Application of acacia auriculae AaRD22 protein and its coding gene in improving drought and salt tolerance of plants

Abstract

The application provides application of Acacia auriculae-deciduate dehydration response protein AaRD22 and a coding gene thereof in improving drought resistance and salt tolerance of plants. The dehydration response protein AaRD22 provided by the application is obtained from Acacia auriculae-deciduata, the AaRD22 of the Acacia auriculae-deciduata has the nucleotide sequence shown in SEQ ID NO. 1, and the encoded protein has the amino acid sequence shown in SEQ ID NO. 2. By means of genetic transformation technology, the AaRD22 gene is heterologously overexpressed, and the drought resistance and salt tolerance of the Acacia auriculae-deciduata are significantly improved. Therefore, the AaRD22 gene of the Acacia auriculae-deciduata and the encoded protein are new gene resources for drought resistance and salt tolerance of the Acacia auriculae-deciduata, will play an important role in genetic improvement of drought resistance and salt tolerance varieties of the Acacia auriculae-deciduata, and have a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering, specifically relating to the dehydration response protein of Acacia confusa (…). A cacia a uriculiformis R esponsive to d Applications of ehydration (AaRD22) and its encoding gene AaRD22 in improving plant drought resistance and salt tolerance. Background Technology

[0002] The continued deterioration of the global climate not only threatens the human living environment but also brings about various frequent natural disasters such as drought, floods, high temperatures, frost damage, and outbreaks of pests and diseases. Due to their sessile growth characteristics, plants cannot avoid sudden adverse factors in a timely manner like animals, resulting in repeated interference from various abiotic stresses during their growth and development. Extreme drought, high salinity, and other abiotic stresses are important environmental factors that inhibit the normal growth and development of plants. Currently, nearly 50% of the annual yield loss of major cultivated crops worldwide is closely related to existing abiotic stresses (Valliyodan B, Nguyen HT. Understanding regulatory networks and engineering for enhanced drought tolerance in plants. Curr Opin Plant Biol. 2006, 9(2): 189-195). In order to cope with severe environmental changes, plants have evolved a series of defense measures to mitigate the damage caused by various abiotic stresses. Abiotic stress factors often cause physiological and biochemical metabolic disorders in plants, leading to differential expression of stress response proteins and their encoding genes. Therefore, discovering plant abiotic stress response proteins and their encoding gene resources, genetically improving crops under abiotic stress, and creating / breeding new varieties with high stress resistance is of great scientific value.

[0003] Acacia auriculiformis is an evergreen tree belonging to the genus Acacia in the legume family. It is a fast-growing tree species widely cultivated in tropical and subtropical regions, and it has value in terms of timber, ornamental value, soil and water conservation, water source conservation, and medicinal value. Due to its rapid growth, strong adaptability, and tolerance to drought and poor soil, it can grow in acidic coarse skeletal soil, sandy soil, and heavy clay soil with severe soil erosion (Wong MM, Cannon CH, Wickneswari R. Identification of lignin genes and regulatory sequences involved in secondary cell wall formation in Acacia auriculiformis and Acacia mangium via de novo transcriptome sequencing. BMCGenomics. 2011, 12:342). In South my country, it grows rapidly and yields abundantly, making it a pioneer tree species for afforestation in difficult sites. In low-lying plains, hilly areas with soil erosion, and coastal aeolian sandy areas, the tall and upright Acacia macrocarpa can still be seen everywhere, making it one of the main tree species for afforestation, soil and water conservation, and soil improvement.

[0004] Responsive to dehydration (RD22) protein 22 is a member of the plant-specific RUBP protein family (comprising four subfamilies: USP, RD22, PG1β, and BNM2). It is closely related to plant responses to soil drought and salinization, and is an important candidate gene for plant stress tolerance. In Arabidopsis thaliana, the RD22 protein is a multifactorial inducible protein, responding to low temperature, salt stress, drought, and the presence of Cu. 2+Induced by exogenous treatments, AtRD22 expression was upregulated. Under drought and salt stress, the ABA-mediated ABA response element (RYACGTGGYR) was activated and regulated by the ABA signaling pathway, and interacted with transcription factors AtMYC2 / rd22BP1 and AtMYB2 proteins, enhancing the drought tolerance of AtRD22 mutants (Abe H, Urao T, Ito T, Seki M, Shinozaki K, Yamaguchi-Shinozaki K. Arabidopsis AtMYC2(bHLH) and AtMYB2(MYB) function as transcriptional activators in abscisic acid signaling. Plant Cell. 2003, 15(1):63-78). Studies have found that the expression of dehydration response proteins is closely related to plant drought resistance and salt tolerance; however, their specific biological functions and mechanisms of action remain unclear. In addition, there are many types of dehydration response proteins, and they play different functions in response to various abiotic stresses. Therefore, the role of the AaRD22 protein and its encoding gene in *Acacia confusa* needs further investigation. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a dehydration response protein AaRD22 of Acacia confusa and its encoding gene AaRD22, as well as its application in improving plant drought resistance and salt tolerance.

[0006] The first object of the present invention is to provide a dehydration response protein AaRD22, the nucleotide sequence of which is shown in SEQ ID NO.1 and the encoded amino acid sequence of which is shown in SEQ ID NO.2.

[0007] The Acacia confusa dehydration response protein AaRD22 of this invention has a nucleotide length of 1116 bp, encoding 371 amino acids, a protein molecular weight of 42.07 kDa, and contains 30 acidic amino acids and 35 basic amino acids. Leucine (Leu) accounts for the largest proportion at 9.2%, followed by valine (Val) at 8.9%. The isoelectric point of the RUBP-RD22 protein is 8.68, and its chemical formula is Ca. 1909 H 2976 N 498 O 535 S 19 The protein has a lipid solubility index of 93.99 and an overall average hydrophilicity of -0.018, ranging from -0.5 to 0.5, indicating it is a hydrophilic protein. The Plant-mPLoc online database predicts that the RUBP-RD22 protein is located in the cell wall. The RUBP-RD22 protein possesses two conserved domains: one located at positions 7-51 (E-value 1.1 × e^(-1 / 2)).-3 The AgrB structural domain (PF04647) is located at positions 158-367 (E-value is 4.1 × e). -76 ) of the RUBP structure domain (PF03181).

[0008] The present invention also provides a recombinant vector containing the aforementioned Acacia confusa dehydration response protein AaRD22.

[0009] Preferably, the recombinant vector is pCAMBIA1301-AaRD22.

[0010] The present invention also provides engineered bacteria containing the recombinant vector described above.

[0011] Preferably, the engineered bacteria is Agrobacterium GV3101 (pSoup-p19) containing pCAMBIA1301-AaRD22.

[0012] The present invention also provides the application of the aforementioned AaRD22 in improving plant drought resistance and salt tolerance.

[0013] Preferably, the application is the use of the Acacia confusa dehydration response protein AaRD22 in improving the drought resistance and salt tolerance of Acacia confusa.

[0014] The beneficial effects of this invention are as follows: This invention successfully discovered and cloned a dehydration response protein from Acacia confusa, named AaRD22. Through genetic transformation technology, it was revealed that AaRD22 has drought and salt tolerance response elements (e.g., dehydration response element DRE, ABA-induced response element ABRE, drought stress-induced element MBS, and abiotic stress-induced element STRE). Its application in improving the drought and salt tolerance of transgenic Acacia confusa plants contributes to a deeper understanding of the biological functions of the Acacia confusa dehydration response protein AaRD22 in plant drought and salt tolerance. It can also provide genetic resources for research on drought and salt tolerance in woody plants and will play a role in research on improving plant drought and salt tolerance through plant genetic engineering. Attached Figure Description

[0015] Figure 1 Amplification of the open reading frame of AaRD22, the dehydration response protein of Acacia confusa.

[0016] Figure 2 The secondary and tertiary structures of the Acacia confusa dehydration response protein AaRD22 are shown.

[0017] Figure 3 Phylogenetic analysis was performed on the dehydration response protein AaRD22 from *Acacia confusa* and other previously reported dehydration response proteins. Amino acid sequence alignment was performed using ClustalX2, and phylogenetic tree construction was conducted using nearest neighbor (NJ) analysis in MEGA 7.0 software.

[0018] Figure 4 The expression pattern of the AaRD22 gene in *Acacia confusa*. (A) AaRD22 in the roots of *Acacia confusa*. R oot, stem S tem, leaf L (A) Expression level in eaves; (B) Expression level of AaRD22 under 100 μM MABA treatment; (C) Expression level of AaRD22 under 20% (w / v) PEG simulated drought treatment; (D) Expression level of AaRD22 under 150 μM NaCl treatment. CK was the control group. Bar represents ± standard error (n=6). Statistical analysis compared the significance between each treatment group and the control group. Different lowercase letters abc or ** indicate statistically significant differences between each treatment group and the control group at p<0.01.

[0019] Figure 5 This is a schematic diagram of the plant recombinant expression vector pCAMBIA1301-AaRD22 used.

[0020] Figure 6 Analysis of AaRD22 expression level and water loss rate in transgenic lines. (A) Expression level of AaRD22 in transgenic lines. (B) Water loss rate in transgenic lines. MOCK is the control group, and OE1 and OE2 are transgenic lines. Bar represents ± standard error (n=3) for each group. ** indicates that there is a statistically significant difference between each treatment group and the control group at p<0.01.

[0021] Figure 7 Fresh weight was determined in AaRD22 overexpressing and wild-type plants under normal, drought, and salt stress conditions. (A) Fresh weight of MOCK, OE1, and OE2 plants under normal conditions. (B) Fresh weight of MOCK, OE1, and OE2 plants under drought stress (20% PEG). (C) Fresh weight of MOCK, OE1, and OE2 plants under salt stress (150 μM NaCl). MOCK served as the control group, and OE1 and OE2 were transgenic lines. Bar represents ± standard error (n = 3). ** indicates a statistically significant difference between the treatment group and the control group at p < 0.01.

[0022] Figure 8Survival rates of AaRD22 gene overexpressing and wild-type plants were determined under normal, drought, and salt stress conditions. (A) Survival rate of MOCK, OE1, and OE2 plants under normal conditions. (B) Survival rate of MOCK, OE1, and OE2 plants under drought stress (20% PEG). (C) Survival rate of MOCK, OE1, and OE2 plants under salt stress (150 μM NaCl). MOCK served as the control group, and OE1 and OE2 were transgenic lines. Bar represents ± standard error (n = 3). ** indicates a statistically significant difference between the treatment group and the control group at p < 0.01.

[0023] Figure 9 To investigate the overexpression of the AaRD22 gene and wild-type plants under normal, drought, and salt stress conditions. Content determination. (A) MOCK, OE1 and OE2 plants under normal conditions Content determination. (B) MOCK, OE1 and OE2 plants under drought stress (20% PEG) conditions. Content determination. (C) MOCK, OE1 and OE2 plants under salt stress (150 μM NaCl) conditions. Content determination. MOCK was the control group, and OE1 and OE2 were transgenic lines. Bar represents ± standard error for each group (n=3). ** indicates that there is a statistically significant difference between each treatment group and the control group at p<0.01. Detailed Implementation

[0024] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0025] Unless otherwise specified in the following examples, all experimental methods can be performed using conventional methods, such as those described in J. Sambrook et al.'s *Molecular Cloning: A Laboratory Manual*, F. Osborne et al.'s *A Concise Laboratory Manual of Molecular Biology*, or the manufacturer's instructions for use of the products employed.

[0026] The *Acacia auriculiformis* used in this example was planted in the experimental forest farm of the Zhejiang Academy of Forestry Sciences (N40°01′, E116°25′; Hangzhou, China); the polysaccharide and polyphenol plant RNA extraction kit was purchased from Beijing Huayueyang Biotechnology Co., Ltd. (Catalog No.: 0416-50); and the reverse transcriptase PrimeScrip was used. TM The RTreagent Kit with gDNAEraser was purchased from Takara (item number: RR047Q). The HD Cloning Kit was purchased from Takara (item number: 639648); the pMD18-T Vector was purchased from Takara (item number: D101A); and the SYBR Premix Ex Taq... TM Kit was purchased from Takara (catalog number: DRR420A); GeneJET Plasmid Miniprep Kit was purchased from Thermo Scientific (catalog number: K0502); ApexHF HS DNA polymerase premix was purchased from Hunan Aikerui Biotechnology Co., Ltd. (catalog number: AG12206); LB and MS media were commonly used media in this field, and their formulations were based on J. Sambrook et al.'s *Molecular Cloning: A Laboratory Manual*. Unless otherwise specified, all materials and reagents used in the following examples were commercially available.

[0027] Example 1: Cloning and phylogenetic analysis of the AaRD22 gene of Acacia confusa.

[0028] (1) Extraction of total RNA from Acacia confusa leaves and synthesis of the first strand of cDNA

[0029] 100 mg of Acacia confusa leaves were collected and ground into powder under liquid nitrogen. Total RNA was extracted from the Acacia confusa leaves using a polysaccharide and polyphenol plant RNA extraction kit (Beijing Huayueyang Biotechnology Co., Ltd., Beijing, China). NanoDrop was used to extract the RNA. TM The content and purity of total RNA in *Acacia confusa* leaves were determined using a 2000°C ultra-micro spectrophotometer (Thermo Scientific, Wisconsin, USA) and a 1.0% agarose gel electrophoresis apparatus (Biorad, California, USA). 1000 ng of purified total RNA was taken and analyzed using reverse transcriptase PrimeScrip. TM Use the RT reagent kit with gDNA Eraser (Takara, Dalian, China) according to the instruction manual to synthesize the first strand of cDNA. Dilute the product to the required concentration and store at -80°C.

[0030] (2) Amplification and sequence analysis of the AaRD22 gene of Acacia confusa.

[0031] Based on the annotated sequences in the publicly available Acacia confusa transcriptome data (NCBI Accession: PRJNA66725), nested PCR was used. Using cDNA from Acacia confusa leaves obtained through reverse transcription as a template, primers ATGATCACCACCTATCATCGGGTC and TTAGTTTTGTTTGGCAACCCACAT were designed using Primer Premier 5.0 primer design software (Premier Biosoft, California, USA). The AaRD22 gene of Acacia confusa was amplified using ApexHF HS DNA polymerase (Aikerui Biotechnology, Changsha, China). The PCR reaction system (50 μL) consisted of: 1.0 μL cDNA template, 25 μL 2×ApexHF FS PCR Master Mix, 1 μL upstream primer, 1 μL downstream primer, and 22 μL sterile water. PCR reaction procedure: 94℃ pre-denaturation for 30 s, followed by 30 cycles (98℃ 10 s, 55℃ 10 s, 72℃ 10 s), and a final extension at 72℃ for 5 min. PCR products were detected using a 1.0% agarose gel electrophoresis system (Biorad, California, USA) and recovered using the GeneJET Plasmid Miniprep Kit (Thermo Scientific, Wisconsin, USA). The ligation product was then ligated into pMD18-T Vector (Takara, Dalian, China), and transformed into *E. coli* DH5α (Aikerui Biotechnology, Changsha, China), and plated onto a plate containing 100 μg / mL of agarose gel. -1 The ampicillin antibiotic was incubated overnight at 37°C on LB agar plates. Single colonies were picked as templates for colony PCR verification. Positive clones were sent to Zhejiang Shangya Biotechnology Co., Ltd. for sequencing. The nucleotide sequence of Acacia confusa AaRD22 was obtained as shown in SEQ ID NO.1.

[0032] The nucleotide length of the Acacia confusa dehydration response protein AaRD22 is 1116 bp. Figure 1 The protein RUBP-RD22, encoding 371 amino acids, has a molecular weight of 42.07 kDa and contains 30 acidic amino acids and 35 basic amino acids. Leucine (Leu) is the most abundant amino acid at 9.2%, followed by valine (Val) at 8.9%. The isoelectric point of RUBP-RD22 is 8.68, and its chemical formula is C2. 1909 H 2976 N 498 O 535 S 19The protein has a lipid solubility index of 93.99 and an overall average hydrophilicity of -0.018, ranging from -0.5 to 0.5, indicating it is a hydrophilic protein. The Plant-mPLoc online database predicts that the RUBP-RD22 protein is located in the cell wall. The RUBP-RD22 protein possesses two conserved domains: one located at positions 7-51 (E-value 1.1 × e^(-1 / 2)). -3 The AgrB structural domain (PF04647) is located at positions 158-367 (E-value is 4.1 × e). -76 ) of the RUBP structure domain (PF03181).

[0033] The secondary structure of the AaRD22 protein, a dehydration response protein from *Acacia confusa*, was predicted using the bioinformatics software SOPMA. The results showed that the AaRD22 protein contained 26.42% α-helices, 22.91% β-sheets, 4.31% β-turns, and 46.36% random coils. Furthermore, the tertiary structure of the AaRD22 protein was constructed using the Swiss-Model, revealing a GMQE value of 0.45 and a Seq Identity of 58.05%. Figure 2 ).

[0034] Furthermore, a phylogenetic tree was constructed using the NJ method. The results showed that plant dehydration response proteins were divided into four subfamilies: R-group, U-group, B-group, and P-group. The Acacia confusa dehydration response protein AaRD22 clustered in the R-group, and was most closely related to the proteins in rapeseed (Brassica napus) AAQ57584, Arabidopsis thaliana (Arabidopsis thaliana) BAA01546, soybean (Glycine max) ACD49739, and cotton (Gossypium hirsutum) AAL67991. Figure 3 Their variable regions are mostly repeating sequences of 20 amino acids in varying numbers, primarily belonging to the RD22 family of proteins.

[0035] Example 2: Analysis of AaRD22 gene expression pattern in Acacia confusa

[0036] Different parts of *Acacia confusa* (roots, stems, and leaves) were harvested, and RNA extraction and reverse transcription were performed according to the method in Example 1-(1). For quantitative real-time PCR, primers GTGCAGATGGGAGCAGAATTA and AGTCCCTGGTTTCACTTTGAG were designed based on the AaRD22 gene, and SYBR Premix Ex Taq was used. TMThe kit (Takara Bio Inc., Dalian, China) was used for real-time PCR amplification. The reaction program was 95℃ denaturation for 2 min, followed by 40 cycles (95℃ for 15 s, 60℃ for 1 min). The internal control gene was EF-1α. The reaction was carried out at... Data were obtained from a 480 Instrument real-time quantitative PCR run (Roche Diagnostics, Mannheim, Germany), using 2 -ΔΔCT The relative expression levels of the samples were calculated using the method. The Acacia da Silva JA, Wen D. Identification of aquaporin members in Acacia auriculiformis and functional characterization of AaPIP1-2 involved in drought stress. Environ Exp Bot. 2021, 185:104425 primers used were GCTCTGTGTTGAGAAGAGCTATG and CGATCATGGAAGGCTGAAAGA.

[0037] The results showed that the AaRD22 gene was most highly expressed in the leaves of *Acacia confusa*, followed by the stems and leaves. Figure 4 A). Furthermore, treatment with 100 μM abscisic acid (ABA), 20% (w / v) polyethylene glycol (PEG6000), and 150 μM sodium chloride (NaCl) can all induce significant upregulation of AaRD22 expression. Figure 4 (BD). Therefore, the AaRD22 gene of Acacia confusa can be significantly upregulated in response to ABA, drought and salt stress treatments, and is an important gene resource for drought and salt tolerance.

[0038] Example 3: Overexpression of the AaRD22 gene enhances the drought and salt tolerance of Acacia confusa.

[0039] (1) Construction of Acacia confusa AaRD22 overexpression vector

[0040] Based on the pCAMBIA1301 plant expression vector ( Figure 5 Primers were designed at the BamHI and HindIII restriction sites based on the open reading frame sequences of AaRD22 and Acacia confusa.

[0041] CGGTACCCGGGGATCCATGATCACCACCTATCATCGGG and

[0042] GGCCAGTGCCAAGCTTGTTTTGTTTGGCAACCCAC

[0043] Using *Acacia confusa* cDNA as a template, the AaRD22 sequence was amplified with high fidelity. The amplified product was purified, and the target fragment was recovered. pCAMBIA1301 was digested with BamHI and HindIII. The HD Cloning Kit (Takara Bio Inc., Dalian, China) was used to construct plant expression vectors. For detailed instructions, please refer to the instruction manual. The pCAMBIA1301 vector contains the CaMV 35S promoter. AaRD22 was constructed by retaining the ATG and removing the stop codon, then ligating it to the 35S promoter to create an AaRD22 overexpression vector. This recombinant plant expression vector was named pCAMBIA1301-AaRD22.

[0044] (2) The recombinant plasmid pCAMBIA1301-AaRD22 was transformed into Agrobacterium GV3101 (pSoup-p19).

[0045] The recombinant plasmid pCAMBIA1301-AaRD22 was transformed into Agrobacterium GV3101 (pSoup-p19) using a heat shock method. The specific procedure was as follows: 1 μg of recombinant plasmid pCAMBIA1301-AaRD22 was mixed with 100 μL of Agrobacterium GV3101 (pSoup-p19) competent cells, incubated on ice for 5 min, flash-frozen in liquid nitrogen for 5 min, and then rapidly transferred to a 37°C water bath for 5 min, followed by incubation on ice for 5 min. 700 μL of antibiotic-free LB medium was added, and the mixture was incubated on a shaker at 28°C and 100 rpm for 2–3 h. The culture was then plated onto 25 mL LB agar plates (containing 50 mg / mL of the medium). -1 Kanamycin). Invert the plate and incubate at 28°C until colonies grow (approximately 2 days). Pick single clones for colony PCR identification. Agrobacterium GV3101 (pSoup-p19) transformed with the recombinant plasmid pCAMBIA1301-AaRD22 was selected as a positive clone.

[0046] (3) Agrobacterium-mediated overexpression of AaRD22 gene in Acacia confusa.

[0047] Agrobacterium tumefaciens activated with pCAMBIA1301-AaRD22 was picked and a single colony was placed in 100 mL of LB liquid medium (containing 50 mg / mL). -1 Kanamycin), incubate overnight (approximately 16 hours) at 28°C with shaking at 180 rpm until OD reaches 100%. 600=0.6~0.8. Collect the bacterial culture by centrifugation at 5000×g for 5 min at room temperature. Resuspend the precipitated Agrobacterium in 100 mL of osmotic buffer (0.2 mM acetylsalicylic acid, 10 mM MgCl2, 10 mM MES, pH 5.7), centrifuge at 5000×g for 5 min, discard the supernatant, and adjust the OD with osmotic buffer. 600 ≈0.6, activated at 25℃ in the dark for 2 hours. Healthy Acacia pubescens leaves were selected, immersed in osmotic buffer containing the AaRD22 gene, vacuum-sealed for 5 minutes, and then air-dried. After 24 hours of dark culture in a tissue culture room at 23±2℃, they were cultured under light for 2 days, and leaves showing positive resistance were selected. Acacia pubescens leaves that overexpressed AaRD22 were selected for subsequent testing.

[0048] The results showed that the expression of the AaRD22 gene was significantly upregulated in the transgenic lines OE1 and OE2 of *Acacia confusa* that overexpressed AaRD22. Figure 6 A), compared with the control group MOCK, the water loss rate was significantly reduced in the transgenic lines OE1 and OE2 that overexpressed AaRD22. Figure 6 B). Therefore, the transgenic lines carrying the AaRD22 gene have stronger drought and salt tolerance, and the transgenic lines are better able to maintain water content to cope with environmental stress, thereby alleviating the damage of environmental stress to the plants.

[0049] Furthermore, using wild-type MOCK and OE1 and OE2 plants overexpressing the AaRD22 gene as research subjects, the study investigated their fresh weight, survival rate, and corresponding O₂ content under normal conditions, drought stress (20% PEG), and salt stress (150 μM NaCl). The results showed that compared with normal conditions, both drought and salt stress treatments significantly reduced the plant's fresh weight. However, compared with MOCK, the OE1 and OE2 lines transgenic with the AaRD22 gene maintained a higher fresh weight. Figure 7 This indicates that the drought resistance and salt tolerance of the AaRD22 gene lines are significantly higher than those of the wild-type lines. Similarly, compared with normal conditions, both drought and salt stress treatments significantly reduced plant survival rates, but compared with MOCK, the OE1 and OE2 lines transgenic with the AaRD22 gene maintained a higher survival rate. Figure 8 The results indicate that the drought resistance and salt tolerance of the AaRD22 gene line are significantly higher than those of the wild line.

[0050] Furthermore, compared to normal conditions, both drought stress and salt stress treatments increase the levels of reactive oxygen species (ROS) in plants. The levels of ROS (measured by content) were significantly increased, but compared with MOCK, the OE1 and OE2 lines transgenic with the AaRD22 gene significantly reduced the levels of ROS in vivo. Figure 9 This indicates that the AaRD22 gene transgenic lines maintain a high level of drought and salt tolerance because they may activate the ABA signaling pathway, thereby maintaining a high relative water content, survival rate, and plant fresh weight, activating the antioxidant enzyme system, promoting the clearance of ROS caused by drought and salt stress, and improving the drought and salt tolerance of the AaRD22 gene transgenic lines.

[0051] SEQ ID NO.1 (AaRD22 gene sequence)

[0052]

[0053] SEQ ID NO.2 (AaRD22 protein sequence)

[0054] MITTYHRVISYQLEHFISPIMKFPLLSIAFVTVSLTPFLYVHIFFFPLRRINVNLYLYMIHTQLQVMLGAMHAALPPEIYWNSTLPNTPMPELLKELLHPIVNVGDSSGGTTSRSVRGWNDQKAYIPTYKPATYNIGYTRYSSANVTQLLHNSNETIFFLEKDLNAGRKMNLHFTHTIIKNASF LPRQVVKSIPFSSSKMDVILNNFNVERGSVVAEVMKNTINICEELGIKGEERYCATSLESMVDFVTSKLGKSVQAWSLEATNQTKEAEYKIMHGVKKVGDTEVVCHKIDYVYAVFLCHKIDNTVAYTVPLEGADGSRIKALCVCHRDTSNWSPEHLSFQLLKVKPGTVPICHFLSQDGVMWVAKQN

[0055] Nest-PCR amplification of the nucleotide sequence of the AaRD22 gene of *Acacia confusa*.

[0056] ATGATCACCACCTATCATCGGGTC

[0057] Nest-PCR amplification of the nucleotide sequence of the AaRD22 gene of *Acacia confusa*.

[0058] TTAGTTTTGTTTGGCAACCCACAT

[0059] Real-time RT-PCR detection of AaRD22 gene expression in Acacia confusa.

[0060] GTGCAGATGGGAGCAGAATTA

[0061] Real-time RT-PCR detection of AaRD22 gene expression in Acacia confusa.

[0062] AGTCCCTGGTTTCACTTTGAG

[0063] β-actin, a reference gene for Acacia confusa

[0064] GCTCTAGTGTTGAGAAGAGCTATG

[0065] β-actin, a reference gene for Acacia confusa

[0066] CGATCATGGAAGGCTGAAAGA

[0067] Construction of AaRD22-pCAMBIA1301 recombinant vector

[0068] CGGTACCCGGGGATCCATGATCACCACCTATCATCGGG

[0069] Construction of AaRD22-pCAMBIA1301 recombinant vector

[0070] GGCCAGTGCCAAGCTTGTTTTGTTTGGCAACCCAC.

Claims

1. Acacia confusa dehydration response protein AaRD22, characterized in that, The amino acid sequence is shown in SEQ ID NO.

2.

2. The gene encoding the AaRD22 dehydration response protein of Acacia confusa as described in claim 1.

3. The gene according to claim 2, characterized in that, The nucleotide sequence is shown in SEQ ID NO.

1.

4. A recombinant vector containing the gene of the dehydration response protein AaRD22 as described in claim 2 or 3.

5. The recombinant vector according to claim 4, characterized in that, The recombinant vector is pCAMBIA1301-AaRD22.

6. Engineered bacteria containing the recombinant vector of claim 4.

7. The engineered bacteria according to claim 6, characterized in that, The engineered bacteria is Agrobacterium GV3101 (pSoup-p19) containing pCAMBIA1301-AaRD22.

8. The application of the Acacia confusa dehydration response protein AaRD22 as described in claim 1 in improving the drought resistance and salt tolerance of plants, wherein the plant is Acacia confusa.

9. The application according to claim 8, characterized in that, This involves the application of overexpressing the gene encoding the dehydration response protein AaRD22 in plants to improve their drought resistance and salt tolerance.

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