PtoABF2.1 gene for improving drought resistance and salt resistance of poplar and application of PtoABF2.1 gene

Abstract

The invention discloses a PtoABF2.1 gene for improving drought resistance and salt resistance of poplar and application of the PtoABF2.1 gene, the nucleotide sequence of the gene is as shown in SEQ ID NO.1, a silent transgenic plant obtained by introducing an interference fragment of the PtoABF2.1 gene into the poplar has the growth vigor obviously superior to that of a wild type and an overexpression plant under the conditions of salt stress, drought stress and salt-drought composite stress, and the growth vigor of the silent transgenic plant is obviously higher than that of the wild type and the overexpression plant. According to the method, the MDA content and the H2O2 content are lower than those of wild plants, and the POD content and the SOD content are higher than those of the wild plants, so that the drought resistance and the salt resistance of the poplars are remarkably improved, technical support is provided for further cultivation of new poplar strains, meanwhile, the stability of wood production under the adverse situation is guaranteed, and the method has important significance for improving the ecological environment and maintaining biological diversity.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and specifically relates to a poplar stress resistance gene, and in particular to a PtoABF2.1 gene for improving the drought resistance and salt tolerance of poplar and its application. Background Art

[0002] Under natural conditions, plants are often harmed by a variety of abiotic stresses during their growth and development, such as salinity, drought, low temperature, and high temperature. These stresses not only restrict the normal growth and development of plants, but also have a significant impact on crop yields and forestry production benefits. Drought stress is one of the most widespread factors among many abiotic stresses. When encountering drought, the water balance in the plant body is broken, and a series of complex physiological and biochemical reactions are rapidly initiated. In order to reduce water loss, the stomata of plants close quickly, resulting in obstruction of leaf gas exchange and insufficient carbon dioxide intake, which in turn affects the dark reaction phase of photosynthesis. In addition, drought can also cause a decrease in plant cell pressure, causing leaves to wilt and curl, further weakening the efficiency of photosynthesis. Under long-term drought stress, the growth and development process of plants is significantly delayed, root growth is restricted, and the ability to absorb nutrients is reduced, ultimately leading to slow plant growth and reduced crop yields.

[0003] Soil salinization is also a major challenge facing agricultural and forestry development. Excessive salt in the soil not only creates a hypertonic environment, hindering water absorption by plant roots 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 normal plant metabolism and imposes multiple restrictions on plant growth and development.

[0004] Poplars are 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 perennial woody plants, they are pioneers in forest cultivation and are well-suited for afforestation, plant ecological restoration, and soil improvement. Research on drought and salt tolerance in poplars is of great significance for afforestation in arid and saline-alkali regions of my country, expanding poplar cultivation areas, and improving the ecological environment.

[0005] AREB / ABFs transcription factors belong to the bZIP (basic leucine zipper) protein subfamily and play a key role in the plant's response to stress. 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 little research on the function of ABFs in poplar drought and salt tolerance. Moreover, as a woody model plant, poplar is different from Arabidopsis thaliana in that its growth and development regulation mechanism is unique, and the functions of many important regulatory factors may change.

[0006] Therefore, it is necessary to study the regulatory role of ABFs in poplar's stress-resistant growth and screen specific ABFs genes that regulate poplar's drought resistance and salt tolerance, so as to promote poplar stress-resistant molecular breeding, ensure wood production 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 in poplar, and found that it was involved in salt stress and drought stress through fluorescence quantitative expression analysis; then, by constructing a PtoABF2.1 overexpression plant vector and a PtoABF2.1-RNAi plant vector, and introducing them into Populus tomentosa, they obtained PtoABF2.1 overexpression and silenced transgenic plants, respectively. Through stress phenotype analysis of the plants, it was found that under normal conditions, the PtoABF2.1 overexpressing transgenic poplars were taller, but there was no obvious difference in growth between the silent transgenic poplars and the wild type; under stress conditions, the PtoABF2.1-RNAi plants grew significantly better than the wild type and overexpressing plants, with reduced MDA content and H2O2 content, and significantly increased POD content and SOD content, indicating that the PtoABF2.1 gene and the protein it encodes can significantly improve the drought and salt resistance of poplars, providing technical support for further breeding new poplar varieties, while ensuring the stability of wood production under adverse conditions, which is of great significance to improving the ecological environment and maintaining biodiversity, thereby completing the present invention.

[0008] Specifically, the purpose 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 drought resistance and salt tolerance in poplar trees, wherein the nucleotide sequence of the PtoABF2.1 gene is shown in SEQ ID NO.1.

[0010] In a second aspect, the present invention provides the use of the PtoABF2.1 gene in improving the drought resistance and salt tolerance of poplar. The nucleotide sequence of the PtoABF2.1 gene is shown in SEQ ID NO.1; the amino acid sequence of the protein encoded by the PtoABF2.1 gene is shown in SEQ ID NO.3.

[0011] In a third aspect, the present invention provides a method for regulating the drought resistance and salt tolerance of poplars, wherein the method regulates the drought resistance and salt tolerance of poplars by regulating the expression of the PtoABF2.1 gene in poplars.

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

[0013] The beneficial effects of the present invention include:

[0014] (1) The PtoABF2.1 gene and its encoded protein provided by the present invention for improving the drought resistance and salt tolerance of poplars can significantly improve the drought resistance and salt tolerance of poplars, providing technical support for further breeding 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, is of great significance to improving the ecological environment and maintaining biodiversity, and opens up a new path for the sustainable development of forestry. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 A comparison chart of the relative expression levels of the PtoABF2.1 gene of Populus tomentosa detected by RT-qPCR at different salt treatment times (0-48 h) in Example 1 is shown; Figure 2 The comparison chart of the expression levels of the PtoABF2.1 gene of Populus tomentosa detected by RT-qPCR under different drought treatment times (0-6d) in Example 1 is shown; Figure 3 A comparison chart showing the expression levels of the PtoABF2.1 gene in different tissues of Populus tomentosa detected by RNA-seq in Example 1 is shown;

[0017] Figure 4 The transcription level of the PtoABF2.1 gene in the overexpression plants detected by RT-qPCR in Example 5 is shown, and the error bars represent the standard deviation; Figure 5 The transcription level of the PtoABF2.1 gene in silenced plants detected by RT-qPCR in Example 5 is shown, and the error bars represent standard deviations; Figure 6 The morphological phenotypes of WT, PtoABF2.1 overexpressing plants (OE-1, OE-6), and silenced (RNAi-2, RNAi-3) transgenic plants in Example 6 before and after salt stress, drought stress, and salt-drought combined stress are shown; Figure 7 a to d show the analysis of malondialdehyde (MDA), hydrogen peroxide (H2O2), peroxidase (POD), and superoxide dismutase (SOD) content in WT, PtoABF2.1 overexpression (OE-1, OE-6), and silenced (RNAi-2, RNAi-3) transgenic plants before and after 200 mM NaCl treatment; Figure 8 a to d show the analysis of malondialdehyde (MDA), hydrogen peroxide (H2O2), peroxidase (POD), and superoxide dismutase (SOD) content in WT, PtoABF2.1 overexpression (OE-1, OE-6), and silenced (RNAi-2, RNAi-3) transgenic plants before and after 20% PEG6000 treatment, respectively; Figure 9 a to d show the analysis of malondialdehyde (MDA), hydrogen peroxide (H2O2), peroxidase (POD), and superoxide dismutase (SOD) content in WT, PtoABF2.1 overexpression (OE-1, OE-6), and silenced (RNAi-2, RNAi-3) transgenic plants before and after treatment with 200 mM NaCl + 20% PEG6000. DETAILED DESCRIPTION

[0018] The present invention will be further described in detail below through preferred embodiments and examples. Through these descriptions, the characteristics and advantages of the present invention will become more clear and distinct.

[0019] The word “exemplary” is used exclusively herein to mean “serving as an example, example, or illustration.” Any embodiment described herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments.

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

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

[0022] The second aspect of the present invention provides the use of the PtoABF2.1 gene in improving the drought resistance and salt tolerance of poplar trees. The nucleotide sequence of the PtoABF2.1 gene is shown in SEQ ID NO.1.

[0023] Wherein, the poplar is white poplar, preferably white poplar.

[0024] Preferably, the PtoABF2.1 gene is located on chromosome 14 of the Populus tomentosa reference genome, with a starting position of 1561778 and an ending 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 as SEQ ID NO.2.

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

[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 as SEQ ID NO.3.

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

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

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

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

[0033] Preferably, the method of regulating the expression of the PtoABF2.1 gene in poplar includes overexpressing or silencing the PtoABF2.1 gene, the nucleotide sequence of the gene is shown in SEQ ID NO.1, and the poplar is Populus tomentosa.

[0034] Among them, when the PtoABF2.1 gene is overexpressed, the drought resistance and salt tolerance of poplar are reduced; when the PtoABF2.1 gene is silenced, the drought resistance and salt tolerance of poplar are improved.

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

[0036] Step I, obtain the CDS and interference fragments 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 the primer PtoABF2.1-OE-F is shown in SEQ ID NO.5, and the nucleotide sequence of the primer PtoABF2.1-OE-R is shown in SEQ ID NO.6.

[0038] Preferably, the nucleotide sequence of the amplified CDS sequence is 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 amplifying primers PtoABF2.1-RNAi-F and PtoABF2.1-RNAi-R, the nucleotide sequence of the primer PtoABF2.1-RNAi-F is shown in SEQ ID NO.7, and the nucleotide sequence of the primer PtoABF2.1-RNAi-R is shown in SEQ ID NO.8.

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

[0041] Step II, construct PtoABF2.1 overexpression recombinant vector and silencing recombinant 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 is obtained by constructing an interference fragment of the PtoABF2.1 gene into a pBI121 vector.

[0044] More preferably, the ligated recombinant vector is tested to obtain a positive clone, and then the plasmid is extracted and sequenced. If the sequence is correct, the vector construction is completed.

[0045] Step III: perform genetic transformation and identify overexpression plants and silenced plants.

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

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

[0048] Furthermore, DNA level identification and transcription level identification were performed on intact poplar plants to obtain PtoABF2.1 overexpression plants and PtoABF2.1 silenced plants, respectively.

[0049] Step IV: stress treatment and phenotypic analysis of the overexpressing plants and silenced plants.

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

[0051] Preferably, the salt stress treatment is as follows: one-month-old wild-type tissue culture seedlings (about 10 cm in height), the agar culture medium at the root of the seedlings is cleaned in water, and then transplanted into a soil culture medium mixed with substrate and vermiculite in a ratio of 1:1.5, and the light conditions are 8 hours of dark culture and 16 hours of light culture; cultured in a constant temperature culture room at 25°C under the same light conditions for 20 days, and then irrigated with 200mM NaCl solution for 20 days;

[0052] The drought stress treatment is as follows: one-month-old wild-type tissue culture seedlings (about 10 cm in height) are cleaned with water after the agar culture medium at the root of the seedlings is transplanted into a soil culture medium containing a mixture of substrate and vermiculite in a ratio of 1:1.5, and the light conditions are 8 hours of dark culture and 16 hours of light culture; the seedlings are cultured in a constant temperature culture room at 25°C under the same light conditions for 20 days, and then irrigated with a 20% PEG 6000 solution for 10 days;

[0053] The salt-drought combined stress treatment comprises the following steps: washing the agar culture medium at the roots of one-month-old wild-type tissue culture seedlings (about 10 cm in height) with water, and then transplanting the seedlings into a soil culture medium containing a mixture of substrate and vermiculite in a ratio of 1:1.5, with a light intensity of 8 hours in the dark and 16 hours in the light; culturing the seedlings in a constant temperature culture room at 25°C under the same light intensity for 20 days, and then watering the seedlings with a mixed solution of 20% PEG 6000 and 200 mM NaCl for 7 days.

[0054] The morphological phenotypes and physiological indexes of the poplar plants treated with the above stresses were analyzed.

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

[0056] In the present invention, the morphological phenotypes and physiological traits of overexpressed and silenced plants were analyzed and it was found that under normal conditions, there was no significant difference in growth state and growth rate between the PtoABF2.1 gene silenced plants and the wild-type Populus tomentosa plants;

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

[0058] From the above, it can be seen that the drought resistance and salt tolerance of poplar trees can be regulated by regulating the expression of the PtoABF2.1 gene in poplar trees. Specifically, the PtoABF2.1 gene can be overexpressed in poplar plants to reduce their drought resistance and salt tolerance; and the PtoABF2.1 gene can be silenced in poplar plants to improve their drought resistance and salt tolerance. This indicates that the PtoABF2.1 gene and the protein it encodes provided by the present invention can significantly improve the drought resistance and salt tolerance of poplar trees, providing technical support for the further development of new poplar varieties.

[0059] A fourth aspect of 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 a reverse sequence of the 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 comprises the following steps:

[0062] Step 1: construct a 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 interference fragment PtoABF2.1-RNAi of the PtoABF2.1 gene is obtained by amplifying primers PtoABF2.1-RNAi-F and PtoABF2.1-RNAi-R, wherein the nucleotide sequence of primer PtoABF2.1-RNAi-F is shown as SEQ ID NO.7, and the nucleotide sequence of primer PtoABF2.1-RNAi-R is shown as SEQ ID NO.8.

[0066] Step 1-2: enzyme-cut the vector and connect the interference fragment to the basic vector to obtain the RNAi silencing recombinant vector.

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

[0068] Step 2: transform the constructed silencing recombinant vector into Escherichia coli to obtain the silencing recombinant vector with the interference fragment correctly inserted.

[0069] The recombinant vector was transformed into Escherichia coli DH5α competent cells to obtain single colonies, which were then amplified and identified by PCR.

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

[0071] Step 3: extract the silent recombinant vector plasmid obtained in step 2 and transform it into poplar plants through Agrobacterium-mediated transformation.

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

[0073] Step 3-1, transforming the silent recombinant vector plasmid into Agrobacterium.

[0074] Preferably, the Agrobacterium is EHA105.

[0075] Step 3-2, activating Agrobacterium.

[0076] Step 3-3, using the leaf disc method to infect poplar plants.

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

[0078] Step 4: Obtain complete poplar plants transformed with the silent recombinant plasmid, and identify the positive plants, which are poplars with high drought resistance and salt tolerance.

[0079] Preferably, the infected poplar leaves are subjected to co-culture, resistance culture and selective subculture to obtain adventitious buds, and the adventitious buds are made to grow adventitious roots through rooting culture to obtain complete poplar plants.

[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 the identification at the DNA level is: 95°C for 2 min; 35 cycles of (94°C for 30 s; 55°C for 30 s; 72°C for 60 s); 72°C for 5 min; 4°C∞.

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

[0083] 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, is of great significance to 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 to the scope of protection of the present invention.

[0086] Unless otherwise specified, the reagents involved in the following examples are all conventional reagents available on the market, and the methods used are all commonly used methods in this technical field.

[0087] Example 1 Fluorescence quantitative detection of PtoABF2.1 gene

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

[0089] The RNA obtained above was used as a template and reverse transcription was performed using HiScript IV All-in-One Ultra RT SuperMix for qPCR-R433 (Nanjing Novozymes Biotech Co., Ltd.) to obtain cDNA. The reaction system (20 μL) is shown in Table 1:

[0090] Table 1

[0091]

[0092] The reaction procedure was: 50°C for 5 min; 85°C 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 internal reference primers Actin-F and Actin-R are shown in SEQ ID NO.11 and SEQ ID NO.12, respectively.

[0094] Fluorescence quantitative PCR was used to determine the expression level of the PtoABF2.1 gene under different stress times. 2×ChamQSYBR Color qPCR Master Mix was used. The reaction system (20 μL) is shown in Table 2:

[0095] Table 2

[0096]

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

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

[0099] (2) In order to further explore 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 different tissues such as roots, stems, cambium, immature xylem, and mature xylem were systematically sampled and RNA-seq detection was performed. The reaction system and procedure were the same as above. The results are shown in Figure 2. Figure 3 The results show that the expression levels of the PtoABF2.1 gene are relatively consistent across all tissues of Populus tomentosa, with no significant differences. After normalization, the average log2 expression level fluctuated minimally across tissues, and statistically, no significant differences were found between tissues (P>0.05). This result suggests that the PtoABF2.1 gene may not be specifically involved in the developmental processes of a particular tissue, but rather plays a relatively stable and broad role in the overall growth and development of Populus tomentosa.

[0100] Example 2 Obtaining the CDS sequence and interfering fragments of the PtoABF2.1 gene

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

[0102] (1) RNA template denaturation

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

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

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

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

[0107] (2) Removal of genomic DNA

[0108] ① Add 2 μl of 5x gDNA wiper mix to the above reaction tube;

[0109] ②The reaction conditions are 42℃, 120s.

[0110] (3) cDNA synthesis

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

[0112] Table 3

[0113]

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

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

[0116] (2) Using cDNA as a template, referring to the genome file of Populus tomentosa (http: / / db.cngb.org / cnsa / project / CNP0004290_40760cfc / reviewlink / ), and considering all the principles of primer design, primer blast tool (NCBI, https: / / blast.ncbi.nlm.nih.gov) was used to design primers for the PtoABF2.1 overexpression sequence (CDS sequence) and silencing sequence (partial reverse sequence of CDS). The primers for amplifying the CDS sequence of the PtoABF2.1 gene 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 primers for amplifying the silencing sequence (interference fragment of the PtoABF2.1 gene, PtoABF2.1-RNAi) were PtoABF2.1-RNAi-F and PtoABF2.1-RNAi-R, and their nucleotide sequences are shown in SEQ ID NO. NO.7 and SEQ ID NO.8.

[0117] Among them, the gene PtoABF2.1 is located on chromosome 14 of the Populus tomentosa genome (http: / / db.cngb.org / cnsa / project / CNP0004290_40760cfc / reviewlink / ), with the starting position 1561778 and the ending position 1565624.

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

[0119] Table 4

[0120]

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

[0122] The PCR products were run on a 2% agarose gel and recovered after detection. They were then purified using a DNA Clean-up Kit from Cosmos. After purification, the purity and concentration of the purified DNA products were determined using an instrument.

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

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

[0125] (1) Enzyme digestion of vector

[0126] The selected overexpression and silencing vectors are both pBI121 vectors, totaling 13629 bp, which contain a strong 35S promoter (CaMV35S), are resistant to kanamycin (Kana), and can be 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°C water bath for 1-2 hours.

[0131] The digested product was purified by gel recovery using the Kangwei Gel Extraction Kit and purified using Kangwei DNA Clean-up Kit. After purification, it was used as a vector backbone and stored at -20°C.

[0132] (2) Connection conversion

[0133] (2.1) Ligation of pBI121 vector

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

[0135] Then, ligation was performed at 50°C for 15 min, and the ligation product was used to transform Escherichia coli DH5α competent cells.

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

[0137] Take 50μl DH5α competent cells, add 10μl ligation product, flick to mix, and let it stand on ice for 30 minutes. Subsequently, place the mixture in a 42°C water bath for 45 seconds, quickly transfer it to ice and cool for 2 minutes, and avoid shaking during the process to ensure transformation efficiency. Next, add 700μl of sterile LB liquid culture medium without antibiotics, blow to mix, and place it on a shaker at 37°C and 220rpm for 45 minutes to revive the bacteria. After recovery, collect the bacteria by centrifugation at 6000rpm for 1 minute, retain 100μl of supernatant to resuspend the bacteria, spread it on LB solid plate culture medium containing kanamycin (100mg / ml), and culture it upside down at 37°C for 14-16 hours.

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

[0139] Using a sterile pipette tip, single colonies were picked from the plates and placed in 250 μl of LB liquid medium containing kanamycin. Cultured at 37°C with shaking at 200 rpm / min for approximately 3 hours to serve as templates for amplification. Positive and negative controls were also set up using the purified PCR product and ddH2O (double-distilled water) as templates. 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 procedure was as follows: 95°C for 2 min; 35 cycles of (94°C for 30 s; 55°C for 30 s; 72°C for 60 s); 72°C for 5 min; and 4°C∞.

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

[0144] The PCR products were detected by 1% agarose gel electrophoresis, and the colonies that could amplify the band with the same size as the positive control were considered positive clones.

[0145] (2.4) Extraction of positive clone plasmids

[0146] PCR-positive clones were transferred to 6 ml of LB liquid medium containing kanamycin and cultured overnight at 37°C with shaking at 200 rpm. Plasmids were extracted using a plasmid extraction kit from Jiangsu Kangwei Reagent Co., Ltd. and sequenced by Beijing Ruibo Xingke Biotechnology Co., Ltd. After sequence alignment, the construction of the overexpression and silencing recombinant vectors was completed.

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

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

[0149] (1.1) Add approximately 1 μg of the PtoABF2.1 overexpression vector plasmid and silencing vector plasmid to 100 μL of Agrobacterium EHA105 competent cells and mix gently.

[0150] (1.2) Place on ice for 5 minutes, quick-freeze in liquid nitrogen for 5 minutes, immediately place in a 37°C water bath for 5 minutes, and then place in an ice bath for 5 minutes.

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

[0152] (1.4) After single colonies have grown, several monoclonal plaques were picked with a sterile pipette tip and placed in a 2 mL centrifuge tube containing 250 μL of YEP liquid medium (supplemented with Rif and Kana at a 1:1000 ratio). The culture was shaken at 200 rpm at 30°C for 2 h. After completion of the culture, the bacterial suspension was aspirated and identified by PCR using the same method as described in Example 3. Positive bacterial suspensions were added to 50% glycerol, quickly frozen in liquid nitrogen, and stored at -80°C for subsequent genetic transformation experiments.

[0153] (2) Activation of Agrobacterium

[0154] (2.1) Remove the Agrobacterium tumefaciens suspension containing the overexpression vector plasmid and the silencing vector plasmid from the -80°C freezer, streak each onto YEP solid medium (1:1000 supplemented with Rif and Kana), seal the plate, and incubate inverted in a dark incubator at 28°C for 2–3 days.

[0155] (2.2) Use a sterile pipette to pick a single colony from the plate and inoculate it into 3 mL of YEP liquid medium (1:1000 supplemented with Rif and Kana). Incubate overnight on a shaker at 30°C, 200 rpm. Transfer 1 mL of the bacterial suspension to a 250 mL sterile Erlenmeyer flask containing 100 mL of YEP liquid medium (1:1000 supplemented with Rif and Kana). Incubate on a shaker at 30°C, 200 rpm, for 4-5 hours, to an OD600 of 0.6-0.8.

[0156] (2.3) Pour the Agrobacterium solution into a sterile 50ml centrifuge tube in a clean bench. Centrifuge at 3500-5000 rpm for 7-10 minutes at room temperature. Collect the culture solution and pour an equal volume of resuspension solution into the tube. Shake the tube to resuspend the cells. Centrifuge again and rinse the cells with the resuspension solution. This culture solution will be used for subsequent infection experiments.

[0157] The composition of the resuspension is: 1 / 2MS + 20g sucrose + 100mM AS (added immediately upon use), pH = 5.6. After preparation, it is sterilized at 121°C for 20min under high temperature and high pressure.

[0158] (3) Leaf disc infection

[0159] (3.1) Leaf pretreatment

[0160] Select leaves of healthy and sterile Populus tomentosa seedlings (the leaves are dark green, thick in texture, and are generally the 4th to 6th leaves from the top), use a sterile surgical blade to make 2 to 3 horizontal wounds on the main vein of the leaves, spread them face down on a pre-culture medium without antibiotics, and culture them at 25°C for 1 to 2 days.

[0161] The composition of the pre-culture medium is: MS+30g sucrose+5g gelatin pH=5.9, and after preparation, it is sterilized at 121°C for 20 minutes under high temperature and high pressure.

[0162] (3.2) Infection and co-cultivation

[0163] In a clean bench, place the pretreated leaves in the resuspended Agrobacterium solution for 10-15 minutes, gently shaking the leaves to ensure adequate contact with the solution. Infect under incandescent light on the clean bench. Then, remove the leaves with sterile tweezers and place them on pre-sterilized filter paper to absorb excess solution. Finally, inoculate the infected leaves face up onto co-cultivation medium (with As) without antibiotics and incubate in the dark at 25°C for 3-4 days, until bacterial colonies develop near the leaves.

[0164] The composition of the co-culture medium in the co-culture plate is: MS+30g sucrose+7g agar+100mM AS, pH=5.9, which is sterilized by high temperature and high pressure at 121°C for 20min after preparation.

[0165] (3.3) Differentiation culture

[0166] After dark culture, appropriate plant resistance is selected according to the carrier and differentiation medium containing corresponding antibiotics is prepared.

[0167] On a clean bench, place the co-cultured leaves that have been dark-treated for 3 days on pre-sterilized filter paper to absorb excess bacteria, and then transfer them to differentiation medium for differentiation culture. The light cycle is (light 16h / dark 8h) and the temperature is 25℃.

[0168] After 10-15 days of incubation in the differentiation medium, use sterile forceps or a scalpel to cut out the light green, dense callus or leaf disc with adventitious buds in a clean bench and place it on new differentiation medium to provide the callus or adventitious buds with more nutrients to induce differentiation. Generally, the medium should be changed every 10 days.

[0169] The composition of the differentiation medium is: 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 is sterilized at 121°C for 20min under high temperature and high pressure.

[0170] (3.4) Rooting culture

[0171] After two weeks of culture in differentiation medium, when adventitious buds grow to over 3-4 cm, excise them individually in a cleanroom using sterile forceps or a scalpel. These buds will then be placed on rooting medium for rooting. After two weeks, adventitious roots will develop from the buds. Subculture the buds (using a medium containing both timentin and kanamol for the first subculture, and only timentin for subsequent subcultures). Molecular analysis of the leaves can be performed during the first subculture.

[0172] The composition of the rooting medium is: 1 / 2MS + 20g sucrose + 0.05mg / LIBA + 0.02mg / LNAA + 7g agar + 250mg / l TMT + 250MG / L cephalosporin + 20mg / L Kana. After preparation, it is sterilized at 121°C for 20min under high temperature and high pressure.

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

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

[0175] (1.1) Place a leaf sample to be extracted in a 2 mL centrifuge tube and add one grinding bead. Quickly freeze in liquid nitrogen and then crush using a tissue crusher for 5 minutes.

[0176] (1.2) After the powder is crushed, add 500 μL of TBS buffer to the centrifuge tube, mix well, and 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 let it 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 2h.

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

[0182] (2) Identification of PtoABF2.1 gene overexpression and silenced plants

[0183] (2.1) DNA level identification

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

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

[0186] The PCR product was detected by agarose gel electrophoresis to check whether the bands were correct. If correct, the plant was a PtoABF2.1 gene overexpression plant or silenced plant.

[0187] PCR detection showed that the 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, and the 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 transcriptional levels

[0189] RT-qPCR was used to detect the transcription level of PtoABF2.1 in overexpression and silenced plants:

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

[0191] (ii) RT-qPCR experiments were performed using 2× ChamQ SYBR Color qPCR Master Mix. The reaction system was configured according to Table 2. 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°C for 30 s; (95°C for 5 s; 60°C for 35 s) for 40 cycles; 95°C for 15 s; 60°C for 1 min; 95°C for 15 s.

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

[0194] The results of the silent plant test 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, 0.23 times and 0.043 times that of the wild-type plant (WT), respectively.

[0195] Example 6 Analysis of stress phenotypes of PtoABF2.1 gene overexpression and silenced plants

[0196] (1) Stress treatment

[0197] (1.1) Salt stress treatment

[0198] One-month-old wild-type tissue culture seedlings (about 10 cm in height) were cultured. The agar culture medium at the root of the seedlings was cleaned in water and then transferred to a soil culture medium composed of a mixture of substrate and vermiculite at a ratio of 1:1.5. The light conditions were 8 h dark culture and 16 h light culture. The seedlings were cultured in a constant temperature culture room at 25°C under the same light conditions for 20 days, and then irrigated with 200 mM NaCl solution for 20 days.

[0199] (1.2) Drought stress treatment

[0200] One-month-old wild-type tissue culture seedlings (about 10 cm in height) were cultured. The agar medium at the root of the seedlings was cleaned in water and then transplanted into a soil culture medium composed of a 1:1.5 mixture of substrate and vermiculite. The light conditions were 8 h in the dark and 16 h in the light. The seedlings were cultured in a constant temperature culture room at 25°C under the same light conditions for 20 days and then irrigated with a 20% PEG 6000 solution for 10 days.

[0201] (1.3) Salt-drought combined stress treatment

[0202] One-month-old wild-type tissue culture seedlings (about 10 cm in height) were cleaned of the agar medium at the root of the seedlings in water and then transplanted into a soil culture medium composed of a 1:1.5 mixture of substrate and vermiculite, with a light intensity of 8 h in the dark and 16 h in the light. The seedlings were cultured in a constant temperature culture room at 25°C under the same light intensity for 20 days and then irrigated with a mixed solution of 20% PEG 6000 and 200 mM NaCl for 7 days.

[0203] (2) Physiological index measurement

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

[0205] The malondialdehyde (MDA) content determination kit of Suzhou Keming Biological was used for determination. The specific steps are as follows:

[0206] (i) Weigh 0.1 g of stress-treated poplar leaves, add 1 mL of the extract, homogenize in an ice bath, centrifuge at 8000 g for 10 min at 4°C, collect the supernatant, and place on ice for testing;

[0207] (ii) Pipette 0.3 mL of reagent 1 into a 1.5 mL centrifuge tube, add 0.1 mL of sample, and mix thoroughly;

[0208] (iii) Keep in a 95°C water bath for 30 min (cover tightly to prevent water loss), cool in an ice bath, and centrifuge at 10,000 g at 25°C for 10 min;

[0209] (iv) 200 μL of supernatant was transferred to a 96-well plate and the absorbance at 532 nm and 600 nm was measured, recorded as A532 and A600, ΔA = A532 - A600;

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

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

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

[0213] (i) Weigh 0.1 g of stressed poplar leaves, add 1 mL of Reagent 1, and homogenize in an ice bath. Transfer to an EP tube, add reagent 1 to 1 mL, centrifuge at 8000 g for 10 min at 4°C, remove the supernatant, and place on ice for testing.

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

[0215] (iii) Place reagents 2, 3, and 4 in a 25°C water bath for at least 10 minutes;

[0216] (iv) Add the following reagents in the EP tube in order:

[0217] Table 7

[0218]

[0219] After adding Reagent 4 to dissolve the precipitate, 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 processed once. Calculate ΔA = A - A.

[0220] (v) The H2O2 content was calculated 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 0.1 g of stress-treated poplar leaves, add 1 mL of the extract, homogenize in an ice bath, centrifuge at 8000 g for 10 min at 4°C, collect the supernatant, and place on ice for testing;

[0223] (ii) Preparation:

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

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

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

[0227] (iii) Dissolve one bottle of Reagent 4 in 5 mL of distilled water (use within one week after dissolution).

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

[0229] Table 8

[0230]

[0231] Mix thoroughly, let it stand at room temperature for 30 min, and measure the absorbance value A of each tube 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) Peroxidase (POD) content determination

[0235] (i) Weigh 0.1 g of stress-treated poplar leaves, add 1 mL of the extract, homogenize in an ice bath, centrifuge at 8000 g for 10 min at 4°C, collect the supernatant, and place on ice for testing;

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

[0237] (iii) Preparation of working solution: Mix Reagent 1, Reagent 2, and Reagent 3 in a ratio of 2.6 (mL): 1.5 (μL): 1 (μL) and preheat at 25°C for at least 10 min before use. Prepare and use immediately.

[0238] (iv) Add 10 μL of sample and 190 μL of working solution to a 96-well plate, mix thoroughly, and record the absorbance at 470 nm at 1 minute (A1) and 2 minutes (A2) (the time interval between A2 and A1 is 1 minute). 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 in A470 per minute per gram of tissue in per mL of reaction system;

[0241] POD (U / g fresh weight) = ΔA × V total ÷ (W × V sample ÷ V sample total) ÷ ​​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 in the figure, after 200mM NaCl treatment for 20 days, wild-type and PtoABF2.1-overexpressing plants grew slowly, with most leaves drying up and shrinking, and some leaves falling off. PtoABF2.1-RNAi plants, on the other hand, grew relatively well under salt stress, with the lower leaves showing slight yellowing and curling, while the upper leaves remained shiny.

[0245] Physiological index measurement results such as Figure 7 As shown in Figures a to d, under salt stress, the levels of reactive oxygen species (ROS) such as H₂O₂ and MDA in PtoABF2.1-RNAi plants were lower than those in WT plants, while the activities of antioxidant enzymes such as SOD and POD were higher than those in WT plants. This indicates that antioxidant enzyme activity increased in silenced plants under salt stress, effectively slowing the excessive accumulation of ROS and reducing plant damage. PtoABF2.1-overexpressing plants, on the other hand, showed the opposite trend.

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

[0247] like Figure 6 As shown, after 10 days of simulated drought stress with 20% PEG 6000, all leaves of PtoABF2.1-overexpressing plants wilted, with severe lower leaf drop. WT plants experienced lower leaf drop, while upper leaves wilted and chlorotized. PtoABF2.1-RNAi plants showed no significant leaf drop, with only the lower leaves showing slight chlorosis and wilting. During the treatment, PtoABF2.1-overexpressing plants showed leaf wilt symptoms on the 4th to 6th day of drought, while PtoABF2.1-RNAi plants did not begin to wilt until the 7th to 8th day.

[0248] In addition, before drought treatment, the height of PtoABF2.1-overexpressing plants was taller 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 such as Figure 8As shown in Figures a-d, under drought stress, PtoABF2.1-RNAi plants had lower levels of reactive oxygen species, such as H₂O₂ and MDA, and higher antioxidant enzyme activities, such as SOD and POD, than WT plants. This indicates that drought stress-induced antioxidant enzyme activity increased, effectively mitigating excessive reactive oxygen species accumulation and reducing plant damage. PtoABF2.1-overexpressing plants, on the other hand, exhibited the opposite trend, indicating that silencing the PtoABF2.1 gene significantly reduced drought damage in poplar trees.

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

[0251] like Figure 6 As shown in the figure, after 7 days of combined salt and drought stress, PtoABF2.1-overexpressing plants experienced severe water depletion, with severe leaf wilt and dryness, and most leaves falling off, resulting in significant damage. PtoABF2.1-RNAi plants showed significantly better growth than wild-type and PtoABF2.1-overexpressing plants, with significantly improved leaf curling and apical wilting compared to wild-type plants. During the treatment, wilting in PtoABF2.1-RNAi plants was also found to be significantly later than in wild-type and PtoABF2.1-overexpressing plants, demonstrating that PtoABF2.1-RNAi plants exhibited greater tolerance to combined stress.

[0252] Physiological index measurement results such as Figure 9 As shown in Figures a to d, under combined salt-drought stress, the contents of reactive oxygen species (ROS) such as H2O2 and MDA in PtoABF2.1-RNAi plants were lower than those in WT plants, while the activities of antioxidant enzymes (SOD and POD) were higher than those in WT plants. This indicates that the increased antioxidant enzyme activity under combined salt-drought stress effectively mitigated the excessive accumulation of ROS and reduced plant damage. PtoABF2.1-overexpressing plants, on the other hand, 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 are not to be construed as limiting the present invention. Those skilled in the art will appreciate that, without departing from the spirit and scope of the present invention, various equivalent substitutions, modifications, or improvements may be made to the technical solutions and implementations of the present invention, all of which fall within the scope of the present invention.

Claims

1. A PtoABF2.1 gene for regulating drought resistance and salt tolerance in poplar trees, characterized in that: The nucleotide sequence of the PtoABF2.1 gene is shown in SEQ ID NO.

1.

2. Application of the PtoABF2.1 gene in improving drought resistance and salt tolerance of poplar, characterized in that: The nucleotide sequence of the PtoABF2.1 gene is shown in SEQ ID NO.1; The amino acid sequence of the protein encoded by the PtoABF2.1 gene is shown in SEQ ID NO.

3.

3. The use of the PtoABF2.1 gene according to claim 2 in improving drought resistance and salt tolerance of poplar, characterized in that: The drought resistance and salt tolerance of poplar trees can be improved by reducing the expression level of the PtoABF2.1 gene in poplar trees.

4. The use of the PtoABF2.1 gene according to claim 3 in improving drought resistance and salt tolerance of poplar, characterized in that: 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.

5. The use of the PtoABF2.1 gene according to claim 4 in improving drought resistance and salt tolerance of poplar, characterized in that: The silencing was achieved by introducing an interference fragment of the PtoABF2.1 gene into poplar.

6. A method for regulating drought resistance and salt tolerance of poplars, characterized in that: The method regulates the drought resistance and salt tolerance of poplars by regulating the expression of the PtoABF2.1 gene in the poplars.

7. The method for regulating drought resistance and salt tolerance of poplar according to claim 6, characterized in that: The method of regulating the expression of the PtoABF2.1 gene in poplar includes overexpressing the PtoABF2.1 gene or silencing the PtoABF2.1 gene.

8. The method for regulating drought resistance and salt tolerance of poplar according to claim 6, characterized in that: The method for regulating and controlling drought resistance and salt tolerance of poplar comprises the following steps: Step I, obtaining the CDS and interference fragments of the PtoABF2.1 gene; Step II, constructing PtoABF2.1 overexpression recombinant vector and silencing recombinant vector; Step III, performing genetic transformation and identifying overexpression plants and silenced plants; Step IV: stress treatment and phenotypic analysis of the overexpressing plants and silenced plants.

9. A method for improving drought resistance and salt tolerance of poplars, characterized in that: The method silences the expression of the PtoABF2.1 gene by cloning a PtoABF2.1 gene interference fragment.

10. The method for improving drought resistance and salt tolerance of poplar according to claim 9, characterized in that: The nucleotide sequence of the PtoABF2.1 gene interference fragment is shown in SEQ ID NO.4.

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