AfAPX2 drought-resistant gene and its application

Abstract

This invention application belongs to the field of genetic engineering technology, specifically disclosing an AfAPX2 drought-resistant gene and its application. The AfAPX2 drought-resistant gene is derived from *Amorpha fruticosa*, and its nucleotide sequence is shown in SEQ ID NO.1. The protein encoded by the AfAPX2 drought-resistant gene has its amino acid sequence shown in SEQ ID NO.2. This invention utilizes the drought stress tolerance detection of tobacco plants overexpressing AfAPX2 via 35S initiation. The overexpressing plants showed enhanced tolerance to natural drought stress, improving the drought resistance of tobacco. This indicates that the AfAPX2 provided by this invention plays an important regulatory role in responding to drought stress, providing a new gene resource for molecular design breeding of plant stress resistance.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, and specifically discloses an AfAPX2 drought-resistant gene and its application. Background Technology

[0002] Plant growth and development under adverse conditions mainly depend on their mechanisms of continuous adaptation to environmental changes, particularly the activation of drought-specific defense mechanisms, proactive physiological adjustments, and the acquisition of structures to cope with environmental changes under environmental stress. Among the biochemical reactions of plants under abiotic stress, a group of metabolites plays a fundamental role: ascorbate peroxidase (APX), an important enzyme component for scavenging reactive oxygen species (ROS). Studies have shown that APX participates in clearing excess ROS produced by oxidative quenching in plant cells under stress. While genetic engineering can be used to selectively improve plants, the current number of drought-resistant genes screened is still limited and cannot meet the needs of scientific research and production. *Amorpha fruticosa*, a deciduous shrub, can tolerate arid soils and exhibits high tolerance and strong reproductive capacity in various habitat conditions. Therefore, understanding the drought resistance mechanism of *Amorpha fruticosa* is of great significance for research on plant tolerance and improving crop yields under abiotic stress. Summary of the Invention

[0003] The purpose of this invention is to provide an AfAPX2 drought-resistant gene and its application, providing new gene resources for molecular design breeding of plant stress resistance.

[0004] To achieve the above objectives, the technical solution of the present invention is as follows: an AfAPX2 drought-resistant gene, the AfAPX2 drought-resistant gene being derived from Amorpha fruticosa, and the nucleotide sequence of the AfAPX2 drought-resistant gene being shown in SEQ ID NO.1.

[0005] The working principle of this technical solution is as follows:

[0006] Drought stress tolerance of tobacco plants overexpressing AfAPX2 initiated at 35S was investigated. Overexpressing plants showed enhanced tolerance to natural drought stress, improving the drought resistance of tobacco. AfAPX2 plays an important regulatory role in the response to drought stress.

[0007] The beneficial effects of this technical solution are as follows:

[0008] (1) It will provide a candidate gene for drought-resistant breeding, and lay a solid theoretical foundation for the genetic improvement of Amorpha fruticosa varieties and other plants and the improvement of resource utilization.

[0009] (2) It provides new gene resources for molecular design breeding of plant stress resistance, which is of great significance for saving farmland water, reducing agricultural input, and mitigating environmental pollution.

[0010] The present invention also provides a protein encoded by the AfAPX2 drought resistance gene, characterized in that the amino acid sequence of the protein is shown in SEQ ID NO.2.

[0011] This invention also provides a method for the AfAPX2 drought-resistant gene, comprising the following steps: extracting total Amorpha fruticosa RNA, reverse transcribing it into cDNA and diluting it 50 times as a template, designing specific primers for APX family genes and internal reference gene Afqublin primers, and performing PCR amplification; the PCR reaction program is 95℃ denaturation for 30s, 58℃ annealing for 30s, 72℃ extension for 30s, for 40 cycles.

[0012] This invention also provides the application of the above-mentioned AfAPX2 drought-resistant gene, the above-mentioned protein, or the AfAPX2 drought-resistant gene obtained by the above-mentioned method in improving the drought resistance of plants, including: Amorpha fruticosa and / or tobacco. Attached Figure Description

[0013] Figure 1 This is a schematic diagram of the Gateway system plant expression vector pGWB18-AfAPX2, which is an example of the AfAPX2 drought resistance gene and its application.

[0014] Figure 2 This is a schematic diagram comparing the growth characteristics of tobacco plants overexpressing the AfAPX2 gene in terms of tolerance to natural drought stress. Detailed Implementation

[0015] The following detailed description illustrates the specific implementation method:

[0016] This invention provides an AfAPX2 drought-resistant gene and its application. The AfAPX2 drought-resistant gene is derived from Amorpha fruticosa, and its nucleotide sequence is as follows:

[0017] ATGGGAAAGTCTTACCCATCTGTGAGTGCTGATTACCAGAAGGCCGTTGAGAAGGCCAAGCAGAAGCTCAGGGGTTTGATCGCTGAGAAACGCTGCGCTCCTCTCATGCTCCGTTTGGCATGGCACTCGGCTGGTACCTATGACGTGAAGACGAAGACCGGTGGTCCCTTCGGAACCATCAAGCATCCCGCTGAGCTTGCTCACGGTGCTAACAACGGGCTCGATATCGCTGTGAGGCTGTTGGAGCCACTGAAGGAGCAGTTCCCTACCTTGAGCTACGCTGATTTCTACCAGTTGGCTGGTGTTGTTGCTGTTGAGGTGACTGGTGGACCTGAAGTTCCTTTCCACCCAGGAAGACAGGACAAGCCTGAGTCACCACCCGAGGGTCGCTTGCCTGATGCAACAAAGGGTTCTGATCACCTGAGGGATGTGTTTGGCAAGGCTATGGGGCTTAGTGATCAGGATATTGTTGCTTTGTCTGGCGGTCACACCCTTGGAGCAGCACACAAGGAGCGTTCTGGATTTGAGGGGCCCTGGACCTCTAACCCTCTCATTTTTGATAACTCATACTTCACGGAGCTTTTGAGTGGTGAGAAGGAAGGCCTCCTTCAGCTGCCATCTGACAAGGCACTTTTGTCAGACCCTGTTTTCCGCCCTCTTGTTGAGAAATATGCTGCGGATGAAGATGCATTCTTTGCTGATTATGCTGAGGCTCACCTAAAGCTTTCCGAGCTTGGGTTTGCTGAAGCCTAA;

[0018] The protein encoded by the AfAPX2 drought resistance gene, and the amino acid sequence of the protein is specifically as follows:

[0019] MGKSYPSVSADYQKAVEKAKQKLRGLIAEKRCAPLMLRLAWHSAGTYDVKTKTGGPFGTIKHPAELAHGANNGLDIAVRLLEPLKEQFPTLSYADFYQLAGVVAVEVTGGPEVPFHPGRQDKPES PPEGRLPDATKGSDHLRDVFGKAMGLSDQDIVALSGGHTLGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVEKYAADEDAFFADYAEAHLKLSELGFAEA.

[0020] The specific implementation process is as follows:

[0021] Four-week-old Amorpha fruticosa seedlings grown in sand culture were treated with a 20% PEG6000 solution for root irrigation. Roots, stems, and leaves of the treated seedlings were flash-frozen in liquid nitrogen at 0h, 6h, 12h, 24h, and 48h. Total RNA was extracted, reverse transcribed into cDNA, and diluted 50-fold as a template. Data were collected on an MxPro-Mx3000P system. Specific primers for APX family genes and primers for the internal reference gene Afqublin were designed. Primer details are shown in Table 1. The PCR reaction program was 95℃ for 30s, 58℃ for 30s, and 72℃ for 30s, for 40 cycles. PCR reactions were performed using 2×Brilliant III SYBR Green qPCR Master Mix (Agilent) to detect the relative expression levels of genes after PEG6000-simulated drought stress.

[0022] Table 1. Details of qRT-PCR primers

[0023]

[0024] Four-week-old Amorpha fruticosa seedlings were treated with 150 mM NaCl and 60 mM NaHCO3 solutions. At the same time point, the above-ground stems and leaves were harvested together, while the underground roots were frozen in liquid nitrogen. The relative expression level of the AfAPX2 gene was detected by qRT-PCR.

[0025] This protocol uses cDNA reverse transcribed from the RNA of the entire *Amorpha fruticosa* plant treated with PEG6000 as a template for guided amplification. The ORF region of the AfAPX2 gene is amplified by PCR using KOD FX polymerase. The reaction system and procedure are performed according to the KOD FX polymerase instructions. The PCR amplification products are subjected to 0.1% agarose gel electrophoresis, and the electrophoresis results are detected using a gel imaging system. The target gene is recovered using a gel extraction kit and ligated into the pBQ-V3 vector using T4 DNA ligase. The ligation product is then sent to Kumei Biotechnology Co., Ltd. for sequencing verification.

[0026] (1) Construction of plant expression vectors

[0027] Combination Figure 1 As shown, in this protocol, pGWB18 and pQB-V3-AfAPX2 plasmid DNA were mixed at a molar ratio of 1:3, and then LR ligase and Gateway reaction buffer were added. The mixture was incubated at 25°C for 2 hours, followed by heat shock transformation into E. coli JM109. The mixture was plated on LB selection medium containing 50 mg / L antibiotics, and single clones were isolated and cultured. After expansion culture, the plasmid was extracted, and PCR amplification was performed using AfAPX2 primers. Electrophoresis confirmed successful ligation of the pGWB18-AfAPX2 vector.

[0028] (2) Acquisition of tobacco through overexpression

[0029] Agrobacterium tumefaciens EHA105 was transformed by electroporation. Agrobacterium containing the pGWB18-AfAPX2 plasmid DNA was cultured to an ODλ of 600 (0.5). The culture was then used to infect 0.5 cm² leaves of wild-type tobacco (Nicotiana tabacum) for 10-20 min. The bacterial culture was blotted dry on filter paper and co-cultured in the dark for 48 h on MS medium (MS + 0.5 mg / L 6-BA + 0.1 mg / L NAA + 20 mg / AS). The bacteria were then destered with carbenicillin solution and further differentiated under hygromycin (50 mg / L) to obtain resistant shoots. These shoots were rooted, cultured into seedlings, transplanted into flowerpots, and allowed to flower and bear fruit. Seeds from the T1 generation transformants were harvested. PCR was used to detect the genomic integration of the 35S-AfAPX2 gene; the seeds were then sown on a medium containing 50 mg / L hygromycin and germinated, and the T2 generation was selected. The T3 generation of transgenic tobacco seeds was harvested and the expression level of the AfAPX2 gene in the transformed tobacco was detected by qPCR using primers.

[0030] (3) Analysis of drought stress tolerance during flowering in tobacco plants overexpressing AfAPX2

[0031] Overexpressing AfAPX2 tobacco and wild-type seeds were sown in pots mixed with vermiculite and peat moss. After reaching the 4-leaf stage, they were transplanted into 7cm×7cm pots and cultured in a plant culture room for another 3 weeks. Water was withheld and allowed to dry naturally when individual plants showed buds. Phenotypic data were collected by taking photos at 7, 10, and 14 days of drought. Watering was resumed after 14 days of natural drought, and the fresh weight of individual plants that had recovered their phenotype was measured 7 days later.

[0032] This study used a natural water deprivation method to test the tolerance of wild-type (WT) Nicotiana benthamiana and Nicotiana plants overexpressing the AfAPX2 gene to water scarcity. Combined with... Figure 2As shown, after 7 days of drought stress, the lower leaves of the plants turned yellow and the upper leaves wilted. The degree of wilting of the top leaves of the overexpression line was lower than that of the WT line. After 14 days of continued natural drought stress, there was no significant difference in drought damage between the WT and AfAPX2 overexpression tobacco plants. Both lines had wilted leaves and the lower leaves were highly yellowed and turned white. Rehydration began on the 14th day. After the first saturated watering, water was added every 2 days. After 14 days of restored water supply and growth, there were significant phenotypic differences between the WT and overexpression lines. The leaves and the vigor of the plants in the AfAPX2 overexpression line were significantly greater than those in the WT line.

[0033] The above descriptions are merely embodiments of the present invention, and common knowledge regarding specific structures and characteristics in the solutions is not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the structure of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent.

Claims

1. An AfAPX2 drought-resistant gene, characterized in that, The gene is derived from Amorpha fruticosa, and its nucleic acid sequence is shown in SEQ ID NO.

1.

2. A protein encoded by the AfAPX2 drought-resistant gene as described in claim 1, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. The application of the AfAPX2 drought-resistant gene according to claim 1 in the preparation of transgenic plants with improved drought resistance; characterized in that, The application is achieved by overexpressing the AfAPX2 drought-resistant gene as described in claim 1; the plant is tobacco.

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