Ptoabf2.1 gene for improving drought resistance and salt tolerance of poplar and application thereof

Abstract

The application discloses a PtoABF2.1 gene for improving drought resistance and salt tolerance of poplar and application of the PtoABF2.1 gene, a nucleotide sequence of the PtoABF2.1 gene is shown as SEQ ID NO. 1, and the silencing transgenic plant obtained by introducing an interference fragment of the PtoABF2.1 gene into the poplar is obviously superior to a wild type and an overexpression plant in growth under conditions of salt stress, drought stress and salt-drought combined stress, the MDA content and the H2O2 content are lower than those of the wild type plant, the POD content and the SOD content are higher than those of the wild type plant, and the drought resistance and the salt tolerance of the poplar are significantly improved, technical support is provided for further cultivation of a new poplar strain, wood production under adversity is ensured, and the application has important significance for improving an ecological environment and maintaining biological diversity.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to poplar stress resistance genes, and more particularly to a PtoABF2.1 gene that improves the drought resistance and salt tolerance of poplar and its application. Background Technology

[0002] Under natural conditions, plants are frequently subjected to various abiotic stresses during their growth and development, such as salinity, drought, low temperature, and high temperature. These stresses not only restrict normal plant growth and development but also significantly impact crop yields and forestry production efficiency. Drought stress is one of the most widespread abiotic stresses. When plants encounter drought, their water balance is disrupted, rapidly initiating a series of complex physiological and biochemical reactions. To reduce water loss, plant stomata close rapidly, hindering gas exchange in leaves and resulting in insufficient carbon dioxide uptake. This, in turn, affects the dark reaction stage of photosynthesis. Furthermore, drought causes a drop in plant cell pressure, leading to leaf wilting and curling, further weakening photosynthetic efficiency. Under prolonged drought stress, plant growth and development are significantly slowed, root growth is restricted, and nutrient absorption capacity decreases, ultimately resulting in slow plant growth and reduced crop yields.

[0003] Soil salinization is also a major challenge for agricultural and forestry development. Excessive salt in the soil not only creates a hypertonic environment, hindering plant roots from absorbing water and causing osmotic stress, but also the accumulation of harmful ions such as sodium and chloride ions disrupts the ion balance within plant cells, causing ion toxicity. This dual stress effect severely interferes with the normal physiological metabolism of plants, resulting in multiple restrictions on plant growth and development.

[0004] Poplar is one of the most important afforestation and greening tree species in northern China, playing an irreplaceable role in ecological protection, timber production, and landscaping. As a perennial woody plant, it is a pioneer species in forest cultivation and is suitable for afforestation, ecological restoration of plants, and improvement of soil environments. Research on drought and salt tolerance in poplar is of great significance for afforestation and greening in arid and saline-alkali areas of my country, expanding the cultivation area of ​​poplar, and improving the ecological environment.

[0005] AREB / ABF transcription factors belong to the bZIP (basic leucine zipper) protein subfamily and play a crucial role in plant stress response mechanisms. Related studies have shown that in the model plant Arabidopsis thaliana, the transcription factor AREB 1 can significantly enhance the plant's tolerance to drought stress by activating the expression of downstream genes in the abscisic acid (ABA) signaling pathway. Currently, there is limited research on the function of ABFs in the drought and salt tolerance of poplar. Furthermore, as a woody model plant, poplar differs from Arabidopsis thaliana in its unique growth and development regulation mechanisms, and the functions of many important regulatory factors may be altered.

[0006] Therefore, it is necessary to study the regulatory role of ABFs in the stress-resistant growth of poplar and to screen specific ABF genes that regulate the drought resistance and salt tolerance of poplar, so as to promote molecular breeding of poplar stress resistance, ensure timber yield under adverse conditions, improve the ecological environment and maintain biodiversity. Summary of the Invention

[0007] To overcome the above problems, the inventors isolated and cloned the PtoABF2.1 gene from poplar trees and found that it was involved in salt stress and drought stress through quantitative fluorescence expression analysis. Then, by constructing a PtoABF2.1 overexpression plant vector and a PtoABF2.1-RNAi plant vector and introducing them into white poplar, PtoABF2.1 overexpression and silence transgenic plants were obtained, respectively. Phenotypic analysis of the plants under stress revealed that under normal conditions, PtoABF2.1 overexpressing transgenic poplars were taller, but silent transgenic poplars showed no significant difference in growth compared to wild-type plants. Under stress conditions, PtoABF2.1-RNAi plants exhibited significantly better growth than wild-type and overexpressing plants, with decreased MDA and H2O2 content and significantly increased POD and SOD content. This indicates that the PtoABF2.1 gene and its encoded protein can significantly improve the drought and salt tolerance of poplars, providing technical support for further breeding of new poplar varieties. Simultaneously, it ensures stable timber production under adverse conditions, which is of great significance for improving the ecological environment and maintaining biodiversity, thus completing this invention.

[0008] Specifically, the object of the present invention is to provide the following aspects:

[0009] In a first aspect, the present invention provides a PtoABF2.1 gene for regulating the drought resistance and salt tolerance of poplar trees, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0010] Secondly, the present invention provides the application of the PtoABF2.1 gene in improving the drought resistance and salt tolerance of poplar trees, wherein the nucleotide sequence of the PtoABF2.1 gene is shown in SEQ ID NO.1; and the amino acid sequence of the protein encoded by the PtoABF2.1 gene is shown in SEQ ID NO.3.

[0011] Thirdly, the present invention provides a method for regulating the drought resistance and salt tolerance of poplar trees, wherein the method regulates the drought resistance and salt tolerance of poplar trees by regulating the expression of the PtoABF2.1 gene in poplar trees.

[0012] Fourthly, the present invention provides a method for improving the drought resistance and salt tolerance of poplar trees, wherein the method silences the expression of the PtoABF2.1 gene by cloning a PtoABF2.1 gene interference fragment.

[0013] The beneficial effects of this invention include:

[0014] (1) The PtoABF2.1 gene and its encoded protein provided by this invention can significantly improve the drought resistance and salt tolerance of poplar, providing technical support for further breeding of new poplar varieties;

[0015] (2) The method for improving the drought resistance and salt tolerance of poplar trees provided by the present invention ensures the stability of timber production under adverse conditions, which is of great significance for improving the ecological environment and maintaining biodiversity, and opens up a new path for the sustainable development of forestry. Attached Figure Description

[0016] Figure 1 The diagram shows the comparison of the relative expression levels of the PtoABF2.1 gene in Populus tomentosa under different salt treatment times (0-48h) as detected by RT-qPCR in Example 1. Figure 2 The graph shows a comparison of the expression levels of the PtoABF2.1 gene in Populus tomentosa under different drought treatment times (0-6d) as detected by RT-qPCR in Example 1. Figure 3 This figure shows a comparison of the expression levels of the PtoABF2.1 gene in different tissues of Populus tomentosa detected by RNA-seq in Example 1.

[0017] Figure 4 The transcriptional level of the PtoABF2.1 gene in overexpressing plants was detected by RT-qPCR in Example 5, and the error bar represents the standard deviation. Figure 5 The transcriptional level of the PtoABF2.1 gene in silent plants was detected by RT-qPCR in Example 5, and the error bars represent the standard deviation. Figure 6 The morphological phenotypes of WT and PtoABF2.1 overexpressing plants (OE-1, OE-6) and silent (RNAi-2, RNAi-3) transgenic plants in Example 6 before and after salt stress, drought stress, and combined salt and drought stress are shown. Figure 7 a-d in the figures show the malondialdehyde (MDA), hydrogen peroxide (H2O2), peroxidase (POD), and superoxide dismutase (SOD) content of WT, PtoABF2.1 overexpression (OE-1, OE-6), and silence (RNAi-2, RNAi-3) transgenic plants before and after treatment with 200 mM NaCl, respectively. Figure 8 a-d in the figures show the content of malondialdehyde (MDA), hydrogen peroxide (H2O2), peroxidase (POD), and superoxide dismutase (SOD) in WT, PtoABF2.1 overexpression (OE-1, OE-6), and silence (RNAi-2, RNAi-3) transgenic plants before and after 20% PEG6000 treatment, respectively. Figure 9 Figures a through d show the content of malondialdehyde (MDA), hydrogen peroxide (H2O2), peroxidase (POD), and superoxide dismutase (SOD) in WT, PtoABF2.1 overexpression (OE-1, OE-6), and silence (RNAi-2, RNAi-3) transgenic plants before and after treatment with 200 mM NaCl + 20% PEG6000, respectively. Detailed Implementation

[0018] 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.

[0019] 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.

[0020] In a first aspect, the present invention provides a PtoABF2.1 gene for regulating drought resistance and salt tolerance in poplar trees, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0021] Preferably, the coding region of the gene has a nucleotide sequence as shown in SEQ ID NO.2 and an amino acid sequence as shown in SEQ ID NO.3.

[0022] In a second aspect, the invention provides the application of the PtoABF2.1 gene in improving the drought resistance and salt tolerance of poplar trees, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0023] The poplar tree is a white poplar, preferably a hairy white poplar.

[0024] Preferably, the PtoABF2.1 gene is located on chromosome 14 of the Populus tomentosa reference genome, with a start position of 1561778 and an end position of 1565624 (http: / / db.cngb.org / cnsa / project / CNP0004290_40760cfc / reviewlink / ).

[0025] According to a preferred embodiment of the present invention, the CDS sequence of the PtoABF2.1 gene is shown in SEQ ID NO.2.

[0026] Preferably, the specific location of the PtoABF2.1 gene CDS sequence is as follows: on chromosome 14, the first segment starts at position 1564038 and ends at position 1565078; the second segment starts at position 1563036 and ends at position 1563116; see http: / / db.cngb.org / cnsa / project / CNP0004290_40760cfc / reviewlink / for details.

[0027] According to a preferred embodiment of the present invention, the amino acid sequence of the protein encoded by the PtoABF2.1 gene is shown in SEQ ID NO.3.

[0028] According to a preferred embodiment of the present invention, the application is to improve the drought resistance and salt tolerance of poplar by reducing the expression level of the PtoABF2.1 gene in poplar.

[0029] Preferably, the method for reducing the expression level of the PtoABF2.1 gene in poplar includes silencing the expression of the PtoABF2.1 gene in poplar.

[0030] In a further preferred embodiment, the silencing is achieved by introducing an interference fragment of the PtoABF2.1 gene into poplar trees.

[0031] Preferably, the interfering fragment of the PtoABF2.1 gene is PtoABF2.1-RNAi, and its nucleotide sequence is shown in SEQ ID NO.4.

[0032] A third aspect of the present invention provides a method for regulating the drought resistance and salt tolerance of poplar trees, wherein the method regulates the drought resistance and salt tolerance of poplar trees by regulating the expression of the PtoABF2.1 gene in poplar trees.

[0033] Preferably, the expression of the PtoABF2.1 gene in poplar is regulated by overexpression or silencing of the PtoABF2.1 gene, the nucleotide sequence of which is shown in SEQ ID NO.1, and the poplar is Populus tomentosa.

[0034] Among them, when the PtoABF2.1 gene was overexpressed, the drought resistance and salt tolerance of poplar decreased; when the PtoABF2.1 gene was silenced, the drought resistance and salt tolerance of poplar increased.

[0035] According to a preferred embodiment of the present invention, the method for regulating the drought resistance and salt tolerance of poplar trees includes the following steps:

[0036] Step I: Obtain the CDS and interference fragment of the PtoABF2.1 gene.

[0037] According to a preferred embodiment of the present invention, the CDS sequence of the PtoABF2.1 gene is obtained by amplification using primers PtoABF2.1-OE-F and PtoABF2.1-OE-R. The nucleotide sequence of primer PtoABF2.1-OE-F is shown in SEQ ID NO.5, and the nucleotide sequence of primer PtoABF2.1-OE-R is shown in SEQ ID NO.6.

[0038] Preferably, the amplified CDS sequence has a nucleotide sequence as shown in SEQ ID NO.2.

[0039] According to a preferred embodiment of the present invention, the interference fragment PtoABF2.1-RNAi of the PtoABF2.1 gene is obtained by amplification using primers PtoABF2.1-RNAi-F and PtoABF2.1-RNAi-R. The nucleotide sequence of primer PtoABF2.1-RNAi-F is shown in SEQ ID NO.7, and the nucleotide sequence of primer PtoABF2.1-RNAi-R is shown in SEQ ID NO.8.

[0040] Preferably, the nucleotide sequence of the amplified interfering fragment PtoABF2.1-RNAi is shown in SEQ ID NO.4.

[0041] Step II: Construct the PtoABF2.1 overexpression recombination vector and the silence recombination vector.

[0042] Preferably, the PtoABF2.1 overexpression recombinant vector is obtained by constructing the CDS sequence of the PtoABF2.1 gene into the pBI121 vector;

[0043] The PtoABF2.1 silencing recombinant vector was obtained by constructing an interference fragment of the PtoABF2.1 gene into the pBI121 vector.

[0044] More preferably, the recombinant vector after ligation is tested to obtain positive clones, and then plasmids are extracted and sequenced. If the sequence determination is correct, the vector construction is complete.

[0045] Step III involves genetic transformation to identify overexpressing and silent plants.

[0046] The constructed recombinant vector plasmid is transformed into Agrobacterium and activated to obtain Agrobacterium bacterial culture. Preferably, the Agrobacterium is EHA105.

[0047] Furthermore, using the leaf disc method, Agrobacterium tumefaciens solution was used to infect poplar leaves, and then complete poplar plants were obtained through co-culture, differentiation culture, and rooting culture.

[0048] Furthermore, DNA-level and transcription-level identification were performed on intact poplar plants, resulting in PtoABF2.1 overexpressing plants and PtoABF2.1 silent plants, respectively.

[0049] Step IV: Stress treatment and phenotypic analysis were performed on overexpressing and silent plants.

[0050] According to a preferred embodiment of the present invention, the stress treatment includes salt stress treatment, drought stress treatment, and combined salt and drought stress treatment.

[0051] Preferably, the salt stress treatment is as follows: one-month-old wild-type tissue culture seedlings (approximately 10 cm in height) are cleaned of the agar medium at the roots of the seedlings in water, and then transferred to a soil culture medium containing a 1:1.5 mixture of substrate and vermiculite. The light conditions are 8 hours of dark culture followed by 16 hours of light culture. The seedlings are then cultured for 20 days in a constant temperature incubator at 25°C under the same light conditions, and then irrigated with 200 mM NaCl solution for 20 days.

[0052] The drought stress treatment was as follows: one-month-old wild-type tissue culture seedlings (approximately 10 cm in height) were cleaned of the agar medium from the roots of the seedlings in water, and then transferred to a soil culture medium containing a 1:1.5 mixture of substrate and vermiculite. The light conditions were 8 hours of dark culture followed by 16 hours of light culture. The seedlings were then cultured in a constant temperature incubator at 25°C under the same light conditions for 20 days, and then irrigated with 20% PEG 6000 solution for 10 days.

[0053] The salt and drought combined stress treatment was as follows: one-month-old wild-type tissue culture seedlings (about 10cm in height) were cleaned of the agar medium at the roots of the seedlings in water, and then transferred to a soil culture medium of substrate and vermiculite mixed at a ratio of 1:1.5. The light conditions were 8 hours of dark culture and 16 hours of light culture. The seedlings were cultured for 20 days in a constant temperature culture room under the same light conditions at 25℃, and then watered with a mixed solution of 20% PEG 6000 and 200mM NaCl for 7 days.

[0054] Morphological phenotypic analysis and physiological index determination were performed on poplar plants that underwent the above-mentioned stress treatments.

[0055] Preferably, the physiological indicators include malondialdehyde (MDA) content, hydrogen peroxide (H2O2) content, peroxidase (POD) content, and superoxide dismutase (SOD) content.

[0056] In this invention, through analysis of the morphological phenotype and physiological traits of overexpressing and silent plants, it was found that under normal conditions, there was no significant difference in growth status and growth rate between silent plants of the PtoABF2.1 gene and wild-type Populus tomentosa plants.

[0057] Under salt stress, drought stress, and combined salt and drought stress, compared with wild-type plants and PtoABF2.1 overexpressing plants, PtoABF2.1 silenced plants showed better growth, lower malondialdehyde and hydrogen peroxide content, and higher peroxidase and superoxide dismutase content; the physiological traits of PtoABF2.1 overexpressing plants showed the opposite trend.

[0058] As described above, the drought resistance and salt tolerance of poplar trees can be regulated by controlling the expression of the PtoABF2.1 gene. Specifically, overexpressing the PtoABF2.1 gene in poplar plants reduces their drought resistance and salt tolerance; silencing the PtoABF2.1 gene in poplar plants increases their drought resistance and salt tolerance. This demonstrates that the PtoABF2.1 gene and its encoded protein provided by this invention can significantly improve the drought resistance and salt tolerance of poplar trees, providing technical support for further breeding of new poplar varieties.

[0059] In a fourth aspect, the present invention provides a method for improving the drought resistance and salt tolerance of poplar trees, wherein the method silences the expression of the PtoABF2.1 gene by cloning a PtoABF2.1 gene interference fragment.

[0060] Preferably, the PtoABF2.1 gene interference fragment is the reversed CDS portion of the PtoABF2.1 gene; more preferably, the nucleotide sequence of the PtoABF2.1 gene interference fragment is as shown in SEQ ID NO.4.

[0061] According to a preferred embodiment of the present invention, the method includes the following steps:

[0062] Step 1: Construct the poplar PtoABF2.1 gene RNAi silencing recombinant vector.

[0063] Preferably, step 1 includes the following sub-steps:

[0064] Step 1-1: Obtain the interference fragment PtoABF2.1-RNAi that can specifically silence the PtoABF2.1 gene.

[0065] According to a preferred embodiment of the present invention, the interfering fragment PtoABF2.1-RNAi of the PtoABF2.1 gene is obtained by amplification using primers PtoABF2.1-RNAi-F and PtoABF2.1-RNAi-R, wherein the nucleotide sequence of primer PtoABF2.1-RNAi-F is shown in SEQ ID NO.7, and the nucleotide sequence of primer PtoABF2.1-RNAi-R is shown in SEQ ID NO.8.

[0066] Steps 1-2: Digest the vector with enzymes, ligate the interference fragment into the base vector, and obtain the RNAi silencing recombinant vector.

[0067] Preferably, the base vector is the pBI121 vector, which is 13629 bp in length and contains a strong 35S promoter (CaMV35S). It is resistant to kanamycin and can be digested with XbaI and BamHI.

[0068] Step 2: Transform the constructed silent recombination vector into E. coli to obtain a silent recombination vector with the correct insertion of the interfering fragment.

[0069] In this process, the recombinant vector was transformed into Escherichia coli DH5α competent cells to obtain single colonies, which were then identified by PCR amplification.

[0070] Preferably, the reaction procedure for single colony identification is: 95℃ for 2 min; (94℃ for 30 s; 55℃ for 30 s; 72℃ for 60 s) 35 cycles; 72℃ for 5 min; 4℃ for ∞.

[0071] Step 3: Extract the silencing recombinant vector plasmid obtained in Step 2 and transfer it into poplar plants via Agrobacterium-mediated transformation.

[0072] Preferably, step 3 includes the following sub-steps:

[0073] Step 3-1: Transform the silent recombinant vector plasmid into Agrobacterium.

[0074] Preferably, the Agrobacterium is EHA105.

[0075] Step 3-2: Activate Agrobacterium.

[0076] Step 3-3: Use the leaf disc method to infect poplar plants.

[0077] Preferably, the poplar plant is a white poplar plant, and the leaves of the poplar plant are selected for infection.

[0078] Step 4: Obtain complete poplar plants that have been transferred into the silent recombinant plasmid. Identify positive plants, which are poplars with high drought resistance and salt tolerance.

[0079] Preferably, after infection, poplar leaves are co-cultured, resistance cultured, and selected for subculture to obtain adventitious buds. Adventitious buds are then grown into adventitious roots through rooting culture, thereby obtaining a complete poplar plant.

[0080] According to a preferred embodiment of the present invention, the identification of positive plants includes identification at the DNA level and identification at the transcription level.

[0081] Preferably, the reaction procedure for DNA level identification is as follows: 95℃ for 2 min; (94℃ for 30 s; 55℃ for 30 s; 72℃ for 60 s) 35 cycles; 72℃ for 5 min; 4℃ for ∞.

[0082] More preferably, the identification of the transcription level is performed by RT-qPCR, and the reaction program is: 95℃ for 30s; (95℃ for 5s; 60℃ for 35s) for 40 cycles; 95℃ for 15s; 60℃ for 1 min; 95℃ for 15s.

[0083] The method for improving the drought resistance and salt tolerance of poplar trees provided by this invention ensures the stability of timber production under adverse conditions, which is of great significance for improving the ecological environment and maintaining biodiversity, and opens up a new path for the sustainable development of forestry.

[0084] Example

[0085] 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.

[0086] 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.

[0087] Example 1: Quantitative Detection of PtoABF2.1 Gene

[0088] (1) Two-month-old hydroponically grown Populus tomentosa were subjected to salt stress (150mM NaCl) and simulated drought stress (20% PGE 6000). Populus leaves were collected at 0, 6, 12, 24 and 48 h after 150mM NaCl treatment and at 0, 1, 3 and 6 d after 20% PEG 6000 treatment. RNA was extracted from the collected leaves according to the instructions of the MolPure Plant RNA Kit from Yisheng Biotechnology Co., Ltd.

[0089] Using the RNA obtained above as a template, cDNA was obtained by reverse transcription using HiScript IV All-in-One Ultra RT SuperMix for qPCR-R433 (Nanjing Novizan Biotechnology Co., Ltd.). The reaction system (20 μL) is shown in Table 1.

[0090] Table 1

[0091]

[0092] The reaction procedure was: 50℃ for 5 min; 85℃ for 5 sec.

[0093] Based on the PtoABF2.1 gene sequence of Populus tomentosa, fluorescent quantitative PCR primers PtoABF2.1-qPCR-F and PtoABF2.1-qPCR-R were designed, and their nucleotide sequences are shown in SEQ ID NO.9 and SEQ ID NO.10, respectively; the nucleotide sequences of the internal control primers Actin-F and Actin-R are shown in SEQ ID NO.11 and SEQ ID NO.12, respectively.

[0094] The expression level of PtoABF2.1 gene under different stress times was determined by real-time PCR using 2×ChamQSYBR Color qPCR Master Mix. The reaction volume (20 μL) is shown in Table 2.

[0095] Table 2

[0096]

[0097] The reaction program was as follows: 95℃ for 30s; (95℃ for 5s; 60℃ for 35s) for 40 cycles; 95℃ for 15s; 60℃ for 1min; 95℃ for 15s.

[0098] The results of the real-time PCR reaction are as follows Figure 1 and Figure 2 As shown, it can be seen that the expression level of the PtoABF2.1 gene changes significantly with the extension of salt and drought treatment time, suggesting that the PtoABF2.1 gene plays a role in the response of Populus tortoise shell to salt and drought stress.

[0099] (2) To further investigate the tissue-specific expression pattern of the PtoABF2.1 gene during the growth and development of Populus tomentosa, mature Populus tomentosa plants were selected, and systematic samples were taken from different tissues, including roots, stems, cambium, immature xylem, and mature xylem. RNA-seq detection was performed, using the same reaction system and procedure as above. The results are as follows: Figure 3 As shown, the expression level of the PtoABF2.1 gene in various tissues of Populus tomentosa is relatively consistent, with no significant differences. After standardization, the average log2 expression level showed minimal fluctuations across tissues, and statistical tests confirmed that the expression differences between tissues were not statistically significant (P>0.05). This result suggests that the PtoABF2.1 gene may not specifically participate in a particular developmental process of a specific tissue, but rather plays a relatively stable and widespread role in the overall growth and development of Populus tomentosa.

[0100] Example 2: Obtaining the CDS sequence and interference fragment of the PtoABF2.1 gene

[0101] (1) RNA was extracted from LM50 Populus tomentosa using the plant RNA extraction kit from Beijing TransGen Biotech Co., Ltd.; the RNA was reverse transcribed into cDNA using the HiScript III 1st Strand cDNA Synthesis Kit (Nanjing Novizan Biotechnology Co., Ltd.). The specific steps are as follows:

[0102] (1) RNA template denaturation

[0103] ① Add RNA sample (total amount not exceeding 1 μg) according to RNA concentration;

[0104] ②Use RNase-free dd H2O to bring the total volume to 8 μl;

[0105] ③ The reaction temperature is 65℃, and the reaction time is 5 minutes;

[0106] ④ Quickly place it on ice to cool rapidly, and let it stand on ice for 2 minutes.

[0107] (2) Remove genomic DNA

[0108] ① Add 2 μl of 5x g DNA wiper Mix to the above reaction tube;

[0109] ② The reaction conditions were 42℃ and 120s.

[0110] (3) cDNA synthesis

[0111] ① Add the reaction mixture shown in Table 3 below to the reaction mixture from the previous step:

[0112] Table 3

[0113]

[0114] ② The reaction conditions were 53℃ for 45 min, 85℃ for 5 s.

[0115] ③ The obtained product can be directly used for PCR reaction.

[0116] (2) Using cDNA as a template and referring to the Populus tomentosa genome file (http: / / db.cngb.org / cnsa / project / CNP0004290_40760cfc / reviewlink / ), and considering various principles of primer design, primers for the PtoABF2.1 overexpression sequence (CDS sequence) and the silence sequence (CDS partial reverse sequence) were designed using the Primer BLAST tool (NCBI, https: / / blast.ncbi.nlm.nih.gov). The amplification primers for the PtoABF2.1 gene CDS sequence were PtoABF2.1-OE-F and PtoABF2.1-OE-R, and their nucleotide sequences are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively. The amplification primers for the silence sequence (the interfering fragment PtoABF2.1-RNAi of the PtoABF2.1 gene) were PtoABF2.1-RNAi-F and PtoABF2.1-RNAi-R, and their nucleotide sequences are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively. As shown in NO.7 and SEQ ID NO.8.

[0117] The gene PtoABF2.1 is located on chromosome 14 of the Populus tomentosa genome (http: / / db.cngb.org / cnsa / project / CNP0004290_40760cfc / reviewlink / ), with a start position of 1561778 and an end position of 1565624.

[0118] Using cDNA obtained from reverse transcription as a template, PCR amplification was performed using 2x Phanta Max Master Mix (Dye Plus) from Nanjing Vazyme Company. The PCR reaction system (50 μL) is shown in Table 4.

[0119] Table 4

[0120]

[0121] The reaction program was as follows: 95℃ for 3 min; (95℃ for 25 s; 55℃ for 30 s; 72℃ for 60 s (amplification efficiency 45 s / kb)) 36 cycles; 72℃ for 5 min; 4℃ to infinity.

[0122] The PCR products were subjected to 2% agarose gel electrophoresis. After verification, the DNA was recovered and purified using the Kangwei Century DNA Clean-up Kit. Following purification, the purity and concentration of the purified DNA product were determined using an instrument.

[0123] The final CDS sequence of the PtoABF2.1 gene was obtained, as shown in SEQ ID NO.2, the amino acid sequence of the encoded protein is shown in SEQ ID NO.3, and the nucleotide sequence of the interfering fragment PtoABF2.1-RNAi (CDS partial reverse sequence) is shown in SEQ ID NO.4.

[0124] Example 3: Construction of PtoABF2.1 gene overexpression vector and silencing recombinant vector

[0125] (1) Enzyme digestion vector

[0126] The selected overexpression and silencing vectors were both pBI121 vectors, totaling 13629 bp, containing a strong 35S promoter (CaMV35S), resistant to kanamycin (Kana), and digested with XbaI and BamHI.

[0127] The enzyme digestion reaction system is shown in Table 5:

[0128] Table 5

[0129]

[0130] The enzyme digestion reaction conditions are: 37℃ water bath for 1-2 hours.

[0131] The enzyme digestion products were purified by gel extraction using the Kangwei Century GelExtraction Kit and purified using the Kangwei Century DNA Clean-up Kit. The purified products were then used as vector backbones and stored at -20°C.

[0132] (2) Connection transformation

[0133] (2.1) Connecting the pBI121 vector

[0134] Using the Uniclone One Step Seamless Cloning Kit from Beijing Jinsha Biotechnology Co., Ltd., the CDS and CDS partial reverse sequences of PtoABF2.1 were constructed into the pBI121 vector to obtain overexpression recombinant vector and silence recombinant vector, respectively.

[0135] Then, the cells were ligated at 50°C for 15 minutes, and the ligation product was used to transform E. coli DH5α competent cells.

[0136] (2.2) Transformation of Escherichia coli DH5α competent cells

[0137] Take 50 μl of DH5α competent cells, add 10 μl of ligation product, gently tap to mix, and incubate on ice for 30 minutes. Then, heat shock the mixture in a 42°C water bath for 45 seconds, and quickly transfer it to ice to cool for 2 minutes, avoiding shaking during this process to ensure transformation efficiency. Next, add 700 μl of antibiotic-free sterile LB liquid medium, mix well by pipetting, and incubate at 37°C and 220 rpm on a shaker for 45 minutes to allow the cells to recover. After recovery, collect the cells by centrifugation at 6000 rpm for 1 minute, resuspend the cells in 100 μl of supernatant, and spread them on LB agar plates containing kanamycin (100 mg / ml). Incubate at 37°C upside down for 14-16 hours.

[0138] (2.3) Identification of positive clones by bacterial culture PCR

[0139] Single colonies from the plate were picked sequentially using sterilized pipette tips and added to 250 μl of LB broth containing kanamycin. The culture was incubated at 37°C with shaking at 200 rpm for approximately 3 hours to serve as amplification templates. Separately, the purified gene PCR product and ddH2O (double-distilled water) were used as templates, with positive and negative controls established respectively. PCR amplification was performed using Taq Plus Master Mix from Nanjing Vazyme. The PCR reaction system is shown in Table 6.

[0140] Table 6

[0141]

[0142] The reaction program was as follows: 95℃ for 2 min; (94℃ for 30 s; 55℃ for 30 s; 72℃ for 60 s) for 35 cycles; 72℃ for 5 min; 4℃ for ∞.

[0143] The nucleotide sequence of primer pBI121-R is shown in SEQ ID NO.13.

[0144] PCR products are detected by 1% agarose gel electrophoresis. Colonies that amplify a band of the same size as the positive control are considered positive clones.

[0145] (2.4) Extraction of positive clone plasmids

[0146] Clones that tested positive for PCR were aspirated into 6 ml of LB liquid medium containing kanamycin and incubated overnight at 37°C with shaking at 200 rpm. Plasmids were extracted using a plasmid miniprep kit from Jiangsu Kangwei Reagent Co., Ltd., and sequenced by Beijing Ruiboxingke Biotechnology Co., Ltd. After the sequences were confirmed to be correct, the construction of the overexpression recombinant vector and the silencing recombinant vector was completed.

[0147] Example 4: Genetic transformation of the PtoABF2.1 gene

[0148] (1) Transformation of Agrobacterium tumefaciens with recombinant plasmid

[0149] (1.1) Take about 1 μg of the overexpression vector plasmid and the silencing vector plasmid of PtoABF2.1 and add them to 100 μL of Agrobacterium EHA105 competent cells, and mix gently.

[0150] (1.2) Place on ice for 5 min, freeze in liquid nitrogen for 5 min, immediately place in a 37℃ water bath for 5 min, and then in an ice bath for 5 min.

[0151] (1.3) Add 700 μL of antibiotic-free YEP liquid medium to the bacterial culture, mix thoroughly, and incubate at 28°C and 200 rpm with shaking for 2–3 hours. After the incubation period, centrifuge the bacterial culture at 6000 rpm for 1 minute, discard part of the supernatant, and mix 100 μL of the supernatant with the bacterial culture. Using sterilized and cooled glass beads, evenly spread the bacterial culture on the surface of YEP solid medium containing 50 mg / L rifampicin and 50 mg / L kanamycin, and then incubate upside down in a 28°C incubator for 72–90 hours.

[0152] (1.4) After single colonies have grown, use a sterile pipette tip to pick up several single-clone plaques and place them in a 2 mL centrifuge tube containing 250 μL of YEP liquid medium (1:1000 with added Rif and Kana). Incubate at 30°C and 200 rpm for 2 h on a shaker. After incubation, aspirate the bacterial solution for PCR identification, using the same method as described in Example 3. Correctly identified positive bacterial solutions are then flash-frozen in liquid nitrogen with 50% glycerol and stored at -80°C for subsequent genetic transformation experiments.

[0153] (2) Activation of Agrobacterium

[0154] (2.1) Take out Agrobacterium tumefaciens containing overexpression vector plasmid and silencing vector plasmid from the -80℃ freezer, streak them onto YEP solid medium (1:1000 with added Rif and Kana), seal the plate, and incubate upside down in a 28℃ dark incubator for 2-3 days.

[0155] (2.2) Using a sterile pipette tip, pick a single colony from the plate and inoculate it into 3 mL of YEP liquid medium (1:1000 with added Rif and Kana). Incubate overnight at 30°C and 200 rpm with shaking. Transfer 1 mL of the bacterial culture to a 250 mL sterile Erlenmeyer flask containing 100 mL of YEP liquid medium (1:1000 with added Rif and Kana). Incubate at 30°C and 200 rpm with shaking for 4–5 h until the OD600 reaches 0.6–0.8.

[0156] (2.3) In a clean bench, pour the Agrobacterium tumefaciens solution into a sterile 50ml centrifuge tube, centrifuge at 3500rpm-5000rpm for 7-10 minutes at room temperature, collect the bacterial solution, pour in an equal volume of resuspension, shake to resuspend the bacterial cells, centrifuge again, and rinse the bacterial cells with the resuspension. This bacterial solution is used for subsequent infection experiments.

[0157] The resuspension consisted of 1 / 2 MS + 20 g sucrose + 100 mM AS (added fresh for use) with a pH of 5.6. After preparation, it was sterilized at 121°C for 20 minutes under high temperature and pressure.

[0158] (3) Leaf disc infection

[0159] (3.1) Blade pretreatment

[0160] Select leaves from healthy, sterile poplar seedlings (the leaves are dark green and relatively thick, generally the 4th to 6th leaves from the top). Use a sterile scalpel to make 2 to 3 horizontal cuts on the main vein of the leaf. Lay the leaves face down on a pre-culture medium without antibiotics and incubate at 25°C for 1 to 2 days.

[0161] The pre-culture medium consisted of MS + 30g sucrose + 5g gel, pH = 5.9. After preparation, it was sterilized at 121℃ for 20 minutes under high temperature and high pressure.

[0162] (3.2) Infection and Co-culture

[0163] In a clean bench, pretreated leaves are immersed in a resuspended Agrobacterium solution for 10–15 minutes, gently agitating the leaves to ensure adequate contact with the solution at any damaged areas. Infection is performed under incandescent light on the clean bench. The leaves are then removed with sterile forceps and placed on pre-sterilized filter paper to absorb excess solution. Finally, the infected leaves are inoculated face up onto a co-culture medium (with added antibiotics, As) and incubated in the dark at 25°C for 3–4 days until bacterial colonies appear near the leaves.

[0164] The co-culture medium in the co-culture plates consisted of MS + 30g sucrose + 7g agar + 100mM AS pH = 5.9. After preparation, it was sterilized at 121℃ for 20 minutes by high temperature and high pressure.

[0165] (3.3) Differentiation culture

[0166] After dark culture, select appropriate plant resistance based on the vector and prepare differentiation medium containing the corresponding antibiotics.

[0167] On a clean bench, leaves that have been co-cultured for 3 days under dark conditions are placed on pre-sterilized filter paper to remove excess bacteria, and then transferred to differentiation medium for differentiation culture. The light cycle is 16 hours of light and 8 hours of darkness, and the temperature is 25°C.

[0168] After culturing on differentiation medium for 10–15 days, in a clean bench, use sterile forceps or a scalpel to cut out dense, light green callus tissue or leaf discs with adventitious buds, and place them on fresh differentiation medium to ensure the callus tissue or adventitious buds have sufficient nutrients to induce differentiation. The medium is generally changed approximately every 10 days.

[0169] The differentiation medium consisted of: MS + 30g sucrose + 0.5mg / L 6BA + 0.05mg / L NAA + 7g agar + 250mg / L TMT + 250mg / L cephalosporin + 20mg / L kana, pH = 5.9. After preparation, it was sterilized at 121℃ for 20min under high temperature and high pressure.

[0170] (3.4) Rooting culture

[0171] After two weeks of culture on differentiation medium, when the adventitious buds grow to 3-4 cm or more, they are individually cut off using sterile forceps or a scalpel in a clean bench and placed on rooting medium for rooting culture. Adventitious roots will emerge from the buds after two weeks. Following rooting, subculture is performed (the first subculture medium contains termethin and kanamycin, subsequent subcultures only contain termethin). Molecular analysis of the leaves can be performed during the first subculture.

[0172] The rooting medium consisted of 1 / 2 MS + 20g sucrose + 0.05mg / L IBA + 0.02mg / L NAA + 7g agar + 250mg / L TMT + 250mg / L cephalosporin + 20mg / L Kana. After preparation, it was sterilized at 121℃ for 20 minutes under high temperature and high pressure.

[0173] Example 5: Identification of PtoABF2.1 gene overexpression and silencing plants

[0174] (1) Crude DNA extraction from wild-type Populus tomentosa, PtoABF2.1 gene overexpressing plants and silent plants (RNAi plants):

[0175] (1.1) Take a leaf sample to be extracted, place it in a 2mL centrifuge tube and add a grinding bead. After quick freezing with liquid nitrogen, use a tissue homogenizer to homogenize for 5min.

[0176] (1.2) After the mixture is broken into powder, add 500 μL of TBS buffer to the centrifuge tube, mix well, and then heat in a 65°C water bath for 10 min.

[0177] (1.3) Centrifuge at 12000 rpm for 15 min, transfer the supernatant to a new centrifuge tube, add an equal volume of isopropanol, and allow to settle at room temperature for 30 min. A white flocculent precipitate will be visible.

[0178] (1.4) Centrifuge at 12000 rpm for 15 min, slowly discard the supernatant, and add 1 ml of 75% ethanol to wash the precipitate.

[0179] (1.5) Centrifuge at 7500 rpm for 10 min, slowly pour off the supernatant and remove excess liquid with a pipette.

[0180] (1.6) Place in a 40℃ oven for 2 hours.

[0181] (1.7) Add 50 μL of ddH2O to the centrifuge tube to dissolve the precipitate and store it at -20°C.

[0182] (2) Identification of plants overexpressing and silent PtoABF2.1 gene

[0183] (2.1) Identification of DNA level

[0184] Using crudely extracted gDNA from transgenic plants as a template, PCR identification was performed using Taq Plus Master Mix. The reaction system was prepared according to Table 6.

[0185] The PCR reaction program was as follows: 95℃ for 2 min; (94℃ for 30 s; 55℃ for 30 s; 72℃ for 60 s) for 35 cycles; 72℃ for 5 min; 4℃ for ∞.

[0186] The PCR products were detected by agarose gel electrophoresis to check if the bands were correct. If correct, the plant was either an overexpressing or silenced PtoABF2.1 gene plant.

[0187] PCR detection revealed that plants numbered OE-1, OE-2, OE-4, OE-6, OE-9, OE-14, OE-17, and OE-18 were PtoABF2.1 overexpressing transgenic plants, while plants numbered RNAi-1, RNAi-2, RNAi-3, RNAi-4, RNAi-5, RNAi-10, and RNAi-11 were PtoABF2.1 silenced transgenic plants.

[0188] (2.2) Identification of transcription level

[0189] The transcriptional levels of PtoABF2.1 in overexpressing and silenced plants were detected using RT-qPCR.

[0190] (i) RNA was extracted from the leaves of the plant to be tested, and reverse transcribed to obtain template cDNA. RNA extraction and reverse transcription of cDNA were performed according to the method described in the kit in Example 1.

[0191] (ii) The RT-qPCR experimental reaction system was prepared according to Table 2 using 2×ChamQ SYBR Color qPCR Master Mix. The primers were PtoABF2.1-qPCR-F and PtoABF2.1-qPCR-R, and the internal control primers were Actin-F and Actin-R.

[0192] Reaction program: 95℃ for 30s; (95℃ for 5s; 60℃ for 35s) 40 cycles; 95℃ for 15s; 60℃ for 1min; 95℃ for 15s.

[0193] The detection results of overexpressing plants are as follows Figure 4 As shown, by Figure 4 It can be seen that the PtoABF2.1 overexpressing plants numbered OE-1 and OE-6 had higher expression levels, which were 19.10 times and 23.42 times higher than those of wild-type plants (WT), respectively.

[0194] The test results of silent plants are as follows: Figure 5 As shown, by Figure 5 It can be seen that the expression levels of PtoABF2.1 silenced plants numbered RNAi-2 and RNAi-3 were low, being 0.23 times and 0.043 times that of wild-type plants (WT), respectively.

[0195] Example 6: Stress phenotype analysis of PtoABF2.1 gene overexpression and silencing plants

[0196] (1) Coercive Management

[0197] (1.1) Salt stress treatment

[0198] One-month-old wild-type tissue culture seedlings (approximately 10cm in height) were cleaned of the agar medium from the roots in water and then transferred to a soil culture medium containing a 1:1.5 mixture of substrate and vermiculite. The light conditions were 8 hours of dark culture followed by 16 hours of light culture. The seedlings were then cultured for 20 days in a constant temperature incubator at 25°C under the same light conditions, and then watered with 200mM NaCl solution for 20 days.

[0199] (1.2) Drought stress treatment

[0200] One-month-old wild-type tissue culture seedlings (approximately 10cm in height) were cleaned of the agar medium from their roots in water and then transferred to a soil culture medium containing a 1:1.5 mixture of substrate and vermiculite. The seedlings were cultured under 8 hours of darkness and 16 hours of light. They were then cultured for 20 days in a constant temperature incubator at 25°C under the same light conditions, and then irrigated with a 20% PEG 6000 solution for 10 days.

[0201] (1.3) Treatment under combined salt and drought stress

[0202] One-month-old wild-type tissue culture seedlings (approximately 10cm in height) were cleaned of the agar medium from their roots in water and then transferred to a soil culture medium containing a 1:1.5 mixture of substrate and vermiculite. The seedlings were cultured under 8 hours of darkness and 16 hours of light. They were then cultured for 20 days in a constant temperature incubator at 25°C under the same light conditions, and then watered with a mixed solution of 20% PEG 6000 and 200mM NaCl for 7 days.

[0203] (2) Measurement of physiological indicators

[0204] (2.1) Determination of malondialdehyde (MDA) content

[0205] The content of malondialdehyde (MDA) was determined using the Suzhou Keming Biotechnology Malondialdehyde (MDA) content determination kit. The specific steps are as follows:

[0206] (i) Weigh 0.1g of poplar leaves after stress treatment, add 1mL of extract, homogenize in an ice bath, centrifuge at 8000g 4℃ for 10min, take the supernatant and place it on ice for testing;

[0207] (ii) Pipette 0.3 mL of reagent one into a 1.5 mL centrifuge tube, add 0.1 mL of sample, and mix well;

[0208] (iii) Keep warm in a 95℃ water bath for 30 min (cover tightly to prevent moisture loss), cool in an ice bath, and centrifuge at 10000g, 25℃ for 10 min;

[0209] (iv) Pipette 200 μL of supernatant into a 96-well plate and measure the absorbance at 532 nm and 600 nm, denoted as A532 and A600, ΔA = A532 - A600;

[0210] (v) Calculate the MDA content according to the MDA calculation formula. MDA content (nmol / g fresh weight) = [ΔA × V total ÷ (ε × d) × 109] ÷ (W × V sample ÷ V total sample) = 51.6 × ΔA ÷ W.

[0211] (2.2) Determination of hydrogen peroxide (H2O2) content

[0212] The hydrogen peroxide (H2O2) content was determined using the Suzhou Keming Biotechnology Hydrogen Peroxide (H2O2) Content Assay Kit:

[0213] (i) Weigh 0.1g of poplar leaves after stress treatment, add 1mL of reagent one, and homogenize in an ice bath; transfer to an EP tube, add reagent one to make up to 1mL, centrifuge at 8000g 4℃ for 10min, take the supernatant, and place it on ice for testing;

[0214] (ii) Preheat the microplate reader for at least 30 minutes, adjust the wavelength to 415nm, and zero the instrument with distilled water;

[0215] (iii) Incubate reagents two, three and four in a water bath at 25°C for at least 10 minutes;

[0216] (iv) Add the reagents listed in Table 7 below to the EP tube in the following order:

[0217] Table 7

[0218]

[0219] After dissolving the precipitate with reagent four, let it stand at room temperature for 5 minutes. Transfer 200 μL to a 96-well plate and measure the absorbance A at 415 nm. The control tube only needs to be tested once. Calculate ΔA = Ameasured - Acontrol.

[0220] (v) Calculate the H2O2 content according to the H2O2 calculation formula: H2O2 content (μmol / g fresh weight) = [(ΔA-0.0006)÷0.3744×V1]÷(W×V1÷V2) = 2.67×(ΔA-0.0006)÷W.

[0221] (2.3) Determination of superoxide dismutase (SOD) content

[0222] (i) Weigh 0.1g of poplar leaves after stress treatment, add 1mL of extract, homogenize in an ice bath, centrifuge at 8000g 4℃ for 10min, take the supernatant and place it on ice for testing;

[0223] (ii) Preparations:

[0224] Preheat the microplate reader for at least 30 minutes and adjust the wavelength to 450nm.

[0225] Dilution of Reagent 3: Dilute Reagent 3 with distilled water 50 times (Reagent 3 and distilled water are diluted 1:49).

[0226] Preparation of working solution: Add 100 μL of reagent 2 to reagent 1 and mix thoroughly. The prepared solution can be stored at 4°C protected from light for one week.

[0227] (iii) Dissolve one bottle of reagent four in 5 mL of distilled water (use within one week after dissolution).

[0228] (iv) Sample determination (add the reagents listed in Table 8 below to the 96-well plate in sequence)

[0229] Table 8

[0230]

[0231] After mixing thoroughly and letting stand at room temperature for 30 minutes, the absorbance value A of each tube was measured at 450 nm.

[0232] (v) Calculate the SOD content according to the SOD calculation formula: SOD activity (U / g fresh weight) = [inhibition percentage ÷ (1 - inhibition percentage) × V total] ÷ (W × V sample ÷ V total) = 20 × inhibition percentage ÷ (1 - inhibition percentage) ÷ W;

[0233] Inhibition percentage = (A control tube - A test tube) / A control tube × 100%.

[0234] (2.4) Determination of peroxidase (POD) content

[0235] (i) Weigh 0.1g of poplar leaves after stress treatment, add 1mL of extract, homogenize in an ice bath, centrifuge at 8000g 4℃ for 10min, take the supernatant and place it on ice for testing;

[0236] (ii) Preheat the microplate reader for at least 30 minutes, adjust the wavelength to 470nm, and zero the instrument with distilled water;

[0237] (iii) Preparation of working solution: Before use, mix reagent 1, reagent 2 and reagent 3 in a ratio of 2.6 (mL): 1.5 (μL): 1 (μL), preheat at 25℃ for at least 10 minutes; prepare and use immediately.

[0238] (iv) Add 10 μL of sample and 190 μL of working solution to a 96-well plate, mix well, and record the absorbance value A1 at 470 nm for 1 min and the absorbance value A2 at 2 min (the time interval between A2 and A1 is 1 min). Calculate ΔA = A2 - A1;

[0239] (v) Calculate the POD content according to the POD calculation formula.

[0240] Unit definition: One unit of enzyme activity is defined as a change of 0.005 A470 per minute per gram of tissue in a reaction system per mL.

[0241] POD (U / g fresh weight) = ΔA × Vtotal ÷ (W × Vsample ÷ Vtotal) ÷ 0.005 ÷ T = 4000 × ΔA ÷ W.

[0242] (3) Stress phenotype analysis

[0243] (3.1) Phenotypic analysis of salt stress treatment

[0244] like Figure 6 As shown, after 20 days of treatment with 200 mM NaCl, wild-type and PtoABF2.1 overexpressing plants grew slowly, with most leaves drying out and wrinkling, and some leaves falling off. In contrast, PtoABF2.1-RNAi plants grew relatively well under salt stress, with the lower leaves showing slight yellowing and curling, while the upper leaves remained glossy.

[0245] Physiological index measurement results as follows Figure 7 As shown in figures a-d: Under salt stress, the content of reactive oxygen species (ROS) such as H2O2 and MDA in PtoABF2.1-RNAi plants was lower than that in WT plants, while the activities of antioxidant enzymes such as SOD and POD were higher than those in WT plants. This indicates that under salt stress, the activity of antioxidant enzymes in silent plants increased, effectively mitigating the excessive accumulation of ROS and reducing the degree of plant damage. In contrast, PtoABF2.1 overexpression plants showed the opposite trend.

[0246] (3.2) Phenotypic analysis of drought stress treatment

[0247] like Figure 6 As shown, after 10 days of treatment with 20% PEG 6000 simulating drought stress, all leaves of PtoABF2.1 overexpressing plants showed wilting, with severe lower leaf drop; lower leaves of WT plants began to drop, while upper leaves showed wilting and yellowing; leaves of PtoABF2.1-RNAi plants did not show significant drop, with only slight yellowing and wilting of lower leaves. During the treatment, it was observed that PtoABF2.1 overexpressing plants showed leaf wilting symptoms as early as day 4-6 of the drought, while PtoABF2.1-RNAi plants only began to show leaf wilting symptoms on day 7-8 of the drought.

[0248] Furthermore, before drought treatment, the height of PtoABF2.1 overexpressing plants was higher than that of WT and PtoABF2.1-RNAi plants. After drought treatment, the height of PtoABF2.1 overexpressing plants gradually became the same as that of WT and PtoABF2.1-RNAi plants, indicating that drought stress has a stronger inhibitory effect on the growth and development of PtoABF2.1 overexpressing plants.

[0249] Physiological index measurement results as follows Figure 8As shown in figures a-d, under drought stress, PtoABF2.1-RNAi plants had lower levels of reactive oxygen species (ROS) such as H2O2 and MDA than WT plants, while exhibiting higher levels of antioxidant enzymes such as SOD and POD. This indicates that drought stress increased antioxidant enzyme activity, effectively mitigating the excessive accumulation of ROS and reducing plant damage. In contrast, PtoABF2.1 overexpression plants showed the opposite trend, demonstrating that silencing the PtoABF2.1 gene significantly reduced the damage to poplar trees under drought stress.

[0250] (3.3) Phenotypic analysis of salt-drought combined stress treatment

[0251] like Figure 6 As shown, after 7 days of combined salt and drought stress treatment, PtoABF2.1 overexpressing plants reached a state of severe dehydration, with extremely severe leaf drying and wilting, and most leaves falling off, indicating significant damage. The growth of PtoABF2.1-RNAi plants was significantly better than that of wild-type and PtoABF2.1 overexpressing plants, with significantly improved leaf curling and apical wilting compared to wild-type. During the treatment, it was also observed that the wilting of PtoABF2.1-RNAi plants occurred significantly later than that of wild-type and PtoABF2.1 overexpressing plants, indicating that PtoABF2.1-RNAi plants exhibit strong tolerance under combined stress.

[0252] Physiological index measurement results as follows Figure 9 As shown in figures a-d: Under combined salt and drought stress, the reactive oxygen species (ROS) content, such as H2O2 and MDA, in PtoABF2.1-RNAi plants was lower than that in WT plants, while the activities of antioxidant enzymes, such as SOD and POD, were higher than those in WT plants. This indicates that the increased antioxidant enzyme activity under combined salt and drought stress effectively mitigated the excessive accumulation of ROS and reduced the degree of plant damage. In contrast, PtoABF2.1 overexpression plants showed the opposite trend.

[0253] 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. PtoABF2.1 The application of genes in improving drought resistance and salt tolerance of poplar, characterized in that, The PtoABF2.1 The nucleotide sequence of the gene is shown as SEQ ID NO. 1; the coding region nucleotide sequence of the gene is shown as SEQ ID NO. 2; The PtoABF2.1 The amino acid sequence of the encoded protein of the gene is shown as SEQ ID NO. 3; By reducing the poplar PtoABF2.1 The expression level of genes is used to improve the drought resistance and salt tolerance of poplar trees; the reduction of gene expression in poplar trees... PtoABF2.1 Methods for measuring gene expression include silencing gene expression in poplar trees. PtoABF2.1 Gene expression; the silencing is achieved through... PtoABF2.1 This was achieved by introducing a gene interference fragment into poplar trees; The PtoABF2.1 The interfering fragment of the gene is PtoABF2.1-RNAi, and its nucleotide sequence is shown as SEQ ID NO.

4.

2. A method of modulating drought resistance and salt tolerance in a poplar plant, comprising, The method involves regulating the poplar tree... PtoABF2.1 Gene expression regulates the drought resistance and salt tolerance of poplar trees; PtoABF2.1 The nucleotide sequence of the gene is shown in SEQ ID NO.1; the nucleotide sequence of the coding region of the gene is shown in SEQ ID NO.

2.

3. The method for regulating drought resistance and salt tolerance of Populus according to claim 2, characterized in that, The means for modulating the expression of the genes in the poplar PtoABF2.1 includes overexpression PtoABF2.1 of the genes or silencing PtoABF2.1 of the genes.

4. The method for regulating drought resistance and salt tolerance of Populus according to claim 2, characterized in that, The method for regulating drought resistance and salt tolerance of poplar comprises the following steps: Step I, obtaining PtoABF2.1 CDS and interfering fragments of genes; Step II, construction PtoABF2.1 Overexpression of recombinant vectors and silencing of recombinant vectors; Step III, genetic transformation is carried out, and overexpression plants and silenced plants are identified; Step IV, stress treatment and phenotype analysis are carried out on the overexpression plants and the silenced plants.

5. A method for improving drought resistance and salt tolerance of poplar, characterized by, The method involves cloning. PtoABF2.1 To silence gene interference fragments PtoABF2.1 Gene expression; the PtoABF2.1 The nucleotide sequence of the gene is shown in SEQ ID NO.1; the nucleotide sequence of the coding region of the gene is shown in SEQ ID NO.

2.

6. The method for improving drought resistance and salt tolerance of poplar according to claim 5, characterized in that, The PtoABF2.1 PtoABF2.1 The nucleotide sequence of the gene interference fragment is shown as SEQ ID NO. 4.

Images