant vector is 35S-PZP211-3Flag vector; The sequence of the gene is shown as SEQ ID NO. 1.

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