Cloning of a gene of sophora alopecuroides saathb7 and application thereof

Abstract

This invention relates to the field of plant genetic engineering technology and provides a bitter bean. SaATHB7 The cloning and application of genes involved transcriptome sequencing of *Bletilla striata* seedlings subjected to simulated abiotic stress. Differentially expressed genes were analyzed to screen for *Bletilla striata* genes associated with salt stress. The sequencing results revealed that these genes likely belong to the homologous domain leucine zipper family of transcription factors, and they were named... SaATHB7 Cloning and quantitative primers were designed based on the sequence obtained from sequencing. RT-PCR was used to detect the expression changes of this gene in different tissues of Sophora flavescens under abiotic stress conditions, in order to preliminarily study the corresponding role of this gene in abiotic stress of Sophora flavescens. At the same time, a plant overexpression vector of this gene was constructed and successfully transformed into wild-type Arabidopsis thaliana for preliminary functional verification. The results showed that overexpression of this gene in Arabidopsis thaliana can improve the salt tolerance and drought tolerance of Arabidopsis thaliana.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, and in particular relates to the cloning of the SaATHB7 gene of bitter bean and its application. Background Technology

[0002] Soil salinization has long been a global problem. Therefore, studying the effects of salt and drought stress on plants, exploring the mechanisms of plant salt and drought tolerance, identifying valuable salt and drought-tolerant genes, developing and utilizing salt and drought-tolerant plant resources, and improving crop salt and drought tolerance are of great significance for the improvement of saline-alkali land.

[0003] Sophora alopecuroides L., a plant belonging to the genus Sophora in the legume family, is also known as bitter bean grass or bitter licorice. It is a perennial herbaceous plant with underground buds and is drought-tolerant and salt-tolerant. Its significant drought and salt tolerance makes it a rich gene pool of resistance genes. Therefore, screening and cloning salt- and drought-related genes from Sophora alopecuroides, analyzing their salt and drought tolerance, and elucidating their related functions will contribute to the further utilization of stress-resistance genes.

[0004] Homologous domain leucine zipper (HD-Zip) transcription factors have been found only in plants and are considered potentially plant-specific transcription factors, possessing homologous domains tightly linked to leucine zipper sequences. Based on sequence homology, conserved sequences, and specific intron and exon positions of the HD-Zip domain, 48 Arabidopsis HD-Zip transcription factor genes can be divided into four families: HD-Zip I (17 members), HD-Zip II (10 members), HD-Zip III (5 members), and HD-Zip IV (16 members). The HD-Zip transcription factor family is one of the largest plant-specific superfamilies and plays a crucial role in responding to abiotic stresses.

[0005] By utilizing transcriptome data obtained from *Bletilla striata* seedlings subjected to saline-alkali and drought treatments, differentially expressed genes were analyzed, and genes related to salt and drought tolerance in *Bletilla striata* were screened. A homologous domain leucine zipper-like transcription factor gene was obtained and named SaATHB7. To date, the role of the SaATHB7 gene in *Bletilla striata* has not been reported. Summary of the Invention

[0006] The purpose of this invention is to provide a cloning of the SaATHB7 gene from bitter bean and its application, aiming to solve the problems mentioned in the background art.

[0007] This invention is implemented as follows: a clone of the *Sophora flavescens* SaATHB7 gene, the amino acid sequence of which is shown in SEQ ID NO.2, and the nucleotide sequence of the plant stress-resistance-related protein SaATHB7, as shown in SEQ ID NO.1. Transcriptome sequencing was performed on *Sophora flavescens* seedlings treated with simulated abiotic stresses (NaCl, Na2HCO3, and PEG6000), and differentially expressed genes were analyzed to screen for *Sophora flavescens* genes related to salt stress. Bioinformatics analysis of the gene's nucleic acid sequence revealed that it may belong to the homologous domain leucine zipper-like transcription factor family, and it was named SaATHB7. The *Sophora flavescens* SaATHB7 gene was then applied to plant salt and drought tolerance. Cloning and quantitative primers were designed based on the sequence obtained from sequencing. RT-PCR was used to detect the expression changes of this gene in different tissues of Sophora flavescens under abiotic stress (NaHCO3, NaCl and PEG) to preliminarily study the corresponding role of this gene in abiotic stress of Sophora flavescens. At the same time, a plant overexpression vector of this gene was constructed and successfully transformed into wild-type Arabidopsis thaliana for preliminary functional verification. The results showed that overexpression of this gene in Arabidopsis thaliana can improve the salt tolerance and drought tolerance of Arabidopsis thaliana.

[0008] This invention provides a cloning and application of the SaATHB7 gene from *Bletilla striata*. In this invention, *Arabidopsis thaliana* transgenic with pCAMBIA3301-SaATHB7 exhibited significantly higher salt and drought tolerance compared to the wild type. Furthermore, in the native plant *Bletilla striata*, the expression of the SaATHB7 gene also showed a significant response to salt and drought stress, indicating that overexpression of the SaATHB7 gene can enhance plant stress resistance, particularly its resistance to salt and drought stress. This provides a new resource for improving crop stress resistance through genetic engineering. Attached Figure Description

[0009] Figure 1 The expression level of SaATHB7 (FPKM) in the root transcriptome of Sophora flavescens under control and abiotic stress (NaCl, Na2CO3 and NaCl) treatments;

[0010] Figure 2 The relative expression levels of the SaATHB7 gene in Sophora flavescens at 0 h, 4 h, and 72 h after treatment with 1.2% NaCl;

[0011] Figure 3 The relative expression levels of the SaATHB7 gene in Sophora flavescens at 0h, 4h and 72h after treatment with 8% PEG6000;

[0012] Figure 4 SaATHB7 gene clone gel image;

[0013] Figure 5 PCR results for constructing the pCAMBIA3301-SaATHB7 vector in Escherichia coli DH5α;

[0014] Figure 6 PCR results of constructing the pCAMBIA3301-SaATHB7 vector in Agrobacterium EHA105;

[0015] Figure 7 Relative quantitative analysis of different lines of Arabidopsis thaliana transgenic with SaATHB7 gene;

[0016] Figure 8 The germination phenotypes of wild-type Arabidopsis thaliana and SaATHB7 transgenic Arabidopsis thaliana under control and 100mM NaCl treatment conditions are shown.

[0017] Figure 9 The chlorophyll content of wild-type Arabidopsis thaliana and SaATHB7 transgenic Arabidopsis thaliana was statistically analyzed under control and 100mM NaCl treatment conditions.

[0018] Figure 10 Root length during the germination stage of wild-type Arabidopsis thaliana and SaATHB7 transgenic Arabidopsis thaliana under control and 100mM NaCl treatment conditions;

[0019] Figure 11 Root length statistics of wild-type Arabidopsis thaliana and SaATHB7 transgenic Arabidopsis thaliana during the germination stage under control and 100mM NaCl treatment conditions;

[0020] Figure 12 Root length of wild-type Arabidopsis thaliana and SaATHB7 transgenic Arabidopsis thaliana seedlings under control and 100mM NaCl treatment conditions;

[0021] Figure 13 Statistical analysis of root length in wild-type Arabidopsis thaliana and SaATHB7 transgenic Arabidopsis thaliana seedlings under control and 100mM NaCl treatment conditions;

[0022] Figure 14 The germination phenotypes of wild-type Arabidopsis thaliana and SaATHB7 transgenic Arabidopsis thaliana under control and 200 mM mannitol treatment conditions;

[0023] Figure 15 Statistical analysis of leaf chlorophyll content in wild-type Arabidopsis thaliana and Arabidopsis thaliana transgenic SaATHB7 under control and 200mM mannitol treatment conditions;

[0024] Figure 16 Root length of wild-type Arabidopsis thaliana and SaATHB7 transgenic Arabidopsis thaliana during the germination stage under control and 200mM mannitol treatment conditions;

[0025] Figure 17 Root length of wild-type Arabidopsis thaliana and SaATHB7 transgenic Arabidopsis thaliana seedlings under control and 200mM mannitol treatment conditions;

[0026] Figure 18 Statistical analysis of root length in wild-type Arabidopsis thaliana and SaATHB7 transgenic Arabidopsis thaliana seedlings under control and 200mM mannitol treatment conditions. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0028] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0029] like Figure 1-18 As shown in the figure, an embodiment of the present invention provides a cloning of the SaATHB7 gene from bitter bean, wherein the amino acid sequence of the SaATHB7 gene is shown in SEQ ID NO.2, and the nucleotide sequence of the plant stress resistance-related protein SaATHB7 is shown in SEQ ID NO.1. The cloning includes the following steps:

[0030] Step 1: Cultivation and Transcriptome Sequencing of Sophora flavescens Seedlings

[0031] 10g of plump *Sophora flavescens* seeds were selected and soaked in 5ml of 98% concentrated sulfuric acid for 20min. After washing the seeds, they were sown in potting soil. The cultivation conditions were: 16h light, 26℃, 65% humidity, and 30000 lux light intensity. After four weeks of germination, the seedlings were transferred to Hoagland nutrient solutions with 200mmol NaCl, 200mmol NaHCO3, and 8% PEG6000 concentration for 72h each. Total RNA (RNA) was extracted from the roots of *Sophora flavescens* under each treatment. The RNA extracted from the roots of the four treatments was used for transcriptome sequencing. The expression level of SaATHB7 in the root transcriptome results is shown below. Figure 1 The relative expression levels of the SaATHB7 gene in *Bletilla striata* at 0h, 4h, and 72h after 1.2% NaCl treatment are shown in the figure (where CK is the control; ST is 1.2% NaCl treatment; AST is 1.2% NaHCO3 treatment; DT is 8% PEG6000 treatment). Figure 2As shown (where "**" indicates a highly significant difference compared to the control (p<0.01)). The relative expression levels of the SaATHB7 gene in *Bletilla striata* at 0h, 4h, and 72h after treatment with 8% PEG6000 are shown in the figure. Figure 3 As shown (where CK is the control; DR4 is the simulated drought treatment for 4 h; DR72 is the simulated drought treatment for 72 h; "**" indicates a highly significant difference compared to the control (p<0.01)).

[0032] Total RNA extraction includes the following steps:

[0033] (1) Take 50-100mg of sample into a pre-cooled mortar, add liquid nitrogen and grind thoroughly into powder;

[0034] (2) Quickly add the ground powder into a 1.5 mL centrifuge tube containing 1 mL TransZol Up, shake well, let stand at room temperature for 5 min, and centrifuge at 10,000 × g for 5 min at 4 °C.

[0035] (3) Take the supernatant into a new centrifuge tube, add 200 μL of chloroform, shake vigorously for 30 seconds, mix well, incubate at room temperature for 5 min, and centrifuge at 10,000 × g for 15 min at 4 °C.

[0036] (4) Take the supernatant into a new centrifuge tube, add an equal volume of isopropanol, invert and mix well, incubate at room temperature for 10 min, centrifuge at 10,000×g for 10 min at 4℃, and discard the supernatant.

[0037] (5) Add 1 mL of pre-cooled 75% ethanol, vortex vigorously with a vortex shaker, and centrifuge at 7500×g for 5 min at 4℃.

[0038] (6) Discard the supernatant and dry at room temperature for 5 minutes;

[0039] (7) Dissolve in 50 μL of RNase-free water, incubate at 58°C for 10 min, and store at -80°C.

[0040] Library Construction and Quality Control: The constructed RNA was total RNA. All mRNAs with polyA tails were enriched using Oligo(dT) magnetic beads, and then the obtained mRNAs were randomly fragmented in Fragmentation Buffer with divalent cations. Using the fragmented mRNA as a template and random oligonucleotides as primers, the first cDNA strand was synthesized using the M-MuLV reverse transcriptase system. The RNA strand was then degraded with RNase H, and the second cDNA strand was synthesized using DNA polymerase I. After purification and end-repaired double-stranded cDNA, A tails were added, and sequencing adapters were ligated. CDNAs with a size of approximately 370–420 bp were screened using AMPure XP beads, and PCR amplification was performed. The PCR products were purified again using AMPure XP beads to obtain the final library.

[0041] After the library was constructed, preliminary quantification was performed using a Qubit 2.0 Fluorometer to dilute the library to 1.5 ng / µL. Subsequently, the insert size was detected using an Agilent 2100 Bioanalyzer. Once the insert size met expectations, the effective concentration of the library was quantified using qRT-PCR (the effective concentration should be higher than 2 nM) to ensure the library quality met requirements. The qRT-PCR system is shown in Table 1, and the qRT-PCR program is shown in Table 2.

[0042] Table 1 Real-time quantitative PCR system

[0043]

[0044] Table 2 Real-time Quantitative PCR Procedure

[0045]

[0046]

[0047] Transcriptome sequencing analysis:

[0048] (1) Sequencing: After the library quality check is qualified, the library is pooled according to the effective concentration and the target data volume requirements, and then Illumina sequencing is performed to generate 150bp paired end reads. The basic principle of sequencing is that synthesis and sequencing are basically synchronized (Sequencing by Synthesis). Fluorescently labeled dNTPs, DNA polymerase and adapter primers are added to the sequencing flow cell for amplification. Each sequencing cluster extends the complementary strand. Each fluorescently labeled dNTP added will release fluorescence. The sequencer can capture the fluorescence signal and use computer software to convert the light signal into sequencing peaks, thereby obtaining the sequence information of the fragment to be tested.

[0049] (2) Data Quality Control: Image data of sequencing fragments detectable by high-throughput sequencers are converted into sequence data (reads) via CASAVA base identification in FASTQ format. These reads primarily contain sequence information and corresponding quality information. The raw sequencing data includes some reads with sequencing adapters and poor sequencing quality. To ensure data analysis quality and reliability, the raw data needs to be filtered. This mainly includes removing reads with indeterminate base information, reads with adapters, and low-quality reads (e.g., reads with Qphred <= 20 accounting for more than 50% of the total read length). Simultaneously, Q20, Q30, and GC content are calculated for the clean data. All subsequent analyses are based on high-quality analysis using the clean data.

[0050] (3) Transcript splicing: Trinity is a highly efficient and stable software for splicing RNA-seq data. It combines three independent software modules: Inchworm, Chrysalis, and Butterfly, to process and splice large amounts of RNA-seq data sequentially. The main processes are as follows:

[0051] The inchworm process begins by reading the fq file containing all reads, converting it to fa format, and merging the 3' and 5' reads to obtain both.fa. The reads are then decomposed according to k-mers, counting the types and number of each type, and sorting them by frequency. The k-mer with the highest frequency is selected as the starting point for extension towards the 3' end, extending one base at a time. The frequency of each k-mer is then counted after extension, and the path with the highest frequency of that k-mer is selected as the extension path. Finally, the overlap relationship is used to extend the k-mers, forming a contig sequence.

[0052] Chrysalis: Cluster all contigs with similar regions higher than k-1-mers to form components. Construct a de Bruijn graph based on different components and verify the reads by comparing them with the components.

[0053] Butterfly: Statistical Transcript (butterfly) splits the graph into linear sequences, eliminates all erroneous sequences using reads and pairs relationships, simplifies the de Bruijn graph of each component, outputs the full-length transcript, and finally obtains the spliced ​​result file.

[0054] (4) Transcript quality assessment: Benchmarking Universal Single-Copy Orthologs (BUSCO) assessment utilizes a single-copy orthologous gene database, combined with software such as Augustus, tblastn, and hmmer, to assess transcript integrity. BUSCO software was used to assess the splicing quality of Trinity.fasta, cluster.fasta, and unigene.fa. The completeness and accuracy of the spliced ​​results were evaluated based on the proportion and completeness of the alignment results.

[0055] (5) Gene Function Annotation: Gene function annotation is based on the following databases: Nr: NCBI official protein sequence database, including PDB (Protein DataBank) protein database, GenBank gene protein coding sequence, SwissProt protein sequence, PRF (Protein Research Foundation) and PIR (Protein Information resource) protein sequences. Nt: NCBI official nucleic acid sequence database, including GenBank, EMBL and DDBJ nucleic acid sequences, excluding EST, PAT, STS, WGS, GSS, TSA and HTG sequences. Pfam: A classification system for protein domain annotation, in which proteins are composed of domains, and the protein sequence of each specific domain has a certain degree of conservation. PFAM classifies protein domains into different protein families and establishes HMM statistical models of amino acid sequences of each family through protein sequence alignment. SwissProt: A collection of protein sequences compiled and studied by experienced biologists. KEGG: A database used to analyze gene products and the metabolic pathways of compounds in cells, as well as the functions of these gene products. It integrates data from genomics, chemical molecules, and biochemical systems, including metabolic pathways, drugs, diseases, functional models, gene sequences, and genomes. The KO system links various KEGG annotation systems; KEGG has established a complete KO annotation system capable of functional annotation of genomes or transcriptomes of newly sequenced species. GO: is an internationally standardized classification system for gene function description. PFAM: divided into two main categories based on the reliability of annotation results: the highly reliable Pfam-A family and the automatically generated Pfam-B family. The HMMER3 program can be used to search for established HMM models to annotate genes. KOG: KOG (euKaryotic Ortholog Groups) subdivides homologous genes from different species into different ortholog clusters based on evolutionary relationships; currently, KOG has 4852 categories. Genes from the same ortholog have the same function.

[0056] (6) Reference sequence alignment: The transcript obtained by Trinity assembly was used as the reference sequence (Ref). The clean reads of each sample were mapped onto the Ref, and reads with an alignment quality value of less than 10 and reads that were not successfully aligned were filtered out. RSEM software was used during the alignment process, and bowtie2 was used with default parameters.

[0057] Differential gene analysis:

[0058] RSEM and bowtie2 software were used to calculate the FPKM (Fragments Per Kilobase of Transcript per Million Fragments Mapped) of transcriptome unigenes obtained by Trinity splicing. Differentially expressed genes between sample groups were analyzed using DESeq2 software. After differential analysis, the Benjamini-Hochberg method was used to perform multiple hypothesis testing correction on the hypothesis test probability (P_value) to obtain the False Discovery Rate (FDR). Differentially expressed genes were screened under the condition that |log2Fold Change|>=1 and FDR<0.05, and the obtained differentially expressed genes were annotated and analyzed in the KEGG and GO databases. Based on the hypergeometric test, GOseq and KOBAS software were used to perform KEGG Pathway and GO enrichment analyses, respectively, using the KEGG database Pathway and the GO database GO-Term as units. A corrected p-value less than 0.05 was used as the critical value for significant enrichment analysis.

[0059] Among the differentially expressed genes obtained, a gene annotated as a leucine zipper-like transcription factor with a homologous domain was screened. Sequence alignment analysis was performed using the NCBI database Blast (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome). It was found that it consists of 738 base pairs, with the reading frame from the 1st to the 738th base from the 5' end, encoding a protein composed of 245 amino acid residues. The SaATHB7 protein contains a homologous domain tightly linked to the leucine zipper sequence, indicating that the SaATHB7 gene may have a similar function to genes in the homologous domain leucine zipper-like transcription factor family.

[0060] Step 2: Cloning of the SaATHB7 gene and construction of the plant expression vector:

[0061] Primers for candidate genes were designed using Primer 5.0 (specific primer sequences are shown in Table 3).

[0062] Table 3. Primer sequences for SaATHB7 cloning

[0063]

[0064] Candidate genes were cloned using *Sophora flavescens* root cDNA as a template. The cloning system is shown in Table 4 below:

[0065] Table 4 Candidate gene cloning reaction system

[0066]

[0067] The procedure is shown in Table 5 below:

[0068] Table 5 PCR amplification program

[0069]

[0070]

[0071] Agarose gel electrophoresis was used to validate candidate gene bands. The SaATHB7 gene clone gel image is shown below. Figure 4 As shown (where M1 is a 2000bp marker), for target genes with band sizes meeting expectations, gel extraction and recovery were performed. The specific experimental steps for recovery are as follows:

[0072] 1. Cut a piece of gel containing the target band on a blue light analyzer and place it into a 2mL centrifuge tube;

[0073] 2. Add an equal volume of sol solution PG to a centrifuge tube and incubate in a 50°C water bath until the gel is completely dissolved;

[0074] 3. Transfer the obtained liquid into the adsorption column, let it stand at room temperature for 2 min, centrifuge at 12000 rpm for 1 min, and discard the waste liquid;

[0075] 4. Add 600 μL PW to each tube, incubate at 12000 rpm for 1 min, discard the waste liquid, and put the adsorption column back into the centrifuge tube;

[0076] 5. Repeat the previous step;

[0077] 6. Centrifuge at 12000 rpm for 12 min;

[0078] 7. Prepare a new 1.5 mL centrifuge tube, place the adsorption column into the centrifuge tube, add 50 μL ddH2O dropwise to the middle of the adsorption column, let stand at room temperature for 2 min, centrifuge at 12000 rpm for 2 min, and collect the DNA solution in the centrifuge tube.

[0079] The recovered fragments were tailed with an A-tail, and the system is shown in Table 6 below:

[0080] Table 6 Reaction system for recovered fragments plus A

[0081]

[0082] 72℃, 20 min, 2 min on ice.

[0083] The candidate gene was ligated into the cloning vector pMD18-T, and the ligation system is shown in Table 7 below:

[0084] Table 7 Connection Reaction System

[0085]

[0086] 16℃, 14h, overnight connection.

[0087] Transformation of Escherichia coli DH5α:

[0088] (1) Preparation and transformation of Escherichia coli DH5α

[0089] 1. Streak Escherichia coli culture on LB solid medium and incubate at 37°C for 12-14 h. Pick a single colony and place it in 10 mL of LB liquid medium and incubate at 37°C with shaking at 180 rpm for 10-12 h.

[0090] 2. Add 1 mL of bacterial culture to 50 mL of LB liquid medium and incubate at 37°C with shaking until the OD600 reaches 0.4-0.6;

[0091] 3. After placing the bacterial culture in an ice bath for 10 minutes, place it in a 50 mL centrifuge tube and centrifuge at 5000 rpm for 10 minutes at 4°C. Pour out the liquid from the tube.

[0092] 4. Add 5 mL of pre-cooled 0.1 M CaCl2, resuspend the bacterial culture, and incubate on ice for 30 min;

[0093] Centrifuge at 5000 rpm for 10 min at 5.4℃, then discard the supernatant;

[0094] 6. Take 1 mL of pre-cooled 0.1 M CaCl2 and suspend the cells;

[0095] 7. Add 200 μL of 80% glycerol, mix thoroughly, dispense 100 μL into each tube, flash freeze in liquid nitrogen, and store at -80°C.

[0096] (2) The extracted yeast plasmid was transformed into Escherichia coli DH5α using the freeze-thaw method, as follows:

[0097] 1. Thaw competent cells in an ice box, add 10 μL of plasmid, and incubate on ice for 30 min;

[0098] 2. Heat shock at 2.42℃ for 90s, immediately ice bath for 4min, then add 800μL LB medium, and culture at 37℃ with shaking at 200rpm for 1-2h;

[0099] Centrifuge at 8000 rpm for 5 min, discard the supernatant, and resuspend the bacterial cells in an appropriate amount of LB medium;

[0100] 4. Take 50 μL of bacterial culture and spread it on LB medium (containing Amp, add 1 μL of Amp per milliliter of medium), and incubate upside down at 37°C for 12-16 h.

[0101] 5. Pick a single colony and add it to 1 mL of liquid LB medium (containing Amp). Incubate at 37°C with shaking at 180 rpm for 8 h. Take 1 μL of the bacterial solution as a template for PCR reaction and perform bacterial PCR. The reaction system and conditions are shown in Tables 8 and 9.

[0102] Table 8 PCR amplification reaction system

[0103]

[0104] Table 9 PCR Amplification Conditions

[0105]

[0106] Single-clone colonies were selected for PCR verification. Samples with the target band matching the expectations were sent in 200 μL increments to a biotechnology company for sequencing to further confirm the sequence accuracy of the cloned target gene.

[0107] (3) Plasmid extraction:

[0108] The extraction method followed the method described in the plasmid extraction kit from Sangon Biotech.

[0109] 1. Add 5 mL of Buffer P1 to the precipitated bacterial cells, pipette and vortex until the bacterial cells are completely suspended; 2. Add 5 mL of Buffer P2, immediately gently invert to mix the solution, and let it stand at room temperature for 3-5 minutes;

[0110] 3. Add 7 mL of Buffer P3, immediately invert the container to mix the solution, and let it stand at room temperature for 5 min;

[0111] 4. Incubate in a 90℃ water bath for 10 min, then place at -20℃ for 10 min, and centrifuge at 12000 rpm for 15 min;

[0112] 5. Transfer all the supernatant into the adsorption column, let stand for 5 minutes, centrifuge at 8000 rpm for 2 minutes, discard the waste liquid in the collection tube, and put it back into the adsorption column;

[0113] 6. Add 5 mL of buffer DW1, centrifuge at 8000 rpm for 2 min, discard the waste liquid in the collection tube, and put it back into the adsorption column;

[0114] 7. Add 5 ml of Wash Solution, centrifuge at 8000 rpm for 2 min, discard the waste liquid in the collection tube, put it back into the adsorption column, and repeat once;

[0115] 8. Centrifuge the empty adsorption column at 10,000 rpm for 2 min;

[0116] 9. Place the adsorption column into a 50mL centrifuge tube, add 1mL of Elution Buffe to the adsorption membrane, let stand for 2min, centrifuge at 10000rpm for 2min, and store the collected plasmid DNA solution at -20℃.

[0117] (4) Construction of plant expression vectors:

[0118] After the target gene recombinant cloning vector with correct sequencing was subjected to culture and plasmid extraction, restriction enzyme sites were designed based on the target gene sequence and the multiple cloning site information of the plant expression vector pCAMBIA3301. Primers with double restriction enzyme sites were also designed. The target gene was cloned using the recombinant cloning vector as a template and then recovered.

[0119] The recovered fragments of the target gene and the pCAMBIA3301 vector were digested with double enzymes. The enzyme digestion system is shown in Table 10.

[0120] Table 10 Enzyme digestion reaction system

[0121]

[0122] Enzyme digestion at 37℃ for 3 hours.

[0123] Electrophoresis, gel cutting, recycling and bonding

[0124] Ligating the target gene fragment with the plant expression vector:

[0125] After agarose gel electrophoresis and imaging analysis, the enzyme digestion products were recovered according to the specified recovery method. The recovered products were then ligated; the ligation system is shown in Table 11.

[0126] Table 11 Connection Reaction System

[0127]

[0128] 16℃, overnight connection for 14 hours.

[0129] The preparation and transformation of Escherichia coli DH5α competent cells were carried out according to the above method.

[0130] Universal primers were designed for recombinant vector PCR detection. PCR results of the constructed pCAMBIA3301-SaATHB7 vector in Escherichia coli DH5α are shown below. Figure 5 As shown (where "M" represents a 2000bp Marker; "-" indicates negative; "+" indicates positive), select 200μL of the single clone with the correct band and send it to the biotechnology company for sequencing verification.

[0131] Cultivars were shaken and plasmids were extracted:

[0132] The correctly sequenced single-clone bacterial culture was inoculated into 50 mL of liquid LB medium (containing 50 μg / mL Kana) and incubated overnight at 37°C and 220 rpm for 12 h until the bacterial concentration reached OD500. 600 Plasmid extraction can be performed with a pH of 1.0-1.5. Simultaneously, add 200 μL of 80% glycerol to 700 μL of the solution, mix well, and store at -80°C. Plasmid extraction should follow the method described above.

[0133] Recombinant plasmid was transformed into Agrobacterium tumefaciens EHA105 and validated:

[0134] Preparation of Agrobacterium EHA105 competent cells:

[0135] 1. Activate the EHA105 bacterial culture by streaking it on a YEP solid plate and incubate at 28°C for 2 days;

[0136] 2. Pick a single colony and place it in 50 mL of YEP liquid medium, and incubate overnight at 28°C with shaking at 200 rpm;

[0137] 3. Take 2 mL of the activated bacterial culture and add it to 50 mL of YEP liquid medium. Incubate at 28°C with shaking at 200 rpm until the OD600 is about 0.5.

[0138] 4. Place the above culture on ice for 30 minutes;

[0139] Centrifuge at 3000 rpm for 5 min at 5.4℃, then discard the supernatant;

[0140] 6. Add 1 mL of pre-cooled 0.2 M CaCl2 suspension of bacteria, add 200 μL of glycerol, dispense into smaller containers, flash freeze in liquid nitrogen, and store at -80 °C.

[0141] Recombinant plasmid transformation of Agrobacterium:

[0142] 1. Thaw Agrobacterium competent cells on ice;

[0143] 2. Add 3 μL of plasmid, flash freeze in liquid nitrogen for 5 min, and then incubate in a water bath at 37°C for 5 min;

[0144] 3. Add 1 mL of antibiotic-free YEP liquid medium and incubate at 28°C with shaking at 200 rpm for 2-4 hours;

[0145] 4. Centrifuge at 10,000 rpm for 1 min, discard the supernatant, and resuspend the bacterial cells in 80 μL of liquid YEP;

[0146] 5. Spread 40 μL of bacterial culture onto a YEP plate (containing Rif and Kan) and incubate at 28°C for 2 days.

[0147] 6. After selecting single colonies for PCR verification, glycerol was added and stored at -80℃. The pCAMBIA3301-SaATHB7 vector was constructed and PCR results were obtained in Agrobacterium EHA105. Figure 6 As shown (where "M" is a 2000bp Marker; "-" indicates negative; "+" indicates positive).

[0148] Step 3: Tissue-specific expression of the SaATHB7 gene in bitter beans:

[0149] The *Bletilla striata* was subjected to stress treatment, following the same method as in step 1. After 4 hours of treatment, root tissues were collected, flash-frozen in liquid nitrogen, and stored at -80°C. Total RNA was extracted from the treated material using a column-based plant total RNA extraction and purification kit from Sangon Biotech, and RNA integrity was assessed by 1% agarose gel electrophoresis. cDNA synthesis was performed according to the instructions for Reverse Transcriptase M-MLV (RNase H-). Real-time quantitative PCR was used to detect the expression of the SaATHB7 gene in roots of *Bletilla striata* at different time points after salt and drought treatment. The experimental procedures were performed on an ABI 7500 real-time quantitative PCR instrument according to the instructions for SGExcel FastSYBRMixture (With ROX) from Sangon Biotech. Lectin from *Bletilla striata* was used as an internal reference gene, and the primers are shown in Table 12 below.

[0150] Table 12 Quantitative primers for SaATHB7

[0151]

[0152]

[0153] The PCR reaction system and procedure are shown in Tables 13 and 14 below:

[0154] Table 13 qRT-PCR reaction system

[0155]

[0156] Table 14 qRT-PCR reaction procedure

[0157]

[0158] The 2-ΔΔCT method was used to analyze the data and determine the relative expression levels of genes. The experiment consisted of three technical replicates and three biological replicates.

[0159] The results showed that the SaATHB7 gene expression was significantly increased after 4 h of salt and drought stress treatment, and was relatively downregulated after 72 h, but the expression level was still higher than that at 0 h, indicating that the SaATHB7 gene actively participates in the response of Sophora flavescens to salt and drought stress.

[0160] This invention provides an application of the SaATHB7 gene from *Arabidopsis thaliana*, specifically its application in plant salt and drought tolerance. The expression of SaATHB7 in *Arabidopsis thaliana* and its analysis of salt and drought tolerance are used as verification. Specifically:

[0161] Wild-type Arabidopsis thaliana was transformed using the Agrobacterium-mediated transformation method with the plant expression vector pCAMBIA3301-SaATHB7. The transgenic Arabidopsis were then screened using basta, and the expression levels of the target gene in positive plants were detected. The salt tolerance of the transgenic Arabidopsis was also analyzed. The specific methods are as follows:

[0162] (1) Arabidopsis thaliana sowing and cultivation:

[0163] Select dried Arabidopsis seeds, first disinfect them with 1 mL of 10% NaClO for 5 min, then wash them repeatedly with sterile distilled water until clear (generally 4-6 times). Using a sterile pipette tip or tweezers, sow the disinfected seeds onto 1 / 2 MS solid medium and vernalize them at 4℃ in the dark for 72 h. After reaching the four-leaf stage, transplant them into prepared potting soil (peat moss:vermiculite = 1:1). Place them in an Arabidopsis culture room (day / night, 16h / 8h, 22℃ / 20℃) for 3-4 weeks, watering with 1 / 2×Hoagland nutrient solution every 3 days. Once the Arabidopsis begins to sprout stems, prepare for the next step of infection and transformation.

[0164] (2) Agrobacterium tumefaciens-mediated flower-dipping method for Arabidopsis thaliana infection:

[0165] 1. Agrobacterium EHA105 transformed into the recombinant plant expression vector was streaked on YEP solid medium containing Kan and Rif antibiotics for activation and incubated at 28℃ for 30-48 hours.

[0166] 2. Pick a single colony and inoculate it into YEP liquid medium containing Kan and Rif antibiotics. Incubate at 28°C and 200 rpm for 12-16 h until the bacterial OD600 reaches 0.8-1.0.

[0167] 3. Collect Agrobacterium cells by centrifugation at 5000 rpm for 10 min at 4℃, and resuspend them in 1 / 2 MS liquid medium (add 20 μL Switt77 per 100 μL of medium) until the OD600 is 0.7-0.9;

[0168] 4. Select wild-type Arabidopsis thaliana flower buds that are only showing white, gently dip the inflorescence into the infection solution for 60-90 seconds, and carefully absorb the residual infection solution with filter paper;

[0169] 5. After infection, Arabidopsis thaliana should be cultured in the dark for 24 hours, then cultured under normal conditions, with timely replenishment of nutrient solution;

[0170] 6. Observe the growth of Arabidopsis thaliana. After 12-15 days, you can carry out reinfection. Harvest the seeds after the Arabidopsis thaliana matures and dry them.

[0171] (3) Screening of transgenic Arabidopsis thaliana:

[0172] After sterilization and vernalization, the obtained T0 generation Arabidopsis seeds were sown on 1 / 2 MS solid medium containing Basta (4 mg / L) for screening. Arabidopsis seedlings that grew normally on the screening medium were transplanted, and three days later, they were sprayed with Basta at a concentration of 200 mg / L for a second screening. Arabidopsis seedlings that grew normally after both screenings were considered preliminary positive seedlings.

[0173] (4) Extraction of Arabidopsis DNA using the CTAB method:

[0174] 1. Take an appropriate amount of Arabidopsis thaliana leaves into 2mL centrifuge tubes (each tube contains 2 steel balls), freeze them quickly in liquid nitrogen, and then grind them into powder using a grinder;

[0175] 2. Add 600 μL of CTAB extraction solution (preheated to 65℃) to a centrifuge tube, then add 15 μL of β-mercaptoethanol, mix by inverting, and place in a 65℃ water bath for 40 min, mixing once every 10 min.

[0176] 3. Add 600 μL of chloroform, mix by inverting, and centrifuge at 12,000 rpm for 15 min at 4 °C;

[0177] 4. Transfer the supernatant after centrifugation to a new 1.5 mL centrifuge tube, add 400 μL of isopropanol, mix well, and allow to settle at -20 °C for 30 min.

[0178] Centrifuge at 12,000 rpm for 15 min at 5.4℃, and discard the supernatant;

[0179] 6. Add 1 mL of pre-cooled 75% ethanol, wash the precipitate, centrifuge at 12,000 rpm for 2 min at 4°C, and discard the supernatant.

[0180] 7. Repeat the previous step and dry the precipitate at room temperature.

[0181] 8. Dissolve the DNA precipitate in 30 μL ddH2O and store at -20℃.

[0182] (5) Propagation and detection of transgenic Arabidopsis thaliana:

[0183] Using the selected Arabidopsis DNA as a template, PCR molecular detection was performed. The harvested T1 generation Arabidopsis seeds were sown, screened, and detected according to the above method to obtain T2 generation transgenic Arabidopsis. The above method was then followed to obtain T3 generation transgenic Arabidopsis, and the seeds were harvested for the next step of the experiment.

[0184] (6) Real-time quantitative PCR of overexpression lines:

[0185] RNA was extracted from each of the selected transgenic Arabidopsis lines and reverse transcribed into cDNA. The expression level was analyzed by real-time quantitative PCR using cDNA as a template. The Arabidopsis gene Actin2 was used as an internal reference gene in Arabidopsis. The specific quantitative system and procedure were as described above.

[0186] (7) Salt stress treatment and germination rate statistics of Arabidopsis thaliana during the germination stage:

[0187] Dry T3 generation transgenic and wild-type Arabidopsis seeds were collected, sterilized, and sown on 1 / 2 MS solid medium containing CK, 100 mM NaCl, and 200 mM mannitol. Vernalization was performed at 4°C for 72 hours, followed by culture in a culture room for 3-15 days, with daily observation of Arabidopsis growth. Three parallel experiments were conducted, with three replicates per experiment. During Arabidopsis germination, germination status was promptly recorded, photographs were taken, and root length was measured and statistically analyzed. A relative quantitative analysis of different lines of SaATHB7 transgenic Arabidopsis was presented. Figure 7 As shown, WT is the wild type; L1, L2, L3, L4, L5, L7, L8 and L9 are SaATHB7 transgenic lines.

[0188] (8) Salt stress treatment and root length measurement of Arabidopsis thaliana seedlings:

[0189] One week after sowing, Arabidopsis thaliana seedlings of various lines were transferred to MS solid medium containing 0M (CK), 100mM NaCl, and 200mM mannitol for culture. The growth of each Arabidopsis thaliana line was observed, photographed, and root length was measured. The results are as follows: Figure 8-18 As shown in the figures, WT represents the wild type; OE5 and OE9 represent Arabidopsis thaliana transgenic with the SaATHB7 gene. Figure 11 The asterisk (*) indicates a significant difference between the transgenic lines and the control (p<0.05). Figure 13 The "**" indicates that the transgenic lines are significantly different from the control (p<0.01). Figure 15 and 18In the diagram, "**" indicates that the transgenic lines are significantly different from the control (p<0.01); "*" indicates that the transgenic lines are significantly different from the control (p<0.05).

[0190] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An application of overexpression of the SaATHB7 gene in *Bletilla striata* for salt and drought tolerance in plants, characterized in that... The plants are Sophora flavescens and Arabidopsis thaliana. The amino acid sequence of the protein encoded by the Sophora flavescens SaATHB7 gene is shown in SEQ ID NO.2, and the nucleotide sequence of SaATHB7, a protein related to plant stress resistance, is shown in SEQ ID NO.1.

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