Andrographis paniculata transcription factor ApNAC82 gene and application thereof

Abstract

The invention discloses an andrographis paniculata transcription factor ApNAC82. A nucleotide sequence of a coding region of the andrographis paniculata transcription factor ApNAC82 is as shown in SEQ ID NO: 2. The invention also discloses a cloning method of the andrographis paniculata transcription factor ApNAC82, which comprises the following steps: cloning a coding region fragment of the andrographis paniculata transcription factor ApNAC82 by taking cDNA (complementary deoxyribonucleic acid) synthesized by reverse transcription of total RNA (ribonucleic acid) of andrographis paniculata as a template through PCR (polymerase chain reaction), or cloning a gene fragment of the andrographis paniculata transcription factor ApNAC82 by taking DNA (deoxyribonucleic acid) of the andrographis paniculata as a template through PCR; and recovering a coding region fragment or a gene fragment of the andrographis paniculata transcription factor ApNAC82 from the PCR product by adopting an agarose gel recovery kit. The invention has important significance for understanding the response mechanism of andrographis paniculata to multiple adversity stresses, especially for mining and utilizing multifunctional transcription factors capable of improving multiple adversity stresses, and for genetic improvement of andrographis paniculata and other species and breeding of stress-resistant varieties.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to the application of the transcription factor ApNAC82 in improving the drought tolerance and high temperature tolerance of plants. Specifically, it relates to ApNAC82 and its application in the ability to cultivate plant varieties with drought and high temperature resistance. Background Art

[0002] Andrographis paniculata is a plant of the Acanthaceae family, with effects such as antibacterial and anti-inflammatory, clearing heat and detoxifying, and detumescence and pain relief. It is the main raw material for various traditional Chinese medicine preparations such as Qinghuo Zhimai tablets, Xiaoyan Lidan tablets, and Andrographolide tablets. In addition, andrographolide is known as the "traditional Chinese medicine antibiotic" and is the main raw material for traditional Chinese veterinary medicines such as Lianxiang powder, which is widely used in the livestock and poultry breeding industry to replace antibiotics in the treatment of diseases such as bacterial diarrhea. Andrographis paniculata is a large-scale traditional Chinese medicine, with an annual demand of 15,000 - 20,000 tons in China. High yield, stable yield, and quality improvement are the key technical problems in the process of Andrographis paniculata cultivation. Drought and high temperature stress directly affect the growth and development of the above-ground part of Andrographis paniculata, interfering with the metabolism, transportation, and transformation of substances, and are the most important abiotic stresses during the growth period of Andrographis paniculata. Therefore, systematically exploring genes related to excellent agronomic traits of Andrographis paniculata and analyzing the adaptation mechanism of Andrographis paniculata stress resistance regulation provide a good theoretical basis and gene resources for the genetic improvement and new germplasm creation of Andrographis paniculata.

[0003] Transcription factors can specifically interact with cis - acting elements on the promoters of eukaryotic genes and have activating or inhibitory effects on gene transcription and expression. Transcription factors play important regulatory roles in plant growth, development, and stress defense responses. The NAC transcription factor is one of the largest gene families, with a highly conserved NAC domain at its N - terminus and a variable transcriptional regulatory region at its C - terminus. Although members of the NAC gene family have a high degree of conservation in the N - terminal NAC domain, there are significant differences in the C - terminal sequences during plant evolution and natural selection. This non - conservation of the protein C - terminus endows it with different functions and characteristics, showing diversity due to differences in species, environment, and genotype. For example, overexpression of OsSNAC1, OsSNAC3, and OsONAC022 in rice enhances the drought and salt tolerance of rice (Fang et al., 2015; Hong et al., 2016; Hu et al., 2006), while overexpression of ZmSNAC1, ZmNAC55, ZmNAC84, and ZmNAC111 in maize improves the tolerance to drought (Lu et al., 2012; Mao et al., 2015; Mao et al., 2016; Zhu et al., 2016). Most studies on NAC transcription factors focus on aspects such as drought, cold, and high salt. There are few studies on NAC under high - temperature stress. Overexpression of the Arabidopsis NAC transcription factor JUNGBRUNNEN1 (JUB1; ANAC042) prolongs the survival time of Arabidopsis and increases its tolerance to heat stress (Shahnejat - Bushehri et al., 2012).

[0004] When overexpressed, transcription factors will accumulate a large number of stress - related proteins, thus interfering with the normal metabolic regulation of plants and resulting in the growth inhibition of transgenic plants. For example, tobacco overexpressing cotton GhDREB1 enhances its resistance to low temperature but shows growth stagnation and delayed flowering. Wheat and barley overexpressing TaDREB2 and TaDREB3 improve drought and cold tolerance but exhibit slow growth, delayed flowering, and reduced seed setting. Overexpression of the Arabidopsis NAC transcription factor ATAF1 (At1g01720, ANAC002) improves drought resistance and disease resistance but causes dwarfing and shortening of the primary root. Therefore, when using transcription factors for genetic improvement, their adverse effects need to be considered. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention provides an Andrographis paniculata transcription factor ApNAC82 gene and its cloning method.

[0006] To achieve the above - mentioned object, the present invention adopts the following technical solutions:

[0007] An Andrographis paniculata transcription factor ApNAC82, the nucleotide sequence of the coding region of the Andrographis paniculata transcription factor ApNAC82 is shown in SEQ ID NO: 2. The Andrographis paniculata transcription factor ApNAC82, the nucleotide sequence of the Andrographis paniculata transcription factor ApNAC82 is shown in SEQ ID NO: 1. The application of the Andrographis paniculata transcription factor ApNAC82 in regulating plant drought resistance and high temperature tolerance, or in increasing the expression level of genes related to plant drought resistance and high temperature tolerance.

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

[0009] A recombinant vector, expression cassette, transgenic cell line, host bacterium, plasmid, Agrobacterium competent cell, containing the Andrographis paniculata transcription factor ApNAC82 nucleic acid molecule. The plasmid overexpresses the ApNAC82 gene. The Agrobacterium competent cell overexpresses the ApNAC82 gene. The application of the recombinant vector, expression cassette, transgenic cell line, host bacterium, plasmid, Agrobacterium competent cell in regulating plant drought resistance and high temperature tolerance, or in increasing the expression level of genes related to plant drought tolerance and high temperature tolerance.

[0010] The regulation is positive regulation, that is, the increase in the content of the ApNAC82 gene or ApNAC82 protein makes the drought resistance and high temperature tolerance of the plant increase. According to the nucleotide sequence of NAC82, through codon optimization or codon synonymous substitution, although the nucleotide sequences are different, the actually encoded proteins are the same and the functions are also the same.

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

[0012] Step1: Using the cDNA reverse transcribed from the total RNA of Andrographis paniculata as a template for PCR reaction to clone the coding region fragment of the Andrographis paniculata transcription factor ApNAC82,

[0013] Or using the total DNA of Andrographis paniculata as a template for PCR reaction to clone the Andrographis paniculata transcription factor ApNAC82 gene fragment;

[0014] The PCR reaction primers are ApNAC82-F and ApNAC82-R,

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

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

[0017] Step 2: Use an agarose gel recovery kit to recover the coding region fragment or gene fragment of the Andrographis paniculata transcription factor ApNAC82 from the PCR product.

[0018] In the cloning method of the Andrographis paniculata transcription factor ApNAC82, in Step 1, first take 0.5 g of Andrographis paniculata leaves and grind them in liquid nitrogen;

[0019] Then use the modified CTAB method to extract the total DNA of Andrographis paniculata leaves,

[0020] Or use a plant total RNA extraction kit DP432 to extract the total RNA of Andrographis paniculata leaves, use a micro nucleic acid and protein quantifier and 1% agarose gel electrophoresis to detect the concentration and integrity of the extracted total RNA, and use the Novoprotein HiScriptII 1st Strand cDNA Synthesis Kit R211 to reverse transcribe the qualified RNA into cDNA.

[0021] In the cloning method of the Andrographis paniculata transcription factor ApNAC82, the reaction system for the reverse transcription is shown in Table 1 of the specific implementation manner,

[0022] The reaction conditions for the first stage of the reverse transcription are denaturation at 65 °C for 5 min;

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

[0024] In the cloning method of the Andrographis paniculata transcription factor ApNAC82, in Step 1, the PCR reaction conditions are: pre-denaturation at 95 °C for 3 min; denaturation at 95 °C for 15 s; annealing at 60 °C for 20 s; extension at 72 °C for 60 s; 35 cycles of amplification; final extension at 72 °C for 10 min.

[0025] In the cloning method of the Andrographis paniculata transcription factor ApNAC82, in Step 1, in the PCR reaction system used, the reaction components and their addition amounts are shown in Table 2 of the specific implementation manner,

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

[0027] (1) According to 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 enzyme cleavage sites on the 35S:EGFP vector were selected, and specific primer sequences for homologous recombination were designed to prepare the following primers:

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

[0029] AGAACACGGGGGACGAGCTCATGAAGAAGAGTGGGGAGTTGA;

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

[0031] CCATGTCGACTCTAGAAAAGGGCTTCTGCATGTACC;

[0032] The 35S:EGFP vector is the abbreviation of the pCAMBIA1300-35S:EGFP vector;

[0033] (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 ApNAC82-EGFP-F and ApNAC82-EGFP-R as primers, the ApNAC82-EGFP insertion fragment was prepared by PCR reaction using the Novoprotein high-fidelity enzyme Phanta Max Super-Fidelity DNA Polymerase P505.

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

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

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

[0037] (4) Using the restriction enzymes SacI and XbaI from New England Biolabs, the 35S:EGFP vector was double-digested to prepare the 35S:EGFP linearized vector.

[0038] Restriction enzyme digestion reaction conditions: 37°C, 60 min; 80°C, 20 min;

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

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

[0041] (6) Using the ClonExpress Ultra One Step Cloning Kit V2, the ApNAC82-EGFP insertion fragment was ligated to the 35S:EGFP linearized vector by homologous recombination to obtain the recombinant product.

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

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

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

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

[0046] (1) According to the coding region nucleotide sequence of ApNAC82, the NdeⅠ and EcoRⅠ-HF restriction enzyme sites on the pGBKT7 vector and the BamHⅠ and PstⅠ restriction enzyme sites on the pGBKT7-VP16 vector were selected, and specific primer sequences for homologous recombination were designed, and the following primers were prepared respectively:

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

[0048] AGGAGGACCTGCATATGATGAAGAAGAGTGGGGAGTTGAACT;

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

[0050] GGATCCCCGGGAATTCTTAAAAGGGCTTCTGCATGTACCCAAAG;

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

[0052] GGTGGGCCGGGGATCATGAAGAAGAGTGGGGAGTTGAACT;

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

[0054] TAGTTATGCGGCCGCTTAAAAGGGCTTCTGCATGTACCCAAAG;

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

[0056] Using pGBKT7-ApNAC82-F and pGBKT7-ApNAC82-R as primers, prepare the pGBKT7-ApNAC82 insertion fragment by PCR reaction.

[0057] Using pGBKT7-VP16-ApNAC82-F and pGBKT7-VP16-ApNAC82-R as primers, prepare the pGBKT7-VP16-ApNAC82 fusion insertion fragment by PCR reaction;

[0058] The PCR reaction is carried out using Novoprotein's high-fidelity enzyme Phanta Max Super-Fidelity DNA Polymerase P505.

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

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

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

[0062] (3) Detect the PCR products by 1% agarose gel electrophoresis, and use the agarose gel recovery kit D2500-01 to recover the pGBKT7-ApNAC82 insertion fragment and the pGBKT7-VP16-ApNAC82 fusion insertion fragment respectively;

[0063] (4) Use the restriction enzymes NdeⅠ and EcoRⅠ-HF from New England Biolabs to double-digest the pGBKT7 vector plasmid to prepare a linearized vector;

[0064] The plasmid of pGBKT7-VP16 vector was digested with restriction enzymes BamHⅠ and PstⅠ from New England Biolabs to prepare a linearized vector;

[0065] The reaction conditions for digestion: 37°C, 60 min; 80°C, 20 min;

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

[0067] The reaction system for digesting the pGBKT7-VP16 vector plasmid is shown in Table 9 of the specific implementation manner,

[0068] (5) The digestion products were detected by 1% agarose gel electrophoresis, and the pGBKT7 linearized vector and pGBKT7-VP16 linearized vector were respectively recovered and purified using the Agarose Gel Recovery Kit D2500-01.

[0069] (6) Using the ClonExpress Ultra One Step Cloning Kit V2,

[0070] The pGBKT7-ApNAC82 insertion fragment was ligated to the pGBKT7 linearized vector by homologous recombination to obtain the first recombinant product,

[0071] The pGBKT7-VP16-ApNAC82 fusion insertion fragment was ligated to the pGBKT7-VP16 linearized vector by homologous recombination to obtain the second recombinant product;

[0072] The reaction conditions for homologous recombination were 50°C, 5 min. After the reaction ended, it was immediately placed on ice for cooling,

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

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

[0075] (7) The first and second recombinant products were respectively transformed into Escherichia coli DH5α. After screening with kanamycin antibiotic and sequencing verification, the pGBKT7-ApNAC82 expression vector and pGBKT7-VP16-ApNAC82 expression vector were respectively obtained.

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

[0077] (1) Using the heat shock method, the 35S:ApNAC82-EGFP fusion expression vector was transferred into Agrobacterium tumefaciens GV310 to obtain recombinant Agrobacterium;

[0078] (2) The recombinant Agrobacterium obtained in step (1) was resuspended with 10 mL of MgCl2 solution with a concentration of 10 mM and containing 200 μM AS, and the OD600 was adjusted to about 1.0. It was left standing at 28 °C for 2 - 3 h to obtain an infection solution;

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

[0080] (4) Through hygromycin screening and PCR identification, T3 generation homozygous positive transgenic plants were screened and obtained. The specific method is as follows:

[0081] The T0 generation regenerated plants were identified by PCR to obtain positive T0 generation transgenic plants, and the positive T0 generation transgenic plants were self-crossed to obtain T1 generation seeds;

[0082] The T1 generation seeds were screened by hygromycin and identified by PCR to obtain positive T1 generation transgenic plants, and the positive T1 generation transgenic plants were self-crossed to obtain T2 generation seeds;

[0083] The T2 generation seeds were screened by hygromycin and identified by PCR to obtain positive T2 generation transgenic plants, and the positive T2 generation transgenic plants were self-crossed to obtain T3 generation seeds;

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

[0085] In step (4), the method of PCR identification is as follows: Genomic DNA of plant leaves was extracted, and PCR amplification was carried out using the primer pair dApNAC3-F and d1300-R. If a specific amplification product was obtained, the plant was a transgenic plant;

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

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

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

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

[0090] Compared with the prior art, the beneficial effects of the present invention are:

[0091] The Andrographis paniculata transcription factor ApNAC82 can control the expression of multiple downstream stress-related genes, participate in the physiological and biochemical processes of stress resistance, improve the stress resistance of Andrographis paniculata plants to various stress conditions, and provide a new method for enhancing the stress resistance of Andrographis paniculata by means of genetic engineering. Abiotic stress has an extremely important impact on the growth, development and yield of Andrographis paniculata, and is the main limiting factor in the cultivation of Andrographis paniculata. Plants usually face complex field conditions, and various stresses do not exist alone. The research results under single stress factors are not applicable to complex field conditions. This invention is of great significance for understanding the response of Andrographis paniculata to multiple stress conditions, especially for exploring and utilizing multifunctional transcription factors that can improve various stress resistances, and for cultivating high-yield and broad-spectrum tolerant Andrographis paniculata plants. Brief Description of the Drawings

[0092] Figure 1 It is a comparative analysis diagram of the nucleotide sequence of ApNAC82 as SEQ ID NO:1 and the coding region nucleotide sequence of ApNAC82 as SE QID NO:2. The yellow box indicates the nucleotide sequence and position of the intron. Sequence 1 refers to SEQ ID NO:1, and sequence 2 refers to SEQ ID NO:2.

[0093] Figure 2 It is a comparison diagram of the amino acid sequence of ApNAC82 protein SEQ ID NO:3 and the protein sequences of members of the ATAF subfamily of Arabidopsis thaliana. The yellow underline indicates the NAM domain.

[0094] Figure 3 It is the subcellular localization of the ApNAC82 transcription factor.

[0095] Figure 4 It is the analysis result of the transcriptional activation activity of ApNAC82.

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

[0097] Figure 6 It is the 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#A pNAC82-15, and WT is the wild type.

[0098] Figure 7 It is the growth of the wild type WT and ApNAC82 overexpression lines under normal growth conditions.

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

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

[0101] Figure 10 Statistical results of the germination rate of wild-type WT and ApNAC82 overexpression lines under different concentrations of mannitol treatment.

[0102] Figure 11 Statistical results of the root length of wild-type WT and ApNAC82 overexpression lines under different concentrations of mannitol treatment.

[0103] Figure 12 Growth conditions of wild-type WT and ApNAC82 overexpression lines after natural drought stress.

[0104] Figure 13 Recovery conditions of wild-type WT and ApNAC82 overexpressing line seedlings after heat stress.

[0105] Figure 14 Growth conditions of wild-type WT and ApNAC82 overexpressing lines after heat stress. Detailed implementation manners

[0106] The technical solutions of the present invention will be further elaborated below through examples.

[0107] Example 1

[0108] 1. Cloning of the ApNAC82 sequence

[0109] (1.1) Take 1.0 g of Andrographis paniculata leaves, grind them in liquid nitrogen, and divide them into two portions. One portion is used to extract the total DNA of Andrographis paniculata leaves, and the other portion is used to extract the total RNA of Andrographis paniculata leaves;

[0110] (1.2) Extract the total DNA of Andrographis paniculata leaves using the modified CTAB method. Use spectrophotometry to measure the quality and concentration of the extracted total DNA of Andrographis paniculata leaves. The OD260 / 280 is 1.9, and the concentration is 276 ng / μL, indicating that the extracted DNA has qualified quality and can be used for subsequent experiments.

[0111] (1.3) The total RNA of Andrographis paniculata leaves was extracted using a plant total RNA extraction kit (DP432). The concentration and integrity of the extracted total RNA of Andrographis paniculata leaves were detected using a micro nucleic acid and protein quantifier and 1% agarose gel electrophoresis. The qualified RNA was reverse transcribed into cDNA using the Novoprotein HiScript II 1st Strand cDNA Synthesis Kit (R211). The reaction system for reverse transcription is as follows:

[0112] Table 1. Reaction system for reverse transcription

[0113]

[0114]

[0115] The reaction conditions for the first stage of reverse transcription were denaturation at 65 °C for 5 min;

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

[0117] (1.4) According to the genomic information of Andrographis paniculata (SRP143459), specific primer sequences for amplifying the gene fragment of the Andrographis paniculata transcription factor ApNAC82 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 the extracted DNA and the reverse transcribed cDNA as templates, and ApNAC82-F / ApNAC82-R as primers, the Novoprotein high-fidelity enzyme Phanta Max Super-Fidelity DNA Polymerase (P505) was used for PCR reaction to clone the gene fragment and coding region fragment of the Andrographis paniculata transcription factor ApNAC82. The PCR reaction conditions were: pre-denaturation at 95 °C for 3 min; denaturation at 95 °C for 15 s; annealing at 60 °C for 20 s; extension at 72 °C for 60 s; 35 cycles of amplification; final extension at 72 °C 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 fragments were recovered and purified using an agarose gel recovery kit (D2500-01).

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

[0126] 2. Sequence analysis of ApNAC82 sequence

[0127] Sequence alignment was performed using the MAFFT software. The results showed that in the Andrographis paniculata genomic DNA, the full-length ApNAC82 gene sequence was 1076 bp, as shown in the nucleotide sequence of the coding region of ApNAC82, SEQ ID NO:1; the full-length coding region sequence of the ApNAC82 gene was 897 bp, as shown in the nucleotide sequence of the coding region of ApNAC82, SEQ ID NO:2; and it encoded 298 amino acids, as shown in Sequence 3. After sequence alignment and analysis, the results were as Figure 1 shown. The ApNAC82 gene included 3 exons and 2 introns. The exons were, in turn: nucleotides 1-165, 257-549, and 638-1076 of the reading frame.

[0128] The conserved domain of the ApNAC82 protein was predicted using NCBI Conserved Domain Search. ApNAC82 had a typical NAM domain between amino acids 10-133, and the family classification was the ATAF class. Homologous protein sequences (AtNAC002, AtNAC032, AtNAC081, AtNAC102) of the ATAF family members of Arabidopsis thaliana NAC transcription factors were obtained from the NCBI database, and then protein alignment was performed using the MAFFT software. From Figure 2 it could be seen that the protein sequences of ApNAC82 and the members of the Arabidopsis thaliana ATAF family were more conserved at the N-terminus, both containing the conserved NAM domain, while there were significant differences in the sequences at the C-terminus, indicating that ApNAC82 had undergone adaptive changes during natural selection and evolution. The non-conservatism of this protein at the C-terminus endows it with different functions and characteristics.

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

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

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

[0132] 3. Subcellular localization analysis of ApNAC82

[0133] (3.1) In this experiment, the green fluorescent protein EGFP was selected as the reporter gene. The coding sequence of EGFP was ligated to the coding sequence of the ApNAC82 protein to form a fusion protein. Then, by transfecting the fusion protein into protoplast cells for expression, the fluorescence signal of EGFP would co-localize with the ApNAC82 protein, thereby observing the subcellular localization of the ApNAC82 protein. In this experiment, the known nuclear localization protein NLS-mKATE was also selected as a control.

[0134] (3.2) The plant expression vector used in this experiment is the pCAMBIA1300-35S:EGFP vector, hereinafter referred to as 35S:EGFP. According to the nucleotide sequence SEQ ID NO:2 of the coding region of ApNAC82, the stop codon TGA was removed, and the ApNAC82-EGFP fusion protein was constructed. The specific primer sequences for homologous recombination were designed by selecting the SacI and XbaI restriction enzyme sites on the 35S:EGFP vector, and 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 SEQ ID NO:2 of the coding region of ApNAC82 as a template, and ApNAC82-EGFP-F / ApNAC82-EGFP-R as primers, the insertion fragment of ApNAC82-EGFP was prepared by PCR reaction using the Novoprotein high-fidelity enzyme Phanta Max Super-Fidelity DNA Polymerase (P505). PCR reaction conditions: 95°C, pre-denaturation for 3 min; 95°C, denaturation for 15 s; 60°C, annealing for 20 s; 72°C, extension for 90 s; 35 cycles of amplification; 72°C, 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 insertion fragment

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

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

[0143] (3.5) Using the restriction enzymes SacI and XbaI from New England Biolabs, the 35S:EGFP vector plasmid was double-digested to prepare a linearized vector, and the 35S:EGFP linearized vector was recovered and purified using an agarose gel recovery kit (D2500-01). Restriction enzyme reaction conditions: 37°C, 60 min; 80°C, 20 min. The restriction enzyme reaction system is shown in Table 4.

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

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

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

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

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

[0149] (3.7) The recombinant product was transformed into Escherichia coli DH5α, and the 35S:ApNAC82-EGFP fusion expression vector was obtained through screening with kanamycin antibiotic and sequencing verification.

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

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

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

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

[0154] The results were as Figure 3 shown. Under the EGFP fluorescence channel, the green fluorescence signal of tobacco protoplast cells co-transformed with NLS-mKATE + 35S:EGFP could be clearly observed to be distributed throughout the whole cell, such as in the nucleus, cell membrane, and cytoplasm, while the green fluorescence signal of tobacco protoplast cells co-transformed with NLS-mKATE + 35S:ApNAC82-EGFP was only distributed in the nucleus.

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

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

[0157] In the bright field channel, there is no fluorescence in tobacco protoplast cells co-transformed with NLS-mKATE + 35S:EGFP and NLS-mKATE + 35S:ApNAC82-EGFP.

[0158] In the overlay 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 nucleus, is a nuclear transcription factor, and plays a transcriptional regulatory role in the nucleus.

[0160] 4. Analysis of ApNAC82 transcriptional activity

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

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

[0163] AGGAGGACCTGCATATGATGAAGAAGAGTGGGGAGTTGAACT;

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

[0165] GGATCCCCGGGAATTCTTAAAAGGGCTTCTGCATGTACCCAAAG;

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

[0167] GGTGGGCCGGGGATCATGAAGAAGAGTGGGGAGTTGAACT;

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

[0169] TAGTTATGCGGCCGCTTAAAAGGGCTTCTGCATGTACCCAAAG。

[0170] (4.2) Using the pCE2_TA-Blunt-Zero-ApNAC82 recombinant vector pCE2_TA-Blunt-Zero containing the nucleotide sequence SEQ ID NO:2 of the coding region of ApNAC82 as a template, and using pGBKT7-ApNAC82-F / pGBKT7-ApNAC82-R, pGBKT7-VP16-ApNAC82-F / pGBKT7-VP16-ApNAC82-R as primers respectively, perform PCR reactions using Novoprotein's high-fidelity enzyme Phanta Max Super-Fidelity DNA Polymerase (P505) to prepare the pGBKT7-ApNAC82 insertion fragment and the pGBKT7-VP16-ApNAC82 fusion insertion fragment respectively. PCR reaction conditions: 95°C, pre-denaturation for 3 min; 95°C, denaturation for 15 s; 60°C, annealing for 20 s; 72°C, extension for 90 s; amplify for 35 cycles; 72°C, final extension for 10 min. The PCR reaction systems are shown in Tables 6 and 7.

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

[0172]

[0173]

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

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

[0176] (4.3) Detect the PCR products by 1% agarose gel electrophoresis, and use an agarose gel recovery kit (D2500-01) to recover the pGBKT7-ApNAC82 insertion fragment and the pGBKT7-VP16-ApNAC82 fusion insertion fragment respectively.

[0177] (4.4) Use the restriction enzymes NdeⅠ and EcoRⅠ-HF from New England Biolabs to double-digest the pGBKT7 vector plasmid to prepare a linearized vector; use the restriction enzymes BamHⅠ and PstⅠ from New England Biolabs to double-digest the pGBKT7-VP16 vector plasmid to prepare a linearized vector. Restriction enzyme reaction conditions: 37°C, 60 min; 80°C, 20 min. The restriction enzyme reaction systems are shown in Tables 8 - 9.

[0178] Table 8. Reaction system for digesting pGBKT7 vector plasmid

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

[0180] Table 9. Reaction system for digesting pGBKT7-VP16 vector plasmid

[0181]

[0182]

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

[0184] (4.6) Using the ClonExpress Ultra One Step Cloning Kit V2, the pGBKT7-ApNAC82 insert fragment was respectively ligated to the pGBKT7 linearized vector by homologous recombination to obtain recombinant product one, and the pGBKT7-VP16-ApNAC82 fusion insert fragment was ligated to the pGBKT7-VP16 linearized vector by homologous recombination to obtain recombinant product two. The reaction conditions for homologous recombination were 50 °C for 5 min, and immediately placed on ice for cooling after the reaction. The recombinant reaction systems are shown in Tables 10 - 11.

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

[0186] Reaction components Dosage pGBKT7 linearized vector 2 μL pGBKT7-ApNAC82 insertion fragment 3 μL 2×CE Mix 5 μL

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

[0188] Reaction components Dosage pGBKT7-VP16 linearized vector 2 μL pGBKT7-VP16-ApNAC82 fusion insertion fragment 3 μL 2×CE Mix 5 μL

[0189] (4.7) The recombinant product one and recombinant product two were respectively transformed into Escherichia coli DH5α, and after screening with kanamycin antibiotic and sequencing verification, the pGBKT7-ApNAC82 expression vector and pGBKT7-VP16-ApNAC82 expression vector were respectively obtained.

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

[0191] (4.9) Transcription activity detection

[0192] a. Respectively pick the positive monoclonal colonies obtained from the transformation in (4.8) and culture them in SD / -Trp liquid medium to adjust the OD600 to 0.2.

[0193] b. Dilute them 10-fold, 100-fold, and 1000-fold successively with ddH20, i.e., OD600 = 0.02, 0.002, 0.0002.

[0194] c. Respectively take 5 μL and spot plate them on SD / -Trp, SD / -His / -Trp, and SD / -Ade / -His / -Trp plates.

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

[0196] The results are as Figure 4 shown. Yeast transformed with pGBKT7, yeast transformed with pGBKT7-ApNAC82, yeast transformed with pGBKT7-VP16, and yeast transformed with pGBKT7-VP16-ApNAC82 can all grow normally on the SD / -Trp medium, indicating successful yeast transformation.

[0197] On the SD / -Trp / -His and SD / -Trp / -His / Ade media, yeast transformed with pGBKT7 cannot grow, while yeast transformed with pGBKT7-ApNAC82 can grow normally, indicating that the HIS3 and ADE2 reporter genes in yeast transformed with pGBKT7-ApNAC82 are activated and expressed.

[0198] VP16 is a transcriptional activator. In yeast transformed with pGBKT7-VP16, the transcriptional activation effect of VP16 will activate the expression of the reporter genes HIS3 and ADE2 in yeast, and then yeast transformed with pGBKT7-VP16 can grow normally on the SD / -Trp / -His and SD / -Trp / -His / Ade media.

[0199] Compared with VP16, the yeast transformed with pGBKT7-VP16-ApNAC82 grew better than the yeast transformed with pGBKT7-VP16 on SD / -Trp / -His and SD / -Trp / -His / Ade media, indicating that the fusion expression of ApNAC82 and VP16 enhanced the transcriptional activation activity of VP16.

[0200] The above experimental results indicate that ApNAC82 has transcriptional activation activity.

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

[0202] (5.1) Select Andrographis paniculata plants with consistent growth and normal growth for 60 days for drought and high temperature stress treatments. For drought stress, the natural drought method was used, and according to the relative water content of the leaves, normal growth (90.45%), mild drought stress (80.81%), and severe drought stress (57.54%) were set respectively; for high temperature stress, a treatment at 50 °C was used, and 0 h (normal growth), 3 h, and 6 h were set respectively. Each treatment had 3 replicates, and each replicate had 5 plants. After the stress treatment was completed, Andrographis paniculata leaves were collected and immediately frozen in liquid nitrogen and stored at -80 °C for later use.

[0203] (5.2) For each of the 5 Andrographis paniculata plants in each replicate treatment above, 0.5 g of leaves were taken and mixed, ground in liquid nitrogen, and the total RNA of Andrographis paniculata leaves was extracted using a plant total RNA extraction kit (DP432); the purity and concentration of the extracted RNA were detected using a NanoDrop 2000 spectrophotometer; the integrity of the RNA was detected using a LabChip GX.

[0204] (5.3) The qualified RNA was used to construct an RNA-seq library; the qualified library was sequenced in PE150 mode using an Illumina NovaSeq6000 sequencing platform; the downloaded data was filtered to obtain Clean Data, and sequence alignment was performed according to the genomic information of Andrographis paniculata (SRP143459). After obtaining Mapped Data, library quality assessment was carried out; StringTie was used to perform normalization through the maximum flow algorithm using FPKM (Fragments Per Kilobase of transcript per Million fragments mapped) as an index to measure the gene expression level.

[0205] From Figure 5It can be seen that under drought stress, the expression of ApNAC82 showed an upward trend with the intensification of drought stress; under mild drought stress, the expression of ApNAC82 was up-regulated 4.79-fold compared with normal growth, and under severe drought stress, the expression of ApNAC82 was further increased, which was 9.61-fold that of normal growth.

[0206] Under heat stress, the expression of ApNAC82 showed an upward trend with the prolongation of heat stress. When heat stress lasted for 3 h, the expression of ApNAC82 was up-regulated 5.18-fold compared with normal growth. When heat stress continued for 6 h, the expression of ApNAC82 was up-regulated 9.51-fold compared with normal growth. The above results indicate that ApNAC82 responds to the induction of both drought and heat stress with up-regulated expression.

[0207] 6. Functional analysis of ApNAC82 gene

[0208] 6.1 Obtaining ApNAC82 gene overexpression plants

[0209] (6.1.1) By using the heat shock method, the recombinant plasmid 35S:ApNAC82-EGFP constructed in the above-mentioned 3. Subcellular localization analysis of ApNAC82 was transferred into Agrobacterium tumefaciens GV310 to obtain recombinant Agrobacterium.

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

[0211] (6.1.3) By using the leaf disc method, transform the leaves of Nicotiana benthamiana, and through co-culture, hygromycin resistance screening, differentiation, and rooting, obtain T0 generation regenerated plants.

[0212] (6.1.4) Through hygromycin screening and PCR identification, screen and obtain T3 generation homozygous positive transgenic plants. The specific method is as follows:

[0213] The T0 generation regenerated plants were identified by PCR to obtain positive T0 generation transgenic plants, and the positive T0 generation transgenic plants were self-crossed to obtain T1 generation seeds;

[0214] The T1 generation seeds were screened by hygromycin and identified by PCR to obtain positive T1 generation transgenic plants, and the positive T1 generation transgenic plants were self-crossed to obtain T2 generation seeds;

[0215] The T2 generation seeds were screened by hygromycin and identified by PCR to obtain positive T2 generation transgenic plants, and the positive T2 generation transgenic plants were self-crossed to obtain T3 generation seeds. For the T2 generation plants, if all the T3 generation plants obtained from this line are positive transgenic plants, then the self-crossed offspring of this line are all homozygous transgenic lines.

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

[0217] In step (6.1.4), the method for PCR identification is as follows: Extract the genomic DNA of plant leaves, perform PCR amplification using the primer pair dApNAC3-F and d1300-R. 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°C, pre-denaturation for 3 min; 95°C, denaturation for 15 s; 60°C, annealing for 20 s; 72°C, extension for 20 s; 35 cycles of amplification; 72°C, 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 Figure 6 shown. The genomic DNA of T3 generation transgenic plants was amplified by PCR method. An amplification product with an expected size of 190 bp was obtained in transgenic positive plants, while the corresponding fragment was not detected in wild-type plants, indicating that the ApNAC82 gene has been successfully integrated into Nicotiana benthamiana. Through hygromycin screening and PCR identification, OE#ApNAC82-6, OE#ApNAC82-9, OE#ApNAC82-10, and OE#ApNAC82-11 are all T3 generation homozygous transgenic lines.

[0225] 6.2 Effects of overexpressing ApNAC82 on tobacco growth

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

[0227] (6.2.2) Sow the tested tobacco seeds in planting pots filled with nutrient soil, place them in an incubator at 25 °C, and cultivate them under the conditions of 16 h light / 8 h darkness, with normal watering during the period;

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

[0229] (6.2.4) After 4 weeks of cultivation, judge the effect of overexpressing ApNAC82 on tobacco growth by comparing the growth vigor and plant height of the wild type WT.

[0230] Overexpression of the Arabidopsis ATAF gene usually causes adverse growth reactions in transgenic plants such as dwarfing and weakness of the plants and shortening of the root system. In 1.1, sequence alignment analysis found that ApNAC82 belongs to the ATAF gene family. Compared with the homologous genes of the Arabidopsis ATAF family, the N-terminal structure is relatively conserved, while there are significant differences in the C-terminal sequences. The non-conservatism of the protein C-terminal end endows it with different functions and characteristics. Therefore, it is necessary to explore whether overexpression of ApNAC82 will cause dwarfing and weakness to determine whether there are adverse reactions in the application of ApNAC82 in crop genetic improvement. As Figure 7 shown, compared with the wild type WT, the three overexpressing ApNAC82 lines OE#ApNAC82-6, OE#ApNAC82-9, and OE#ApNAC82-11 grow vigorously and do not show obvious dwarfing symptoms. Therefore, overexpression of ApNAC82 will not cause adverse reactions of dwarfing and weakness to Nicotiana benthamiana.

[0231] 6.3 Drought resistance identification of ApNAC82-overexpressing tobacco

[0232] 6.3.1 Mannitol simulated drought stress treatment

[0233] (6.3.1.1) Tested seeds: Seeds of Nicotiana benthamiana wild type WT and the 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 media 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 s, rinse with sterile water 3 times; then add 1 mL of freshly prepared 12% sodium hypochlorite solution and disinfect for 15 min, rinse with sterile water 5 times;

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

[0237] (6.3.1.5) After spot-sowing, seal it with a sealing film and place it in an incubator at 25 °C, with a light intensity of 8000 Lx, 16 h of light and 8 h of darkness. Observe the growth trend after emergence.

[0238] (6.3.1.6) Germination rate detection: After culturing for 5 days, observe the growth trends of the wild type and transgenic lines on the 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 sown seeds) × 100%

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

[0241] The results are as Figures 8 - 11 shown. On the MS medium without mannitol (0 mM), there were no significant differences in the germination rates and root lengths of the wild type WT and the three transgenic lines.

[0242] Under the treatment of 100 mM mannitol, the germination rate of the wild type WT was 59.43%, and the average root lengths were 0.33 cm. While 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 the treatment of 200 mM mannitol, the germination rate of the wild type WT was 13.17%, and the average root length was 0.13 cm. While 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 the treatment of 300 mM mannitol, 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 Natural drought stress treatment

[0247] (6.3.2.1) Test seeds: Seeds of Nicotiana benthamiana wild type WT and seeds of 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) Sow the test tobacco seeds in planting pots filled with nutrient soil, place them in an incubator at 25 °C, and culture them under the conditions of 16 h light / 8 h darkness, and water normally during the period.

[0249] (6.3.2.3) Select plants with good growth status, consistent growth trend, and a growth cycle of 4 weeks for the drought treatment experiment. Before the experiment starts, stop watering after sufficient irrigation for natural drought stress treatment to keep the soil moisture content consistent. On the 12th day of the drought treatment, observe the growth trend of the plants.

[0250] The results are as Figure 12 shown. After 12 days of drought treatment, the leaves of wild type WT plants showed obvious wilting physiological phenomena, with the lower leaves dry, wrinkled, wilted, and yellowed, while the transgenic plants showed relatively mild wilting symptoms, with a better growth trend than wild type WT plants, and the degree of leaf wilting was significantly less than that of wild type WT plants. These results indicate that overexpression of ApNAC82 can improve the drought tolerance of Nicotiana benthamiana.

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

[0252] 6.4.1 Identification of the recovery ability of seedlings after high-temperature stress treatment

[0253] (6.4.1.1) Test seeds: Seeds of Nicotiana benthamiana wild type WT and seeds of 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: Place the tobacco seeds in a 1.5 mL centrifuge tube, add 1 mL of 70% alcohol and soak for 60 s, then rinse with sterile water 3 times; then add 1 mL of freshly prepared 12% sodium hypochlorite solution and disinfect for 15 min, and rinse with sterile water 5 times.

[0255] (6.4.1.3) Add 500 μL of 0.1% water agar to the centrifuge tube to suspend the seeds, and then spot the seeds on the MS medium with grids. Sow 4 lines on each plate.

[0256] (6.4.1.4) After spotting, seal with a sealing film and place it in an incubator at 25 °C, with a light intensity of 8000 Lx, 16 h of light, and 8 h of darkness for culture.

[0257] (6.4.1.5) After 2 weeks of cultivation, the Petri dishes were treated at a high temperature of 45 °C for 12 h, and then placed at 25 °C to resume growth for 2 d. The high-temperature tolerance was judged according to the re-greening of the leaves.

[0258] The results are as Figure 13 shown. High-temperature stress can cause the chlorophyll in the leaves to be damaged and turn white. The leaves of the wild type WT and transgenic lines were all restored to varying degrees after high-temperature stress. Only a small number of seedlings in the wild type WT had their leaves re-greened, while the probability of re-greening of the transgenic seedlings was significantly higher than that of the wild type WT.

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

[0260] (6.4.2.1) Test seeds: Seeds of Nicotiana benthamiana wild type WT and seeds of 3 T3-generation homozygous transgenic lines OE#ApNAC82-9 and OE#ApNAC82-11 obtained in step 6.1.

[0261] (6.4.2.2) Sow the test tobacco seeds in planting pots filled with nutrient soil, place them in an incubator at 25 °C, and culture them under the conditions of 16 h light / 8 h darkness, and water normally during the period;

[0262] (6.4.2.3) Select plants with good growth status, consistent growth vigor, and a growth period of 4 weeks for high-temperature stress treatment at 45 °C for 24 h, and observe the growth vigor of the plants.

[0263] The results are as Figure 14 shown. After high-temperature stress treatment, the leaves of the wild type WT plants showed obvious wilting physiological phenomena, while only the lower leaves of the transgenic plants showed relatively slight wilting symptoms. These results indicate that overexpression of ApNAC82 can improve the high-temperature tolerance of Nicotiana benthamiana.

Claims

1. A Andrographis paniculata transcription factor ApNAC82, characterized in that, The nucleotide sequence of the coding region of the Andrographis paniculata transcription factor ApNAC82 is shown in SEQ ID NO:

2.

2. The Andrographis paniculata transcription factor ApNAC82 according to claim 1, characterized in that The nucleotide sequence of the Andrographis paniculata transcription factor ApNAC82 is shown in SEQ ID NO:

1.

3. A protein encoded by the andrographis paniculata transcription factor ApNAC82 as described in claim 1 or 2, characterized in that, The amino acid sequence of the ApNAC82 protein is shown in SEQ ID NO:

3.

4. A recombinant vector, expression cassette, transgenic cell line, host bacterium, characterized in that, Containing the Andrographis paniculata transcription factor ApNAC82 nucleic acid molecule.

5. The application of the ApNAC82 gene, the ApNAC82 protein, and the recombinant vector, expression cassette, transgenic cell line, and host bacterium containing the ApNAC82 gene or the ApNAC82 protein in regulating plant drought resistance and high-temperature tolerance, or in increasing the expression level of genes related to plant drought resistance and high-temperature tolerance.

6. A cloning method for the Andrographis paniculata transcription factor ApNAC82, characterized in that, Comprising the following steps: Step1: Using the cDNA reverse-transcribed from the total RNA of Andrographis paniculata as a template, perform a PCR reaction to clone the coding region fragment of the Andrographis paniculata transcription factor ApNAC82, or using the total DNA of Andrographis paniculata as a template, perform a PCR reaction to clone the gene fragment of the Andrographis paniculata transcription factor ApNAC82; The PCR reaction primers are ApNAC82-F and ApNAC82-R, The sequence of ApNAC82-F is: ATGAAGAAGAGTGGGGAGTTGA; The sequence of ApNAC82-R is: TCAAAAGGGCTTCTGCATGTACC; Step2: Use an agarose gel recovery kit to recover the coding region fragment or gene fragment of the Andrographis paniculata transcription factor ApNAC82 from the PCR product.

7. The cloning method of the Andrographis paniculata transcription factor ApNAC82 according to claim 3, characterized in that, In Step1, first take 0.5 g of Andrographis paniculata leaves and grind them in liquid nitrogen; Then use the modified CTAB method to extract the total DNA of Andrographis paniculata leaves, or use the plant total RNA extraction kit DP432 to extract the total RNA of Andrographis paniculata leaves, use a micro nucleic acid and protein quantifier and 1% agarose gel electrophoresis to detect the concentration and integrity of the extracted total RNA, and use the Novizan HiScript II 1st Strand cDNA Synthesis Kit R211 kit to reverse-transcribe the qualified RNA into cDNA; The reaction system of the reverse transcription is shown in Table 1, Table 1. Reaction system of reverse transcription The reaction conditions for the first stage of the reverse transcription are denaturation at 65 °C for 5 min; The reaction conditions for the second stage of the reverse transcription are 25 °C for 5 min; 50 °C for 45 min; 85 °C for 2 min; In Step1, the PCR reaction conditions are: pre-denaturation at 95 °C for 3 min; denaturation at 95 °C for 15 s; annealing at 60 °C for 20 s; extension at 72 °C for 60 s; 35 cycles of amplification; final extension at 72 °C for 10 min. In the PCR reaction system used in Step1, the reaction components and their addition amounts are shown in Table 2, Table 2. PCR reaction system for amplifying the Andrographis paniculata transcription factor ApNAC82 8. A method for preparing a 35S:ApNAC82-EGFP fusion expression vector, characterized in that, Comprising the following steps: (1) According to the nucleotide sequence of the coding region of ApNAC82, removing the stop codon TGA, an ApNAC82-EGFP fusion protein was constructed. The SacI and XbaI restriction enzyme sites on the 35S:EGFP vector were selected, and specific primer sequences for homologous recombination were designed to prepare the following primers: The sequence of ApNAC82-EGFP-F is: AGAACACGGGGGACGAGCTCATGAAGAAGAGTGGGGAGTTGA; The sequence of ApNAC82-EGFP-R is: CCATGTCGACTCTAGAAAAGGGCTTCTGCATGTACC; The 35S:EGFP vector is the abbreviation of the 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 ApNAC82-EGFP-F and ApNAC82-EGFP-R as primers, the Phanta Max Super-Fidelity DNA Polymerase P505 from Novoprotein was used for PCR reaction to prepare the insertion fragment of ApNAC82-EGFP; PCR reaction conditions: 95°C, pre-denaturation for 3 min; 95°C, denaturation for 15 s; 60°C, annealing for 20 s; 72°C, extension for 90 s; 35 cycles of amplification; 72°C, final extension for 10 min; The PCR reaction system is shown in Table 3, Table 3. PCR reaction system for amplifying the ApNAC82-EGFP insertion fragment (3) The PCR product was detected by 1% agarose gel electrophoresis, and the ApNAC82-EGFP insertion fragment was recovered using the Agarose Gel DNA Purification Kit D2500-01; (4) Using the restriction enzymes SacI and XbaI from New England Biolabs, the 35S:EGFP vector was double-digested to prepare the 35S:EGFP linearized vector, Restriction enzyme reaction conditions: 37°C, 60 min; 80°C, 20 min; The restriction enzyme reaction system is shown in Table 4, Table 4. Reaction system for digesting the 35S:EGFP vector plasmid (5) The digested product was detected by 1% agarose gel electrophoresis, and the 35S:EGFP linearized vector was recovered and purified using the Agarose Gel DNA Purification Kit D2500-01; (6) Using the ClonExpress Ultra One Step Cloning Kit V2, the ApNAC82-EGFP insertion fragment was ligated to the 35S:EGFP linearized vector by homologous recombination to obtain the recombinant product; The reaction conditions for homologous recombination were 50°C, 5 min, and immediately placed on ice for cooling after the reaction; The homologous recombination reaction system is shown in Table 5, Table 5. Homologous recombination reaction system for constructing the 35S:ApNAC82-EGFP fusion expression vector (7) Transform the recombinant product into Escherichia coli DH5α, and obtain the 35S:ApNAC82-EGFP fusion expression vector through kanamycin antibiotic screening and sequencing verification.

9. A method for preparing an expression vector containing an ApNAC82 gene fragment, characterized in that, The steps include: (1) According to the nucleotide sequence of the coding region of ApNAC82, select the NdeⅠ and EcoRⅠ-HF restriction enzyme sites on the pGBKT7 vector, and the BamHⅠ and PstⅠ restriction enzyme sites on the pGBKT7-VP16 vector, design the specific primer sequences for homologous recombination, and prepare the following primers respectively: The sequence of pGBKT7-ApNAC82-F is (5'-3'): AGGAGGACCTGCATATGATGAAGAAGAGTGGGGAGTTGAACT; The sequence of pGBKT7-ApNAC82-R is (5'-3'): GGATCCCCGGGAATTCTTAAAAGGGCTTCTGCATGTACCCAAAG; The sequence of pGBKT7-VP16-ApNAC82-F is (5'-3'): GGTGGGCCGGGGATCATGAAGAAGAGTGGGGAGTTGAACT; The sequence of pGBKT7-VP16-ApNAC82-R is (5'-3'): TAGTTATGCGGCCGCTTAAAAGGGCTTCTGCATGTACCCAAAG; (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, Using pGBKT7-ApNAC82-F and pGBKT7-ApNAC82-R as primers, prepare the pGBKT7-ApNAC82 insertion fragment by PCR reaction, Using pGBKT7-VP16-ApNAC82-F and pGBKT7-VP16-ApNAC82-R as primers, prepare the pGBKT7-VP16-ApNAC82 fusion insertion fragment by PCR reaction; The PCR reaction is carried out using the Novoprotein high-fidelity enzyme 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; amplify for 35 cycles; 72℃, final extension for 10 min, The PCR reaction system for amplifying the pGBKT7-ApNAC82 insertion fragment is shown in Table 6, The PCR reaction system for amplifying the pGBKT7-VP16-ApNAC82 fusion insertion fragment is shown in Table 7, Table 6. PCR reaction system for amplifying the pGBKT7-ApNAC82 insertion fragment Table 7. PCR reaction system for amplifying the pGBKT7-VP16-ApNAC82 fusion insertion fragment (3) The PCR products were detected by 1% agarose gel electrophoresis, and the pGBKT7-ApNAC82 insertion fragment and the pGBKT7-VP16-ApNAC82 fusion insertion fragment were respectively recovered using the Agarose Gel DNA Purification Kit D2500-01. (4) The pGBKT7 vector plasmid was double-digested with the restriction enzymes NdeⅠ and EcoRⅠ-HF from New England Biolabs to prepare a linearized vector. The pGBKT7-VP16 vector plasmid was double-digested with the restriction enzymes BamHⅠ and PstⅠ from New England Biolabs to prepare a linearized vector. The digestion reaction conditions were: 37°C, 60 min; 80°C, 20 min. The reaction system for digesting the pGBKT7 vector plasmid is shown in Table 8. The reaction system for digesting the pGBKT7-VP16 vector plasmid is shown in Table 9. Table 8. Reaction system for digesting the pGBKT7 vector plasmid Table 9. Reaction system for digesting the pGBKT7-VP16 vector plasmid (5) The digestion products were detected by 1% agarose gel electrophoresis, and the purified pGBKT7 linearized vector and pGBKT7-VP16 linearized vector were respectively recovered using the Agarose Gel DNA Purification Kit D2500-01. (6) Using the ClonExpress Ultra One Step Cloning Kit V2. The pGBKT7-ApNAC82 insertion fragment was ligated to the pGBKT7 linearized vector by homologous recombination to obtain recombinant product I. The pGBKT7-VP16-ApNAC82 fusion insertion fragment was ligated to the pGBKT7-VP16 linearized vector by homologous recombination to obtain recombinant product II. The reaction conditions for homologous recombination were 50°C, 5 min. After the reaction ended, it was immediately placed on ice for cooling. The homologous recombination reaction system for constructing the pGBKT7-ApNAC82 expression vector is shown in Table 10. The homologous recombination reaction system for constructing the pGBKT7-VP16-ApNAC82 expression vector is shown in Table 11. Table 10. Homologous recombination reaction system for constructing the pGBKT7-ApNAC82 expression vector Table 11. Homologous recombination reaction system for constructing the pGBKT7-VP16-ApNAC82 expression vector (7) The recombinant product I and recombinant product II were respectively transformed into Escherichia coli DH5α. After screening with kanamycin and sequencing verification, the pGBKT7-ApNAC82 expression vector and the pGBKT7-VP16-ApNAC82 expression vector were respectively obtained.

10. A method for preparing a plant overexpressing the ApNAC82 gene, characterized in that, It includes the following steps: (1) Using the heat shock method, the 35S:ApNAC82-EGFP fusion expression vector was transferred into Agrobacterium tumefaciens GV310 to obtain recombinant Agrobacterium. (2) The recombinant Agrobacterium obtained in step (1) was resuspended with 10 mL of MgCl2 solution with a concentration of 10 mM and containing 200 μM AS, and the OD600 was adjusted to about 1.

0. It was left standing at 28°C for 2 - 3 h to obtain an infection solution. (3) The leaf disc method was adopted to transform the leaves of the plants. After co-cultivation, hygromycin resistance screening, differentiation, and rooting, the T0 generation of regenerated plants was obtained; (4) Through hygromycin screening and PCR identification, the T3 generation of homozygous positive transgenic plants was screened and obtained. The specific method is as follows: The regenerated plants of the T0 generation were identified by PCR to obtain the positive transgenic plants of the T0 generation. The positive transgenic plants of the T0 generation were self-crossed to obtain the seeds of the T1 generation; The seeds of the T1 generation were screened by hygromycin and identified by PCR to obtain the positive transgenic plants of the T1 generation. The positive transgenic plants of the T1 generation were self-crossed to obtain the seeds of the T2 generation; The seeds of the T2 generation were screened by hygromycin and identified by PCR to obtain the positive transgenic plants of the T2 generation. The positive transgenic plants of the T2 generation were self-crossed to obtain the seeds of the T3 generation; In step (4), the hygromycin screening concentration was 50 mg / L; In step (4), the method of PCR identification was as follows: The genomic DNA of the plant leaves was extracted, and PCR amplification was carried out using the primer pair of dApNAC3-F and d1300-R. If a specific amplification product was obtained, the plant was a transgenic plant; The sequence of dApNAC82-F is: TCCATTACTACGACAATTCCGGC; The sequence of d1300-R is: TCAAAAGGGCTTCTGCATGTACC; PCR reaction conditions: 95 °C, pre-denaturation for 3 min; 95 °C, denaturation for 15 s; 60 °C, annealing for 20 s; 72 °C, extension for 20 s; 35 cycles of amplification; 72 °C, final extension for 10 min; The PCR reaction system is shown in Table 12, Table 12. PCR reaction system for detecting positive transgenic plants

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