Andrographis transcript factor apnac82 gene and application thereof

Abstract

The application discloses a Andrographis paniculata transcription factor ApNAC82, wherein the coding region nucleotide sequence of the Andrographis paniculata transcription factor ApNAC82 is shown as SEQ ID NO:2. The application further discloses a cloning method of the Andrographis paniculata transcription factor ApNAC82, comprising the following steps: synthesizing cDNA by reverse transcription of total RNA of Andrographis paniculata as a template for PCR reaction to clone a coding region fragment of the Andrographis paniculata transcription factor ApNAC82, or using DNA of the Andrographis paniculata as a template for PCR reaction to clone a gene fragment of the Andrographis paniculata transcription factor ApNAC82. The application has important significance for understanding the response mechanism of the Andrographis paniculata to multiple adversity stresses, especially for mining and utilizing multifunctional transcription factors capable of improving multiple adversity stresses, and for genetic improvement and adversity-resistant variety cultivation of the Andrographis paniculata and other species.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of the transcription factor ApNAC82 in improving plant drought and heat tolerance. Specifically, it relates to ApNAC82 and its application in cultivating plant varieties with drought and heat resistance. Background Technology

[0002] Andrographis paniculata, a plant of the Acanthaceae family, possesses antibacterial, anti-inflammatory, heat-clearing, detoxifying, swelling-reducing, and analgesic effects. It is a major raw material for various traditional Chinese medicine preparations, including Qinghuo Zhimai tablets, Xiaoyan Lidan tablets, and Andrographolide tablets. Furthermore, Andrographis paniculata is known as a "traditional Chinese medicine antibiotic," being a key ingredient in traditional Chinese veterinary medicines such as Lianxiang San, and is widely used in livestock and poultry farming as an alternative to antibiotics in treating bacterial diarrhea and other diseases. As a major traditional Chinese medicinal herb, Andrographis paniculata is in high demand in my country, with an annual demand of 15,000 to 20,000 tons. High yield, stable yield, and improved quality are key technical challenges in its cultivation. Drought and high-temperature stress directly affect the aboveground growth and development of Andrographis paniculata, interfering with metabolism, transport, and transformation of substances, and are the most significant abiotic stresses during its growth period. Therefore, systematically exploring genes related to superior agronomic traits in Andrographis paniculata and elucidating the adaptive mechanisms regulating its stress resistance will provide a sound theoretical foundation and gene resources for the genetic improvement and creation of new germplasm of Andrographis paniculata.

[0003] Transcription factors can specifically interact with cis-acting elements on eukaryotic gene promoters, activating or repressing gene transcriptional expression. They play crucial regulatory roles in plant growth, development, and stress defense responses. NAC transcription factors are one of the largest gene families, containing a highly conserved NAC domain at their N-terminus and a variable transcriptional regulatory region at their C-terminus. Although the NAC gene family members exhibit high conservation of the N-terminal NAC domain, significant differences in the C-terminal sequence have arisen through evolution and natural selection. This non-conservation at the C-terminus leads to diverse functions and properties, resulting in species, environment, and genotype variations. For example, overexpression of OsSNAC1, OsSNAC3, and OsONAC022 in rice enhances its drought and salt tolerance (Fang et al., 2015; Hong et al., 2016; Hu et al., 2006), while overexpression of ZmSNAC1, ZmNAC55, ZmNAC84, and ZmNAC111 in maize improves drought tolerance (Lu et al., 2012; Mao et al., 2015; Mao et al., 2016; Zhu et al., 2016). Most research on NAC transcription factors focuses on drought, cold, and high salinity. Research on the role of NAC in high-temperature stress is relatively limited. Overexpression of the Arabidopsis NAC transcription factor JUNGBRUNNEN1 (JUB1; ANAC042) prolonged the survival time of Arabidopsis and increased its tolerance to heat stress (Shahnejat-Bushehri et al., 2012).

[0004] Overexpression of transcription factors can lead to the accumulation of stress-related proteins, interfering with normal metabolic regulation in plants and inhibiting the growth of transgenic plants. For example, overexpression of cotton GhDREB1 in tobacco enhances resistance to low temperatures but results in growth arrest and delayed flowering. Overexpression of TaDREB2 and TaDREB3 in wheat and barley improves drought and cold tolerance but exhibits slow growth, delayed flowering, and reduced seed setting. Overexpression of the Arabidopsis NAC transcription factor ATAF1 (At1g01720, ANAC002) improves drought and disease resistance but causes dwarfing and shorter taproots. Therefore, the adverse effects of transcription factors must be considered when using them for genetic improvement. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a gene for the Andrographis paniculata transcription factor ApNAC82 and a method for cloning it.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A *Andrographis paniculata* transcription factor, ApNAC82, is disclosed, with its coding region nucleotide sequence shown in SEQ ID NO:2. The nucleotide sequence of the *Andrographis paniculata* transcription factor, ApNAC82, is also shown in SEQ ID NO:1. The application of the *Andrographis paniculata* transcription factor ApNAC82 in regulating plant drought resistance and heat tolerance, or in increasing the expression levels of genes related to plant drought resistance and heat tolerance, is also disclosed.

[0008] A protein encoded by the Andrographis paniculata transcription factor ApNAC82, the amino acid sequence of which is shown in SEQ ID NO:3. The application of ApNAC82 protein in regulating plant drought resistance and heat tolerance, or in increasing the expression levels of genes related to plant drought resistance and heat tolerance.

[0009] A recombinant vector, expression cassette, transgenic cell line, host bacteria, plasmid, and Agrobacterium-competent cells contain the Andrographis paniculata transcription factor ApNAC82 nucleic acid molecule. The plasmid overexpresses the ApNAC82 gene. The Agrobacterium-competent cells overexpress the ApNAC82 gene. The recombinant vector, expression cassette, transgenic cell line, host bacteria, plasmid, and Agrobacterium-competent cells are used in regulating plant drought resistance and heat tolerance, or in increasing the expression levels of genes related to plant drought resistance and heat tolerance.

[0010] The regulation is positive; that is, increased levels of the ApNAC82 gene or ApNAC82 protein increase the plant's drought resistance and heat tolerance. Based on the nucleotide sequence of NAC82, codon optimization or synonymous codon substitution can be used to achieve the same encoded protein and function, even though the nucleotide sequences may differ.

[0011] A method for cloning the Andrographis paniculata transcription factor ApNAC82 includes the following steps:

[0012] Step 1: Using cDNA synthesized from the reverse transcription of total RNA from Andrographis paniculata as a template, a PCR reaction was performed to clone the coding region fragment of the Andrographis paniculata transcription factor ApNAC82.

[0013] Alternatively, the total DNA of Andrographis paniculata can be used as a template to clone the Andrographis paniculata transcription factor ApNAC82 gene fragment by PCR reaction;

[0014] The PCR primers were ApNAC82-F and ApNAC82-R.

[0015] The sequence of ApNAC82-F is: ATGAAGAAGAGTGGGGAGTTGA;

[0016] The sequence of ApNAC82-R is: TCAAAAGGGCTTCTGCATGTACC;

[0017] Step 2: The coding region or gene fragment of the Andrographis paniculata transcription factor ApNAC82 was recovered from the PCR product using an agarose gel extraction kit.

[0018] In the cloning method of the Andrographis paniculata transcription factor ApNAC82, Step 1 involves first taking 0.5g of Andrographis paniculata leaves and grinding them with liquid nitrogen.

[0019] Then, the modified CTAB method was used to extract total DNA from the leaves of Andrographis paniculata.

[0020] Alternatively, total RNA can be extracted from Andrographis paniculata leaves using the DP432 plant total RNA extraction kit. The concentration and integrity of the extracted total RNA can be detected using a micro-nucleic acid protein quantification instrument and 1% agarose gel electrophoresis. The qualified RNA can then be reverse transcribed into cDNA using the Novizan HiScript II 1st Strand cDNA Synthesis Kit R211.

[0021] The cloning method for the Andrographis paniculata transcription factor ApNAC82, and the reaction system for the reverse transcription are shown in Table 1 of the specific embodiments.

[0022] The reaction conditions for the first stage of reverse transcription were denaturation at 65°C and heating for 5 minutes.

[0023] The reaction conditions for the second stage of reverse transcription were 25°C for 5 min; 50°C for 45 min; and 85°C for 2 min.

[0024] The cloning method for the Andrographis paniculata transcription factor ApNAC82, in Step 1, includes the following PCR reaction conditions: 95℃, pre-denaturation for 3 min; 95℃, denaturation for 15 s; 60℃, annealing for 20 s; 72℃, extension for 60 s; amplification for 35 cycles; and 72℃, final extension for 10 min.

[0025] The cloning method for the Andrographis paniculata transcription factor ApNAC82, in Step 1, uses a PCR reaction system, and the components and their amounts are detailed in Table 2 of the specific implementation method.

[0026] A method for preparing a 35S:ApNAC82-EGFP fusion expression vector includes the following steps:

[0027] (1) Based on the nucleotide sequence of the coding region of ApNAC82, the stop codon TGA was removed to construct the ApNAC82-EGFP fusion protein. The SacI and XbaI restriction sites on the 35S:EGFP vector were selected to design specific primer sequences for homologous recombination, and the following primers were prepared:

[0028] The sequence of ApNAC82-EGFP-F is:

[0029] AGAACACGGGGGACGAGCTCATGAAGAAGAGTGGGGAGTTGA;

[0030] The sequence of ApNAC82-EGFP-R is as follows:

[0031] CCATGTCGACTCTAGAAAAGGGCTTCTGCATGTACC;

[0032] The 35S:EGFP vector is an abbreviation for pCAMBIA1300-35S:EGFP vector;

[0033] (2) Using the pCE2_TA-Blunt-Zero recombinant vector pCE2_TA-Blunt-Zero-ApNAC82 containing the coding region nucleotide sequence of ApNAC82 as a template, and using ApNAC82-EGFP-F and ApNAC82-EGFP-R as primers, the ApNAC82-EGFP insert fragment was prepared by PCR reaction using Phanta Max Super-Fidelity DNA Polymerase P505.

[0034] PCR reaction conditions: 95℃, pre-denaturation for 3 min; 95℃, denaturation for 15 s; 60℃, annealing for 20 s; 72℃, extension for 90 s; 35 cycles of amplification; 72℃, final extension for 10 min.

[0035] The PCR reaction system is shown in Table 3 of the specific implementation method.

[0036] (3) The PCR products were detected by 1% agarose gel electrophoresis, and the ApNAC82-EGFP insert fragment was recovered using the agarose gel recovery kit D2500-01.

[0037] (4) The 35S:EGFP vector was prepared by double digestion of the 35S:EGFP vector with the restriction enzymes SacI and XbaI from New England Biolabs.

[0038] Enzyme digestion reaction conditions: 37℃, 60 min; 80℃, 20 min;

[0039] The enzyme digestion reaction system is shown in Table 4 of the specific implementation method.

[0040] (5) The enzyme digestion products were detected by 1% agarose gel electrophoresis, and the 35S:EGFP linearized vector was recovered and purified using the agarose gel recovery kit D2500-01.

[0041] (6) Using the ClonExpress Ultra One Step Cloning Kit V2, the ApNAC82-EGFP insert was ligated into the 35S:EGFP linearized vector via homologous recombination to obtain the recombination product;

[0042] The reaction conditions for homologous recombination were 50°C for 5 min, and the reaction was immediately cooled on ice after completion.

[0043] The homologous recombination reaction system is shown in Table 5 of the specific implementation method.

[0044] (7) The recombinant product was transformed into Escherichia coli DH5α, and the 35S:ApNAC82-EGFP fusion expression vector was obtained by screening with kanamycin and sequencing verification.

[0045] A method for preparing an expression vector containing an ApNAC82 gene fragment includes the following steps:

[0046] (1) Based on the nucleotide sequence of the coding region of ApNAC82, select the NdeⅠ and EcoRI-HF restriction sites on the pGBKT7 vector and the BamHI and PstⅠ restriction sites on the pGBKT7-VP16 vector, and design specific primer sequences for homologous recombination. The following primers were prepared respectively:

[0047] The pGBKT7-ApNAC82-F sequence is (5'-3'):

[0048] AGGAGGACCTGCATATGATGAAGAAGAGTGGGGAGTTGAACT;

[0049] The pGBKT7-ApNAC82-R sequence is (5'-3'):

[0050] GGATCCCCGGGAATTCTTAAAAGGGCTTCTGCATGTACCCAAAG;

[0051] The pGBKT7-VP16-ApNAC82-F sequence is (5'-3'):

[0052] GGTGGGCCGGGGATCATGAAGAAGAGTGGGGAGTTGAACT;

[0053] The pGBKT7-VP16-ApNAC82-R sequence is (5'-3'):

[0054] TAGTTATGCGGCCGCTTAAAAGGGCTTCTGCATGTACCCAAAG;

[0055] (2) Using the pCE2_TA-Blunt-Zero recombinant vector pCE2_TA-Blunt-Zero-ApNAC82, which contains the coding region nucleotide sequence of ApNAC82, as a template,

[0056] Using pGBKT7-ApNAC82-F and pGBKT7-ApNAC82-R as primers, the pGBKT7-ApNAC82 insert fragment was prepared by PCR reaction.

[0057] Using pGBKT7-VP16-ApNAC82-F and pGBKT7-VP16-ApNAC82-R as primers, the pGBKT7-VP16-ApNAC82 fusion insert was prepared by PCR reaction.

[0058] PCR reactions were performed using Phanta Max Super-Fidelity DNA Polymerase P505, a high-fidelity enzyme from Novizan.

[0059] PCR reaction conditions: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s; 60℃ annealing for 20 s; 72℃ extension for 90 s; 35 cycles of amplification; 72℃ final extension for 10 min.

[0060] The PCR reaction system for amplifying the pGBKT7-ApNAC82 insert fragment is shown in Table 6 of the specific implementation method.

[0061] The PCR reaction system for amplifying the pGBKT7-VP16-ApNAC82 fusion insert is shown in Table 7 of the specific implementation method.

[0062] (3) The PCR products were detected by 1% agarose gel electrophoresis. The pGBKT7-ApNAC82 insert and the pGBKT7-VP16-ApNAC82 fusion insert were recovered by the agarose gel recovery kit D2500-01.

[0063] (4) Linearized vectors were prepared by double digestion of pGBKT7 vector plasmid with New England Biolabs endonucleases NdeⅠ and EcoRI-HF.

[0064] Linearized vectors were prepared by double digestion of the pGBKT7-VP16 vector plasmid with BamHI and PstI from New England Biolabs.

[0065] Enzyme digestion reaction conditions: 37℃, 60 min; 80℃, 20 min;

[0066] The reaction system for digesting the pGBKT7 vector plasmid is shown in Table 8 of the specific implementation method.

[0067] The reaction system for enzyme digestion of the pGBKT7-VP16 vector plasmid is shown in Table 9 of the specific implementation method.

[0068] (5) The enzyme digestion products were detected by 1% agarose gel electrophoresis. The pGBKT7 linearized vector and pGBKT7-VP16 linearized vector were recovered and purified by the agarose gel recovery kit D2500-01.

[0069] (6) The ClonExpress Ultra One Step Cloning Kit V2 reagent kit was used.

[0070] The pGBKT7-ApNAC82 insert was ligated to the pGBKT7 linearized vector via homologous recombination to obtain recombination product one.

[0071] The pGBKT7-VP16-ApNAC82 fusion insert was ligated to the pGBKT7-VP16 linearized vector via homologous recombination to obtain recombination product two.

[0072] The reaction conditions for homologous recombination were 50°C for 5 minutes, and the mixture was immediately cooled on ice after the reaction was completed.

[0073] The homologous recombination reaction system for constructing the pGBKT7-ApNAC82 expression vector is shown in Table 10 of the specific implementation methods.

[0074] The homologous recombination reaction system for constructing the pGBKT7-VP16-ApNAC82 expression vector is shown in Table 11 of the specific implementation methods.

[0075] (7) Recombinant product 1 and recombinant product 2 were transformed into Escherichia coli DH5α, and after screening with kanamycin and sequencing verification, pGBKT7-ApNAC82 expression vector and pGBKT7-VP16-ApNAC82 expression vector were obtained, respectively.

[0076] A method for preparing an ApNAC82 gene overexpressing plant includes the following steps:

[0077] (1) The 35S:ApNAC82-EGFP fusion expression vector was transformed into Agrobacterium GV310 by heat shock method to obtain recombinant Agrobacterium;

[0078] (2) Resuspend the recombinant Agrobacterium obtained in step (1) in 10 mL of MgCl2 solution with a concentration of 10 mM and 200 μM AS, adjust the OD600 to about 1.0, and let it stand at 28℃ for 2-3 h to obtain the infection solution;

[0079] (3) Using the leaf disc method, plant leaves were transformed, and after co-culture, hygromycin resistance screening, differentiation, and rooting, T0 generation regenerated plants were obtained;

[0080] (4) Homozygous positive transgenic plants of generation T3 were obtained by screening with hygromycin and PCR identification. The specific methods are as follows:

[0081] T0 generation regenerated plants were identified by PCR to obtain positive T0 generation transgenic plants, and T1 generation seeds were obtained by self-pollination of positive T0 generation transgenic plants.

[0082] T1 generation seeds were screened with hygromycin and identified by PCR to obtain positive T1 generation transgenic plants. The positive T1 generation transgenic plants were then self-pollinated to obtain T2 generation seeds.

[0083] T2 generation seeds were screened with hygromycin and identified by PCR to obtain positive T2 generation transgenic plants. The positive T2 generation transgenic plants were then self-pollinated to obtain T3 generation seeds.

[0084] In step (4), the screening concentration of hygromycin is 50 mg / L;

[0085] In step (4), the PCR identification method is as follows: extract genomic DNA from the plant leaves, and perform PCR amplification using dApNAC3-F and d1300-R primer pairs. If a specific amplification product is obtained, the plant is a transgenic plant.

[0086] The sequence of dApNAC82-F is: TCCATTACTACGACAATTCCGGC;

[0087] The sequence of d1300-R is: TCAAAAGGGCTTCTGCATGTACC;

[0088] PCR reaction conditions: 95℃, pre-denaturation for 3 min; 95℃, denaturation for 15 s; 60℃, annealing for 20 s; 72℃, extension for 20 s; 35 cycles of amplification; 72℃, final extension for 10 min.

[0089] The PCR reaction system is shown in Table 12 of the specific implementation method.

[0090] Compared with the prior art, the present invention has the following beneficial effects:

[0091] The Andrographis paniculata transcription factor ApNAC82 can control the expression of multiple downstream genes related to stress resistance, participate in the physiological and biochemical processes of stress resistance, and improve the resistance of Andrographis paniculata plants to various abiotic stresses. This provides a new method for enhancing the stress resistance of Andrographis paniculata using genetic engineering. Abiotic stress has a crucial impact on the growth, development, and yield of Andrographis paniculata and is a major limiting factor in its cultivation. Plants typically face complex field conditions, and various stresses do not exist in isolation. Research results based on a single stress factor are not applicable to complex field conditions. This invention is of great significance for understanding the response of Andrographis paniculata to multiple abiotic stresses, especially for identifying and utilizing multifunctional transcription factors that can enhance resistance to multiple abiotic stresses, and for cultivating high-yielding, broad-spectrum tolerant Andrographis paniculata plants. Attached Figure Description

[0092] Figure 1 This is a comparative analysis diagram of the nucleotide sequence of ApNAC82, such as SEQ ID NO:1, and the coding region nucleotide sequence of ApNAC82, SEQ ID NO:2. The yellow boxes indicate the nucleotide sequences and positions of the introns; sequence 1 refers to SEQ ID NO:1, and sequence 2 refers to SEQ ID NO:2.

[0093] Figure 2 This is a comparison diagram of the amino acid sequence of the ApNAC82 protein (SEQ ID NO:3) with the protein sequences of members of the Arabidopsis ATAF subfamily. The yellow underlined area represents the NAM domain.

[0094] Figure 3 Subcellular localization of the ApNAC82 transcription factor.

[0095] Figure 4 The results are the analysis results of ApNAC82 transcriptional activation activity.

[0096] Figure 5 The expression of the ApNAC82 gene in Andrographis paniculata in response to drought and high temperature stress.

[0097] Figure 6 PCR identification of ApNAC82 positive transgenic lines. Among them, 1-10 are OE#ApNAC82-6, 11-20 are OE#ApNAC82-9, 21-30 are OE#ApNAC82-10, 31-40 are OE#ApNAC82-11, 41-55 are OE#ApNAC82-15, and WT is wild type.

[0098] Figure 7 The growth of wild-type WT and ApNAC82 overexpression lines under normal growth conditions.

[0099] Figure 8 Photographs showing the germination rates of wild-type WT and ApNAC82 overexpression lines under different concentrations of mannitol treatment.

[0100] Figure 9 Photographs showing the root length results of wild-type WT and ApNAC82 overexpression lines under different concentrations of mannitol treatment.

[0101] Figure 10 The germination rates of wild-type WT and ApNAC82 overexpression lines under different concentrations of mannitol treatment are statistically analyzed.

[0102] Figure 11 The root length of wild-type WT and ApNAC82 overexpression lines under different concentrations of mannitol treatment is statistically analyzed.

[0103] Figure 12 This shows the growth of wild-type WT and ApNAC82 overexpressing lines after natural drought stress.

[0104] Figure 13 This study examines the recovery of wild-type WT and ApNAC82-overexpressing seedlings after high-temperature stress.

[0105] Figure 14 The growth of wild-type WT and ApNAC82-overexpressing lines after high temperature stress. Detailed Implementation

[0106] The technical solution of the present invention will be further illustrated below through embodiments.

[0107] Example 1

[0108] 1. Cloning of the ApNAC82 sequence

[0109] (1.1) Take 1.0g of Andrographis paniculata leaves, grind them with liquid nitrogen, divide them into two portions, one portion for extracting total DNA from Andrographis paniculata leaves, and the other portion for extracting total RNA from Andrographis paniculata leaves;

[0110] (1.2) Total DNA was extracted from Andrographis paniculata leaves using a modified CTAB method. The mass and concentration of the extracted total DNA from the leaves were determined by spectrophotometry. The OD260 / 280 ratio was 1.9 and the concentration was 276 ng / μL, indicating that the extracted DNA was of acceptable quality and could be used for subsequent experiments.

[0111] (1.3) Total RNA was extracted from Andrographis paniculata leaves using the Plant Total RNA Extraction Kit (DP432). The concentration and integrity of the extracted total RNA were detected using a micro-nucleic acid and protein quantification instrument and 1% agarose gel electrophoresis. The extracted RNA was reverse transcribed into cDNA using the Novizan HiScript II 1st Strand cDNA Synthesis Kit (R211). The reverse transcription reaction system is as follows:

[0112] Table 1. Reverse transcription reaction system

[0113]

[0114]

[0115] The reaction conditions for the first stage of reverse transcription are denaturation at 65°C and heating for 5 minutes;

[0116] The reaction conditions for the second stage of reverse transcription were 25℃ for 5 min; 50℃ for 45 min; and 85℃ for 2 min.

[0117] (1.4) Based on the genomic information of Andrographis paniculata (SRP143459), specific primer sequences for amplifying the Andrographis paniculata transcription factor ApNAC82 gene fragment were designed, and the following primers were prepared:

[0118] The sequence of ApNAC82-F is (5'-3'): ATGAAGAAGAGTGGGGAGTTGA;

[0119] The sequence of ApNAC82-R is (5'-3'): TCAAAAGGGCTTCTGCATGTACC.

[0120] (1.5) Using extracted DNA and reverse-transcribed cDNA as templates, and ApNAC82-F / ApNAC82-R as primers, PCR was performed using Phanta Max Super-Fidelity DNA Polymerase (P505) to clone the gene fragment and coding region fragment of the Andrographis paniculata transcription factor ApNAC82. The PCR reaction conditions were: 95℃, pre-denaturation for 3 min; 95℃, denaturation for 15 s; 60℃, annealing for 20 s; 72℃, extension for 60 s; 35 cycles of amplification; and 72℃, final extension for 10 min. The PCR reaction system is shown in Table 2.

[0121] Table 2. PCR reaction system

[0122]

[0123]

[0124] (1.6) The obtained PCR products were subjected to 1% agarose gel electrophoresis, and the target fragment was recovered and purified using an agarose gel recovery kit (D2500-01).

[0125] (1.7) Using the TOPO cloning kit, the target fragment was ligated into the pCE2_TA-Blunt-Zero vector and transformed into Escherichia coli competent cells DH5α. After screening with ampicillin and kanamycin, positive single clones were selected for sequencing to obtain the gene sequence and coding region sequence of Andrographis paniculata transcription factor ApNAC82.

[0126] 2. Sequence analysis of the ApNAC82 sequence

[0127] Sequence alignment using MAFFT software revealed that the full-length ApNAC82 gene sequence in the Andrographis paniculata genomic DNA is 1076 bp, as shown in SEQ ID NO:1; the full-length coding region sequence is 897 bp, as shown in SEQ ID NO:2; encoding 298 amino acids, as shown in sequence 3. Sequence alignment analysis yielded the following results: Figure 1 As shown, the ApNAC82 gene consists of 3 exons and 2 introns. The exons are located at positions 1-165 of the reading frame, positions 257-549, and positions 638-1076.

[0128] The domains of the ApNAC82 protein were predicted using NCBI Conserved Domain Search. ApNAC82 exhibits a typical NAM domain between amino acids 10 and 133, classifying it as an ATAF family member. Homologous protein sequences (AtNAC002, AtNAC032, AtNAC081, AtNAC102) of Arabidopsis NAC transcription factors were obtained from the NCBI database and then aligned using MAFFT software. Figure 2 It can be seen that the protein sequences of ApNAC82 and Arabidopsis ATAF family members are relatively conserved at the N-terminus, both containing a conserved NAM domain, while the sequences at the C-terminus are quite different. This indicates that ApNAC82 has undergone adaptive changes during natural selection and evolution, and this non-conservation at the C-terminus gives it different functions and properties.

[0129] SEQ ID NO:1: The ApNAC82 gene sequence is as follows: ATGAAGAAGAGTGGGGAGTTGAACTTGC CGGCGGGATTCCGATTCCATCCCACCGACGACGAGCTCGTCCAGCATTACCTTTGCCGGAAGTGCGCCGGACAGAGGATTTCCGTCCCCATTATCGCCGAAATCAACCTCTACAACTTCGATCCATGGCAGCTTCCCGGTATGCATATCACATACTAATTAATAGATTAATTGCATAATTAATTGATTGAAAATTGATCTGTATTGATTGATGGATTGATCAATGGCAGGAATGTCTCTGTACGGCGAGAAGGAGTGGTACTTCTTCTCACCGCGGGACCGGAAGTACCCGAACGGTTCGCGGCCGAACCGGGCGGCGGGGACCGGGTACTGGAAGGCGACCGGCGCCGACAAGCCGGTCGGGAAGCCGAAGACTCTGGGGATCAAAAAGGCCCTGGTTTTCTACGCCGGCAAAGCCCCCAGAGGCGTCAAGACCAATTGGATCATGCACGAGTACCGCCTCGCCAATGTCGATCGATCCGCCGACAACAACAAGAAGAAGAACAACAACAATAATCTAAGGGTAATGAACTTTTAACATCCTATCTGCCATTTCGTGTGTCCTTAAAGATTGTCTCAATCGATTGAATTAATGATTTTTTTGCTTGCAGCTTGATGATTGGGTTTTATGCCGGATATACAACAAGAAAGGGACCCTGGAGAGGCACGTCGCCGGCGGCGATGATCAAGGTCCGATCAAGAAGGGGGAGGTCGAATTAATGTCGGAATATATCCAAGAGGAGAAGCCCAATGTGGGTGGCTGCAAGAACAAGACCAGTAGTACATCGCCGGAGTCGTCGAGCATGATGTCCGAGCACGTGCTGTCGCCGGAGGTGGAGAGCGAGCCGAAATGGAGCGACCTGCTGGTGCAAAACGATTTTCAGTTAATGAGTAATAGTAGTGTTAGTTATTTGGATGGGTTCGATCACAATGATCCCTTCGTCTCCAACATGATCCATTACTACGACAATTCCGGCGCCGGAGACCAGTTGTCGGGGCTCTTCTTCCACCCCGACGCCGCCTTTGGGTACATGCAGAAGCCCTTTTGA。

[0130] SEQ ID NO:2: The coding region sequence of ApNAC82 is as follows: ATGAAGAAGAGTGGGGAGTTGAACTT GCCGGCGGGATTCCGATTCCATCCCACCGACGACGAGCTCGTCCAGCATTACCTTTGCCGGAAGTGCGCCGGACAGAGGATTTCCGTCCCCATTATCGCCGAAATCAACCTCTACAACTTCGATCCATGGCAGCTTCCCGGAATGTCTCTGTACGGCGAGAAGGAGTGGTACTTCTTCTCACCGCGGGACCGGAAGTACCCGAACGGTTCGCGGCCGAACCGGGCGGCGGGGACCGGGTACTGGAAGGCGACCGGCGCCGACAAGCCGGTCGGGAAGCCGAAGACTCTGGGGATCAAAAAGGCCCTGGTTTTCTACGCCGGCAAAGCCCCCAGAGGCGTCAAGACCAATTGGATCATGCACGAGTACCGCCTCGCCAATGTCGATCGATCCGCCGACAACAACAAGAAGAAGAACAACAATAATCTAAGGCTTGATGATTGGGTTTTATGCCGGATATACAACAAGAAAGGGACCCTGGAGAGGCACGTCGCCGGCGGCGATGATCAAGGTCCGATCAAGAAGGGGGAGGTCGAATTAATGTCGGAATATATCCAAGAGGAGAAGCCCAATGTGGGTGGCTGCAAGAACAAGACCAGTAGTACATCGCCGGAGTCGTCGAGCATGATGTCCGAGCACGTGCTGTCGCCGGAGGTGGAGAGCGAGCCGAAATGGAGCGACCTGCTGGTGCAAAACGATTTTCAGTTAATGAGTAATAGTAGTGTTAGTTATTTGGATGGGTTCGATCACAATGATCCCTTCGTCTCCAACATGATCCATTACTACGACAATTCCGGCGCCGGAGACCAGTTGTCGGGGCTCTTCTTCCACCCCGACGCCGCCTTTGGGTACATGCAGAAGCCCTTTTGA。

[0131] Sequence 3: The amino acid sequence of the ApNAC82 protein encoded by ApNAC82 is as follows (SEQ ID NO:3): MKK SGELNLPAGFRFHPTDDELVQHYLCRKCAGQRISVPIIAEINLYNFDPWQLPGMSLYGEKEWYFFSPRDRKYPNGSRPNRAAGTGYWKATGADKPVGKPKTLGIKKALVFYAGKAPRGVKTNWIMHEYRLANVDRSADNNKKKNNNNNLRLDDWVLCRIYNKKGTLERHVAGGDDQGPIKKGEVELMSEYIQEEKPNVGGCKNKTSSTSPESSSMMSEHVLSPEVESEPKWSDLLVQNDFQLMSNSSVSYLDGFDHNDPFVSNMIHYYDNSGAGDQLSGLFFHPDAAFGYMQKPF.

[0132] 3. Subcellular localization analysis of ApNAC82

[0133] (3.1) In this experiment, green fluorescent protein (EGFP) was selected as the reporter gene. The coding sequence of EGFP was linked with the coding sequence of ApNAC82 protein to form a fusion protein. This fusion protein was then transfected into protoplast cells for expression. The fluorescent signal of EGFP co-localized with that of ApNAC82 protein, thus allowing the observation of the subcellular localization of ApNAC82 protein. The known nuclear localization protein NLS-mKATE was also selected as a control in this experiment.

[0134] (3.2) The plant expression vector used in this experiment was pCAMBIA1300-35S:EGFP vector, hereinafter referred to as 35S:EGFP. Based on the nucleotide sequence of the coding region of ApNAC82 (SEQ ID NO:2), the stop codon TGA was removed to construct the ApNAC82-EGFP fusion protein. SacI and XbaI restriction sites on the 35S:EGFP vector were selected, and specific primer sequences for homologous recombination were designed. The following primers were prepared:

[0135] The sequence of ApNAC82-EGFP-F is (5'-3'):

[0136] AGAACACGGGGGACGAGCTCATGAAGAAGAGTGGGGAGTTGA;

[0137] The sequence of ApNAC82-EGFP-R is (5'-3'):

[0138] CCATGTCGACTCTAGAAAAGGGCTTCTGCATGTACC.

[0139] (3.3) Using the pCE2_TA-Blunt-Zero recombinant vector pCE2_TA-Blunt-Zero-ApNAC82 containing the nucleotide sequence of the coding region of ApNAC82 (SEQ ID NO:2) as a template, and using ApNAC82-EGFP-F / ApNAC82-EGFP-R as primers, PCR was performed using Phanta Max Super-Fidelity DNA Polymerase (P505) to prepare the ApNAC82-EGFP insert fragment. PCR reaction conditions: 95℃, pre-denaturation for 3 min; 95℃, denaturation for 15 s; 60℃, annealing for 20 s; 72℃, extension for 90 s; 35 cycles of amplification; 72℃, final extension for 10 min. The PCR reaction system is shown in Table 3.

[0140] Table 3. PCR reaction system for amplifying the ApNAC82-EGFP insert fragment

[0141] Reaction components Added amount 2×Phanta Max Buffer 25μL dNTP Mix (10mM each) 1μL Phanta Max Super-Fidelity DNA Polymerase 1μL ApNAC82-EGFP-F (10 μM) 2μL ApNAC82-EGFP-R (10 μM) 2μL pCE2_TA-Blunt-Zero-ApNAC82 plasmid 100ng <![CDATA[ddH20]]> Make up to 50 μL

[0142] (3.4) The PCR products were detected by 1% agarose gel electrophoresis, and the ApNAC82-EGFP insert fragment was recovered using an agarose gel recovery kit (D2500-01).

[0143] (3.5) The 35S:EGFP vector plasmid was double-digested with SacI and XbaI from New England Biolabs to prepare a linearized vector. The linearized 35S:EGFP vector was then purified using an agarose gel extraction kit (D2500-01). The digestion conditions were: 37℃ for 60 min; 80℃ for 20 min. The digestion reaction system is shown in Table 4.

[0144] Table 4. Reaction system for digesting 35S:EGFP vector plasmid

[0145] Reaction components Added amount Restriction endonuclease XbaI (10 U) 1μL Restriction endonuclease SacI (10 U) 1μL 35S:EGFP vector plasmid 1μg 10×NEBuffer Cutsmart 5μL <![CDATA[ddH20]]> Make up to 50 μL

[0146] (3.6) Using the ClonExpress Ultra One Step Cloning Kit V2, the ApNAC82-EGFP insert was ligated into the 35S:EGFP linearized vector via homologous recombination to obtain the recombinant product. The homologous recombination reaction conditions were 50℃ for 5 min, and the reaction was immediately cooled on ice after completion. The recombination reaction system is shown in Table 5.

[0147] Table 5. Homologous recombination reaction system for constructing the 35S:ApNAC82-EGFP fusion expression vector

[0148] Reaction components Added amount 35S:EGFP linearized vector 2μL ApNAC82-EGFP insert fragment 3μL 2×CE Mix 5μL

[0149] (3.7) The recombinant product was transformed into Escherichia coli DH5α, and the 35S:ApNA C82-EGFP fusion expression vector was obtained by screening with kanamycin and sequencing verification.

[0150] (3.8) Preparation and transformation of tobacco leaf protoplasts were performed according to the method of Yoo, Cho, & Sheen, 2007 (Yoo SD, Cho YH, Sheen J. Arabidopsis mesophyll protoplasts: a versatile cell system for transgenic gene expression analysis. Nature protocols, 2007, 2(7): 1565-1572). After transformation, the cells were observed using a Nikon C2-ER laser confocal microscope. The fluorescence parameters are as follows:

[0151] Green fluorescent protein EGFP: excitation light 488nm, emission light 510nm;

[0152] Nuclear localization fusion fluorescent protein mKATE: excitation light 561nm, emission light 580nm;

[0153] Chloroplast autofluorescence: excitation light 640nm, emission light 675nm.

[0154] The results are as follows Figure 3 As shown, under the EGFP fluorescence channel, the green fluorescence signal of tobacco protoplast cells co-transfected with NLS-mKATE+35S:EGFP can be clearly observed distributed throughout the entire cell, including the nucleus, cell membrane, and cytoplasm, while the green fluorescence signal of tobacco protoplast cells co-transfected with NLS-mKATE+35S:ApNAC82-EGFP is only distributed in the nucleus.

[0155] The nuclear marker fluorescent protein mKATE is located in the cell nucleus. Under the mKATE channel, the red fluorescence of tobacco protoplast cells co-transfected with NLS-mKATE+35S:EGFP and NLS-mKATE+35S:ApNAC82-EGFP plasmids is only distributed in the cell nucleus.

[0156] In the chloroplast fluorescence channel, the chloroplasts of tobacco protoplast cells cotransformed with NLS-mKATE+35S:EGFP and NLS-mKATE+35S:ApNAC82-EGFP exhibit autofluorescence on the cell membrane.

[0157] In the bright field channel, tobacco protoplast cells cotransformed with NLS-mKATE+35S:EGFP and NLS-mKATE+35S:ApNAC82-EGFP showed no fluorescence.

[0158] In the superposition field, the fluorescence signal of co-transformed NLS-mKATE+35S:ApNAC82-EGFP overlaps with the position of the nuclear marker fluorescent protein.

[0159] The above results indicate that the ApNAC82 gene is expressed in the cell nucleus, is a nuclear transcription factor, and plays a transcriptional regulatory role in the cell nucleus.

[0160] 4. ApNAC82 transcriptional activity analysis

[0161] (4.1) Based on the nucleotide sequence of the coding region of ApNAC82 (SEQ ID NO:2), select the NdeⅠ and EcoRI-HF restriction sites on the pGBKT7 vector and the BamHI and PstⅠ restriction sites on the pGBKT7-VP16 vector, and design specific primer sequences for homologous recombination. Prepare the following primers respectively:

[0162] The pGBKT7-ApNAC82-F sequence is (5'-3'):

[0163] AGGAGGACCTGCATATGATGAAGAAGAGTGGGGAGTTGAACT;

[0164] The pGBKT7-ApNAC82-R sequence is (5'-3'):

[0165] GGATCCCCGGGAATTCTTAAAAGGGCTTCTGCATGTACCCAAAG;

[0166] The pGBKT7-VP16-ApNAC82-F sequence is (5'-3'):

[0167] GGTGGGCCGGGGATCATGAAGAAGAGTGGGGAGTTGAACT;

[0168] The pGBKT7-VP16-ApNAC82-R sequence is (5'-3'):

[0169] TAGTTATGCGGCCGCTTAAAAGGGCTTCTGCATGTACCCAAAG.

[0170] (4.2) Using the pCE2_TA-Blunt-Zero recombinant vector pCE2_TA-Blunt-Zero-ApNAC82 containing the nucleotide sequence of the coding region of ApNAC82 (SEQ ID NO:2) as a template, PCR reactions were performed using Phanta Max Super-Fidelity DNA Polymerase (P505) with pGBKT7-ApNAC82-F / pGBKT7-ApNAC82-R and pGBKT7-VP16-ApNAC82-F / pGBKT7-VP16-ApNAC82-R as primers to prepare the pGBKT7-ApNAC82 insert and the pGBKT7-VP16-ApNAC82 fusion insert, respectively. PCR reaction conditions: 95℃, pre-denaturation for 3 min; 95℃, denaturation for 15 s; 60℃, annealing for 20 s; 72℃, extension for 90 s; 35 cycles of amplification; 72℃, final extension for 10 min. The PCR reaction system is shown in Tables 6 and 7.

[0171] Table 6. PCR reaction system for amplifying the pGBKT7-ApNAC82 insert fragment

[0172]

[0173]

[0174] Table 7. PCR reaction system for amplifying the pGBKT7-VP16-ApNAC82 fusion insert.

[0175] Reaction components Added amount 2×Phanta Max Buffer 25μL dNTP Mix (10mM each) 1μL Phanta Max Super-Fidelity DNA Polymerase 1μL pGBKT7-VP16-ApNAC82-F(10μM) 2μL pGBKT7-VP16-ApNAC82-R(10μM) 2μL pCE2_TA-Blunt-Zero-ApNAC82 plasmid 100ng <![CDATA[ddH20]]> Make up to 50 μL

[0176] (4.3) The PCR products were detected by 1% agarose gel electrophoresis. The pGBKT7-ApNAC82 insert and the pGBKT7-VP16-ApNAC82 fusion insert were recovered by the agarose gel recovery kit (D2500-01).

[0177] (4.4) The pGBKT7 vector plasmid was prepared by double digestion with the restriction enzymes NdeⅠ and EcoRI-HF from New England Biolabs; the pGBKT7-VP16 vector plasmid was prepared by double digestion with the restriction enzymes BamHI and PstⅠ from New England Biolabs. Digestion conditions: 37℃, 60 min; 80℃, 20 min. The digestion reaction systems are shown in Tables 8 and 9.

[0178] Table 8. Reaction system for enzyme digestion of pGBKT7 vector plasmid

[0179] Reaction components Added amount Restriction endonuclease NdeⅠ (10 U) 1μL Restriction endonuclease EcoRI-HF (10 U) 1μL pGBKT7 vector plasmid 1μg 10×NEBuffer Cutsmart 5μL <![CDATA[ddH20]]> Make up to 50 μL

[0180] Table 9. Reaction system for enzyme digestion of pGBKT7-VP16 vector plasmid

[0181]

[0182]

[0183] (4.5) The enzyme digestion products were detected by 1% agarose gel electrophoresis. The pGBKT7 linearized vector and pGBKT7-VP16 linearized vector were recovered and purified by the agarose gel recovery kit (D2500-01).

[0184] (4.6) Using the ClonExpress Ultra One Step Cloning Kit V2, the pGBKT7-ApN AC82 insert fragment was ligated to the pGBKT7 linearized vector via homologous recombination to obtain recombinant product one, and the pGBKT7-VP16-ApNAC82 fusion insert fragment was ligated to the pGBKT7-VP16 linearized vector via homologous recombination to obtain recombinant product two. The homologous recombination reaction conditions were 50℃ for 5 min, and the reaction was immediately cooled on ice after completion. The recombination reaction system is shown in Tables 10 and 11.

[0185] Table 10. Homologous recombination reaction system for constructing the pGBKT7-ApNAC82 expression vector

[0186] Reaction components Added amount pGBKT7 linearized carrier 2μL pGBKT7-ApNAC82 Insert Fragment 3μL 2×CE Mix 5μL

[0187] Table 11. Homologous recombination reaction system for constructing the pGBKT7-VP16-ApNAC82 expression vector

[0188] Reaction components Added amount pGBKT7-VP16 linearization vector 2μL pGBKT7-VP16-ApNAC82 fused insertion fragment 3μL 2×CE Mix 5μL

[0189] (4.7) Recombinant product 1 and recombinant product 2 were transformed into Escherichia coli DH5α, and after screening with kanamycin and sequencing verification, pGBKT7-ApNAC82 expression vector and pGBKT7-VP16-ApNAC82 expression vector were obtained, respectively.

[0190] (4.8) Using the LiAC / PEG yeast transformation method, pGBKT7, pGBKT7-VP16, pGBKT7-ApNAC82, and pGBKT7-VP16-ApNAC82 were transformed into yeast Y2HGold competent cells to obtain yeast transformed with pGBKT7, yeast transformed with pGBKT7-ApNAC82, yeast transformed with pGBKT7-VP16, and yeast transformed with pGBKT7-VP16-ApNAC82, respectively.

[0191] (4.9) Transcription activity detection

[0192] a. Select positive single clones obtained from (4.8) transformation and culture them in SD / -Trp liquid medium, adjusting OD600 to 0.2;

[0193] b. Dilute with ddH2O sequentially by 10 times, 100 times, and 1000 times, i.e., OD600 = 0.02, 0.002, and 0.0002;

[0194] c. Take 5 μL of the sample and spot it onto SD / -Trp, SD / -His / -Trp and SD / -Ade / -His / -Trp plates respectively;

[0195] d. Incubate at 28-30℃ for 2-3 days, and determine whether ApNAC82 has transcriptional activity based on the growth status of each sample on different screening media.

[0196] The results are as follows Figure 4 As shown, yeast transformed with pGBKT7, yeast transformed with pGBKT7-ApNAC82, yeast transformed with pGBKT7-VP16, and yeast transformed with pGBKT7-VP16-ApNAC82 all grew normally on SD / -Trp medium, indicating that the yeast transformation was successful.

[0197] On SD / -Trp / -His and SD / -Trp / -His / Ade media, yeast transgenic with pGBKT7 could not grow, while yeast transgenic with pGBKT7-ApNAC82 could grow normally, indicating that the HIS3 and ADE2 reporter genes were activated and expressed in yeast transgenic with pGBKT7-ApNAC82.

[0198] VP16 is a transcription activator. In yeast transgenic pGBKT7-VP16, the transcriptional activation of VP16 activates the expression of reporter genes HIS3 and ADE2 in the yeast, thus enabling the yeast transgenic pGBKT7-VP16 to grow normally on SD / -Trp / -His and SD / -Trp / -His / Ade media.

[0199] Compared to VP16, yeast transgenic pGBKT7-VP16-ApNAC82 showed stronger growth on SD / -Trp / -His and SD / -Trp / -His / Ade media than yeast transgenic pGBKT7-VP16, indicating that the fusion expression of ApNAC82 and VP16 enhances the transcriptional activation activity of VP16.

[0200] The above experimental results demonstrate that ApNAC82 possesses transcriptional activation activity.

[0201] 5. ApNAC82 expression analysis in response to drought and high temperature

[0202] (5.1) Selected Andrographis paniculata plants with uniform growth and 60 days of normal growth for drought and high temperature stress treatment. Drought stress was applied using natural drought, and based on the relative water content of the leaves, three treatments were set: normal growth (90.45%), mild drought stress (80.81%), and severe drought stress (57.54%). High temperature stress was applied using 50℃ treatment, with treatments set for 0h (normal growth), 3h, and 6h. Each treatment was set in 3 replicates, with 5 plants per replicate. After the stress treatment was completed, Andrographis paniculata leaves were collected, immediately flash-frozen in liquid nitrogen, and stored at -80℃ for later use.

[0203] (5.2) Take 0.5g of leaves from each of the 5 Andrographis paniculata plants in each of the above replicate treatments, mix them, grind them in liquid nitrogen, and extract total RNA from the Andrographis paniculata leaves using a plant total RNA extraction kit (DP432); use a NanoDrop 2000 spectrophotometer to detect the purity and concentration of the extracted RNA; use LabChip GX to detect the integrity of the RNA.

[0204] (5.3) Qualified RNA was used to construct RNA-seq libraries; qualified libraries were sequenced using the Illumina NovaSe q6000 sequencing platform in PE150 mode; the offline data was filtered to obtain Clean Data, and the sequences were aligned according to the genome information of Andrographis paniculata (SRP143459) to obtain Mapped Data, which was then used for library quality assessment; StandardTie was used to standardize the data using the maximum flow algorithm and FPKM (Fragments Per Kilobase of transcripts per Million fragments mapped) as an indicator of gene expression level.

[0205] from Figure 5It can be seen that under drought stress, the expression of ApNAC82 showed an upregulation trend with the aggravation of drought stress; under mild drought stress, the expression of ApNAC82 was upregulated by 4.79 times compared with normal growth, and under severe drought stress, the expression of ApNAC82 was further increased, reaching 9.61 times that of normal growth.

[0206] Under high-temperature stress, the expression of ApNAC82 showed an upregulation trend with the prolongation of high-temperature stress. After 3 hours of high-temperature stress, the expression of ApNAC82 was upregulated by 5.18 times compared with normal growth, and after 6 hours of continuous high-temperature stress, the expression of ApNAC82 was upregulated by 9.51 times compared with normal growth. These results indicate that ApNAC82 is simultaneously upregulated in response to drought and high-temperature stress.

[0207] 6. ApNAC82 gene functional analysis

[0208] 6.1 Obtaining plants overexpressing the ApNAC82 gene

[0209] (6.1.1) The recombinant plasmid 35S:ApNA C82-EGFP constructed in the subcellular localization analysis of ApNAC82 in section 3 above was transformed into Agrobacterium GV310 by heat shock method to obtain recombinant Agrobacterium.

[0210] (6.1.2) Resuspend the recombinant Agrobacterium obtained in step (1) in 10 mL of MgCl2 solution (10 mM, containing 200 μM AS), adjust the OD600 to about 1.0, and let it stand at 28℃ for 2 to 3 h to obtain the infection solution.

[0211] (6.1.3) The leaf disc method was used to transform tobacco leaves, and after co-culture, hygromycin resistance screening, differentiation and rooting, T0 generation regenerated plants were obtained.

[0212] (6.1.4) Homozygous positive transgenic plants of generation T3 were obtained by screening with hygromycin and PCR identification. The specific methods are as follows:

[0213] T0 generation regenerated plants were identified by PCR to obtain positive T0 generation transgenic plants, and T1 generation seeds were obtained by self-pollination of positive T0 generation transgenic plants.

[0214] T1 generation seeds were screened with hygromycin and identified by PCR to obtain positive T1 generation transgenic plants. The positive T1 generation transgenic plants were then self-pollinated to obtain T2 generation seeds.

[0215] T2 generation seeds were screened using hygromycin and identified by PCR to obtain positive T2 generation transgenic plants. Self-pollination of these positive T2 generation transgenic plants yielded T3 generation seeds. If all T3 generation plants obtained from a T2 generation plant are positive transgenic plants, then the self-pollinated offspring of that line are all homozygous transgenic lines.

[0216] In step (6.1.4), the screening concentration of hygromycin is 50 mg / L.

[0217] In step (6.1.4), the PCR identification method is as follows: extract genomic DNA from the plant leaves, and perform PCR amplification using the dApNAC3-F and d1300-R primer pairs. If a specific amplification product is obtained, the plant is a transgenic plant.

[0218] The sequence of dApNAC82-F is (5'-3'): TCCATTACTACGACAATTCCGGC;

[0219] The sequence of d1300-R is (5'-3'): TCAAAAGGGCTTCTGCATGTACC.

[0220] PCR reaction conditions: 95℃, pre-denaturation for 3 min; 95℃, denaturation for 15 s; 60℃, annealing for 20 s; 72℃, extension for 20 s; 35 cycles of amplification; 72℃, final extension for 10 min. The PCR reaction system is shown in Table 12.

[0221] Table 12. PCR reaction system for detecting positive transgenic plants

[0222]

[0223]

[0224] The results are as follows Figure 6 As shown, genomic DNA of the T3 generation transgenic plants was amplified by PCR. The expected 190 bp amplification product was obtained in the transgenic positive plants, while no corresponding fragment was detected in the wild-type plants, indicating that the ApNAC82 gene had been successfully integrated into Nicotiana benthamiana. Hygromycin screening and PCR identification confirmed that OE#ApNAC82-6, OE#ApNAC82-9, OE#ApNAC82-10, and OE#ApNAC82-11 were all homozygous transgenic lines of the T3 generation.

[0225] 6.2 Effects of ApNAC82 overexpression on tobacco growth

[0226] (6.2.1) Test seeds: wild-type WT seeds of Nicotiana benthamiana, and seeds of the three T3 generation homozygous transgenic lines OE#ApNAC82-6, OE#ApNAC82-9, and OE#ApNAC82-11 obtained in step 6.1.

[0227] (6.2.2) The tested tobacco seeds were sown in planting pots filled with nutrient soil and placed in an incubator at 25℃ for 16h light / 8h darkness, with normal watering during the period;

[0228] (6.2.3) After emergence, retain 4 seedlings in each planting pot and cultivate them in a 25℃ incubator under 16h light / 8h darkness conditions, with normal watering during the period;

[0229] (6.2.4) After 4 weeks of culture, the effect of ApNAC82 overexpression on tobacco growth was determined by comparing the growth and plant height of wild-type WT.

[0230] Overexpression of the Arabidopsis ATAF gene typically leads to adverse growth responses in transgenic plants, such as stunted growth, weak stunted plants, and shortened root systems. In section 1.1, sequence alignment analysis revealed that ApNAC82 belongs to the ATAF gene family. Compared to its Arabidopsis ATAF family homologs, it exhibits relatively conserved N-terminal structure, while showing significant differences in its C-terminal sequence. The non-conservation of the C-terminus of the protein results in different functions and characteristics. Therefore, it is necessary to investigate whether overexpression of ApNAC82 leads to stunted growth and weak stunted plants to determine whether the application of ApNAC82 in crop genetic improvement has adverse effects. Figure 7 As shown, compared with wild-type WT, the three strains OE#ApNAC82-6, OE#ApNAC82-9, and OE#ApNAC82-11 that overexpressed ApNAC82 grew vigorously and did not show obvious dwarfing symptoms. Therefore, overexpression of ApNAC82 will not cause dwarfing and weakness in Nicotiana benthamiana.

[0231] 6.3 Identification of drought resistance in tobacco plants overexpressing ApNAC82

[0232] 6.3.1 Mannitol-simulated drought stress treatment

[0233] (6.3.1.1) Test seeds: wild-type WT seeds of Nicotiana benthamiana and seeds of the three T3 generation homozygous transgenic lines OE#ApNAC82-6, OE#ApNAC82-9 and OE#ApNAC82-11 obtained in step 6.1.

[0234] (6.3.1.2) Prepare MS medium containing 0 mM, 100 mM, 200 mM and 300 mM mannitol;

[0235] (6.3.1.3) Seed disinfection: Take tobacco seeds and place them in a 1.5 mL centrifuge tube. Add 1 mL of 70% alcohol and soak for 60 seconds. Rinse three times with sterile water. Then add 1 mL of freshly prepared 12% sodium hypochlorite solution for disinfection for 15 minutes. Rinse five times with sterile water.

[0236] (6.3.1.4) Add 500 μL of 0.1% water agar to centrifuge tubes to suspend the seeds, and sow the seeds on MS medium containing different concentrations of mannitol. Sow 4 lines on each plate.

[0237] (6.3.1.5) After sowing, seal the seedbed with sealing film and place it in an incubator at 25℃ with a light intensity of 8000Lx, light for 16 hours and darkness for 8 hours. Observe the growth after emergence.

[0238] (6.3.1.6) Germination rate detection: After 5 days of culture, observe the growth of wild-type and transgenic lines on MS medium containing different concentrations of mannitol, count the germinated seeds and calculate the germination rate. The calculation formula is as follows:

[0239] Germination rate = (Number of germinated seeds / Total number of seeds sown) × 100%

[0240] (6.3.1.7) Measure the root length: After 5 days of culture, randomly select 4 seedlings from wild-type and transgenic lines, moisten them, gently spread out the roots, measure the length of the main root with a ruler and record it.

[0241] The results are as follows Figures 8-11 As shown, on MS medium without mannitol (0 mM), there were no significant differences in germination rate and root length between wild-type WT and the three transgenic lines.

[0242] Under 100 mM mannitol treatment, the germination rate of wild-type WT was 59.43%, and the average root length was 0.33 cm. The germination rates of the three lines OE#ApNAC82-6, OE#ApNAC82-9, and OE#ApNAC82-11 were 84.07%, 81.03%, and 80.67%, respectively, and the average root lengths were 1.43 cm, 1.47 cm, and 1.37 cm, respectively.

[0243] Under 200 mM mannitol treatment, the germination rate of wild-type WT was 13.17%, and the average root length was 0.13 cm. The germination rates of the three lines OE#ApNAC82-6, OE#ApNAC82-9, and OE#ApNAC82-11 were 58.00%, 72.33%, and 69.37%, respectively, and the average root lengths were 0.47 cm, 0.57 cm, and 0.67 cm, respectively.

[0244] Under 300 mM mannitol treatment, neither the wild-type WT nor the three transgenic lines germinated.

[0245] The above results indicate that overexpression of ApNAC82 improves the tolerance of Nicotiana benthamiana to mannitol stress.

[0246] 6.3.2 Treatment of Natural Drought Stress

[0247] (6.3.2.1) Test seeds: wild-type WT seeds of Nicotiana benthamiana and seeds of the three T3 generation homozygous transgenic lines OE#ApNAC82-6, OE#ApNAC82-9 and OE#ApNAC82-11 obtained in step 6.1.

[0248] (6.3.2.2) The tested tobacco seeds were sown in planting pots filled with nutrient soil and placed in an incubator at 25℃ for 16h light / 8h darkness, with normal watering during the period;

[0249] (6.3.2.3) Select plants with good growth, uniform growth, and a growth cycle of 4 weeks for drought treatment experiments. Before the experiment, watering was stopped after thorough irrigation to allow for natural drought stress treatment and maintain uniform soil moisture content. On the 12th day of drought treatment, the growth of the plants was observed.

[0250] The results are as follows Figure 12 As shown, after 12 days of drought treatment, the leaves of wild-type WT plants exhibited obvious physiological wilting, with the lower leaves becoming dry, wrinkled, and yellowed. In contrast, the transgenic plants showed milder wilting symptoms, exhibited better growth than wild-type WT plants, and showed significantly less leaf wilting. These results indicate that overexpression of ApNAC82 can improve the drought tolerance of Nicotiana benthamiana.

[0251] 6.4 Identification of high-temperature tolerance in tobacco overexpressing ApNAC82

[0252] 6.4.1 Identification of seedling recovery ability after high temperature stress treatment

[0253] (6.4.1.1) Test seeds: wild-type WT seeds of Nicotiana benthamiana and seeds of the three T3 generation homozygous transgenic lines OE#ApNAC82-6, OE#ApNAC82-10 and OE#ApNAC82-11 obtained in step 6.1.

[0254] (6.4.1.2) Seed disinfection: Take tobacco seeds and place them in a 1.5 mL centrifuge tube. Add 1 mL of 70% alcohol and soak for 60 seconds. Rinse three times with sterile water. Then add 1 mL of freshly prepared 12% sodium hypochlorite solution for disinfection for 15 minutes. Rinse five times with sterile water.

[0255] (6.4.1.3) Add 500 μL of 0.1% water agar to a centrifuge tube to suspend the seeds, and sow the seeds on MS medium with squares, with 4 lines sown on each plate.

[0256] (6.4.1.4) After sowing, seal with sealing film and place in an incubator at 25℃, light intensity of 8000Lx, light for 16h, and darkness for 8h.

[0257] (6.4.1.5) After two weeks of cultivation, the plate was placed at 45℃ for 12 hours and then placed at 25℃ to recover growth for 2 days. The high temperature tolerance was judged based on the leaf regreening.

[0258] The results are as follows Figure 13 As shown, high temperature stress can cause the chlorophyll in leaves to be destroyed and turn white. The leaves of both wild-type WT and transgenic lines recovered to varying degrees after high temperature stress. Only a small number of wild-type WT seedlings had their leaves turn green again, while the probability of transgenic seedlings turning green again was significantly higher than that of wild-type WT.

[0259] 6.4.2 Identification of the high-temperature tolerance of transgenic plants

[0260] (6.4.2.1) Test seeds: wild-type WT seeds of Nicotiana benthamiana and seeds of the three T3 generation homozygous transgenic lines OE#ApNAC82-9 and OE#ApNAC82-11 obtained in step 6.1.

[0261] (6.4.2.2) The tested tobacco seeds were sown in planting pots filled with nutrient soil and placed in an incubator at 25℃ for 16h light / 8h darkness, with normal watering during the period;

[0262] (6.4.2.3) Select plants with good growth, uniform growth, and a growth cycle of 4 weeks and subject them to 45℃ high temperature stress treatment for 24 hours, and observe the growth of the plants.

[0263] The results are as follows Figure 14 As shown, after high-temperature stress treatment, the leaves of wild-type WT plants exhibited obvious wilting, while the transgenic plants only showed mild wilting symptoms in the lower leaves. These results indicate that overexpression of ApNAC82 can improve the high-temperature tolerance of Nicotiana benthamiana.

Claims

1. A method for improving the drought resistance and high-temperature tolerance of plants, characterized in that, The method includes introducing a nucleotide sequence encoding the Andrographis paniculata transcription factor ApNAC82 into a plant and expressing the nucleotide sequence in the plant; The nucleotide sequence is selected from the following group: (a) A nucleotide sequence as shown in SEQ ID NO:1; or (b) The coding region nucleotide sequence as shown in SEQ ID NO:2; The plant in question is Nicotiana benthamiana.

2. The method for improving plant drought resistance and high-temperature tolerance as described in claim 1, characterized in that, A method for introducing a nucleotide sequence encoding the Andrographis paniculata transcription factor ApNAC82 into a plant and expressing the nucleotide sequence in the plant includes the following steps: (1) The 35S:ApNAC82-EGFP fusion expression vector was transformed into Agrobacterium GV310 by heat shock method to obtain recombinant Agrobacterium; (2) Resuspend the recombinant Agrobacterium obtained in step (1) in 10 mL of MgCl2 solution with a concentration of 10 mM and containing 200 μM AS, adjust the OD600 to about 1.0, and let it stand at 28 ℃ for 2-3 h to obtain the infection solution; (3) The leaf disc method was used to transform plant leaves, and after co-culture, hygromycin resistance screening, differentiation and rooting, T0 generation regenerated plants were obtained; (4) Homozygous positive transgenic plants of generation T3 were obtained by screening with hygromycin and PCR identification. The specific methods are as follows: T0 generation regenerated plants were identified by PCR to obtain positive T0 generation transgenic plants, and T1 generation seeds were obtained by self-pollination of positive T0 generation transgenic plants. T1 generation seeds were screened with hygromycin and identified by PCR to obtain positive T1 generation transgenic plants. The positive T1 generation transgenic plants were then self-pollinated to obtain T2 generation seeds. T2 generation seeds were screened with hygromycin and identified by PCR to obtain positive T2 generation transgenic plants. The positive T2 generation transgenic plants were then self-pollinated to obtain T3 generation seeds. In step (4), the screening concentration of hygromycin is 50 mg / L; In step (4), the PCR identification method is as follows: extract genomic DNA from the plant leaves, and perform PCR amplification using dApNAC3-F and d1300-R primer pairs. If a specific amplification product is obtained, the plant is a transgenic plant. The sequence of dApNAC82-F is: TCCATTACTACGACAATTCCGGC; The sequence of d1300-R is: TCAAAAGGGCTTCTGCATGTACC; PCR reaction conditions: 95℃, pre-denaturation for 3 min; 95℃, denaturation for 15 s; 60℃, annealing for 20 s; 72℃, extension for 20 s; 35 cycles of amplification; 72℃, final extension for 10 min. The PCR reaction system was as follows: 12.5 μL of 2× Accurate Taq Master Mix, 1 μL of 10 μM dApNAC82-F, 1 μL of 10 μM d1300-R, 50 ng of genomic DNA from plant leaves, and ddH2O to a final volume of 50 μL.

3. The method for improving plant drought resistance and high-temperature tolerance as described in claim 2, characterized in that, In step (1), the preparation method of the 35S:ApNAC82-EGFP fusion expression vector includes the following steps: (1) Based on the nucleotide sequence of the coding region of ApNAC82, the stop codon TGA was removed to construct the ApNAC82-EGFP fusion protein. The SacI and XbaI restriction sites on the 35S:EGFP vector were selected to design specific primer sequences for homologous recombination. The following primers were prepared: The sequence of ApNAC82-EGFP-F is as follows: AGAACACGGGGGACGAGCTCATGAAGAAGAGTGGGGAGTTGA; The sequence of ApNAC82-EGFP-R is: CCATGTCGACTCTAGAAAAGGGCTTCTGCATGTACC; The 35S:EGFP vector is an abbreviation for pCAMBIA1300-35S:EGFP vector; (2) Using the pCE2_TA-Blunt-Zero recombinant vector pCE2_TA-Blunt-Zero-ApNAC82 containing the nucleotide sequence of the coding region of ApNAC82 as a template, and using ApNAC82-EGFP-F and ApNAC82-EGFP-R as primers, the ApNAC82-EGFP insert fragment was prepared by PCR reaction using Phanta Max Super-Fidelity DNA Polymerase P505. PCR reaction conditions: 95℃, pre-denaturation for 3 min; 95℃, denaturation for 15 s; 60℃, annealing for 20 s; 72℃, extension for 90 s; 35 cycles of amplification; 72℃, final extension for 10 min. The PCR reaction system consisted of 25 μL of 2×PhantaMax Buffer, 1 μL of 10 mMeach dNTP Mix, 1 μL of PhantaMax Super-Fidelity DNA Polymerase, 2 μL of 10 μM ApNAC82-EGFP-F, 2 μL of 10 μM ApNAC82-EGFP-R, 100 ng of pCE2_TA-Blunt-Zero-ApNAC82 plasmid, and ddH2O to a final volume of 50 μL. (3) The PCR products were detected by 1% agarose gel electrophoresis, and the ApNAC82-EGFP insert fragment was recovered using the agarose gel recovery kit D2500-01; (4) The 35S:EGFP vector was prepared by double digestion of the 35S:EGFP vector with the restriction enzymes SacI and XbaI from New England Biolabs. Enzyme digestion reaction conditions: 37℃, 60 min; 80℃, 20 min; The enzyme digestion reaction system is as follows: 1 μL of 10 U restriction endonuclease XbaI, 1 μL of 10 U restriction endonuclease SacI, 1 μg of 35S:EGFP vector plasmid, 5 μL of 10×NEBufferCutsmart, and ddH2O to a final volume of 50 μL. (5) The enzyme digestion products were detected by 1% agarose gel electrophoresis, and the 35S:EGFP linearized vector was recovered and purified using the agarose gel recovery kit D2500-01. (6) Using the ClonExpress Ultra One Step Cloning Kit V2, the ApNAC82-EGFP insert was ligated to the 35S:EGFP linearized vector via homologous recombination to obtain the recombinant product; The reaction conditions for homologous recombination were 50°C for 5 min, and the reaction was immediately cooled on ice after completion. The homologous recombination reaction system was as follows: 2 μL of 35S:EGFP linearized vector, 3 μL of ApNAC82-EGFP insert, and 5 μL of 2×CE Mix were added. (7) The recombinant product was transformed into Escherichia coli DH5α, and the 35S:ApNAC82-EGFP fusion expression vector was obtained by screening with kanamycin and sequencing verification.

4. The use of recombinant vectors, expression cassettes, transgenic cell lines or host bacteria containing the ApNAC82 gene, whose nucleotide sequence is selected from SEQ ID NO:1 or SEQ ID NO:2, in the preparation of products for improving the drought resistance and heat tolerance of tobacco Benzodiaceae.

5. Application of the ApNAC82 gene, selected from nucleotide sequences such as SEQ ID NO:1 or SEQ ID NO:2, in improving drought resistance and heat tolerance in Nicotiana benthamiana.

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