Cloning and application of SaPEI7 gene from Sophora alopecuroides

Abstract

The present invention is applicable to the field of genetic engineering technology and provides a cloning and application of the Sophora alopecuroides (Solanum sophora) SaPEI7 gene. By screening differentially expressed genes from transcriptome sequencing of Sophora alopecuroides under simulated stress conditions, a Sophora alopecuroides gene related to salt tolerance was identified through bioinformatics analysis as the Sophora alopecuroides (Solanum sophora) pectin methylesterase inhibitor protein gene SaPEI7. Quantitative detection using RT-PCR technology revealed a significant increase in gene expression under salt stress conditions. The gene was functionally verified by constructing a plant expression vector and successfully transformed into Arabidopsis thaliana. The results showed that overexpression of the gene in Arabidopsis thaliana improved its salt tolerance, providing a new resource for improving crop stress resistance through genetic engineering technology.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to cloning of a Sophora alopecuroides SaPEI7 gene and application thereof. Background Art

[0002] Soil salinization has long been a global problem. According to incomplete statistics from the Food and Agriculture Organization of the United Nations, the global area of ​​saline-alkali land is approximately 9.5 million square kilometers, of which my country accounts for approximately 10%. my country's salinized land is distributed from coastal areas to inland areas, and from humid to arid regions. According to data released at the 2010 China Symposium on the Utilization of Salt-Alkali Soil Resources, the total area of ​​saline-alkali land in my country exceeds 500 million mu (approximately 1.5 billion hectares), including saline-alkali wasteland and saline-alkali land that affects arable land. Of this, approximately 200 million mu (10% of my country's total arable land) has potential for agricultural development. The accumulation of soil salt is primarily due to low precipitation and high evaporation in most regions, resulting in accumulated salt exceeding the tolerance level of plants, severely impacting crop growth. Therefore, studying the effects of salt stress on plants, exploring plant salt tolerance mechanisms, identifying valuable salt-tolerant genes, developing and utilizing salt-tolerant plant resources, and improving crop salt tolerance are of great significance for improving saline-alkali land and ensuring global food security.

[0003] Pectin methylesterase inhibitors (PEIs) are a multigene family first discovered in kiwifruit by Balestrieri et al. (1990). They inhibit the activity of pectin methylesterase (PME) in plants. Previous structural analysis of PEIs revealed a PEI domain similar to the N-terminal PRO region of many plant PMEs. PEIs interact with endogenous plant PME proteins to form complexes that exert their effects. PEIs have been found in species such as Arabidopsis thaliana, Brassica campestris, flax, Sorghum bicolor, and rice, demonstrating their ubiquity in plants. Furthermore, their gene expression varies depending on plant location and developmental timing. Previous studies have shown that Arabidopsis PEI affects the development of the apical meristem of Arabidopsis, with root cells in transgenic Arabidopsis elongated compared to controls. An et al. (2008) studied Arabidopsis overexpressing CaPEI1 and found that the plants exhibited enhanced tolerance to water stress, increased germination rates, and root elongation in seedlings, as well as enhanced tolerance to oxidative stress. Jolie et al. (2010) have shown that PEI can accelerate plant growth, fruit development, and ripening. In summary, plant PEI has multiple biological functions closely related to plant growth and development. Furthermore, its expression is influenced by a variety of factors, suggesting that PEI may participate in a variety of biological regulatory processes.

[0004] Sophora alopecuroides (Sophora alopecuroides L.), a plant of the genus Sophora in the Leguminosae family, also known as bitter bean grass and bitter liquorice, is a perennial herbaceous, drought-tolerant plant with rhizomes and underground shoots. Its remarkable salt and alkali tolerance makes it a rich reservoir of resistance genes. Therefore, screening and cloning salt-tolerance-related genes from Sophora alopecuroides, analyzing their salt tolerance, and elucidating their related functions will facilitate the further utilization of stress resistance genes. However, existing reports on the role of pectin methylesterase inhibitor proteins in plant salt tolerance are limited, and no reports on pectin methylesterase inhibitor proteins in Sophora alopecuroides have been found.

[0005] Therefore, we used transcriptome data obtained from sophora alopecuroides seedlings exposed to salinity and drought to analyze differentially expressed genes and screen for genes associated with salt tolerance. We identified a pectin methylesterase inhibitor protein gene, named SaPEI7. To date, the role of SaPEI7 in sophora alopecuroides has not been reported. Summary of the Invention

[0006] The purpose of the embodiments of the present invention is to provide a clone of the SaPEI7 gene of Sophora alopecuroides and its application, aiming to solve the problems raised in the above background technology.

[0007] The present invention is achieved by: a sophora alopecuroides SaPEI7 gene, the nucleotide sequence of which is shown in SEQ ID NO. 1, and the amino acid sequence of the plant stress resistance-related protein SaPEI7 is shown in SEQ ID NO. 2. Transcriptome sequencing was performed on sophora alopecuroides seedlings treated with simulated stress (NaCl, Na2HCO3, and PEG6000), and the differentially expressed genes obtained were analyzed to screen out sophora alopecuroides genes associated with salt stress. Bioinformatics analysis of the gene nucleic acid sequence obtained by sequencing revealed that the gene may belong to the pectin methylesterase inhibitor protein family gene and was named SaPEI7. Cloning primers and quantitative primers were designed based on the sequence obtained by sequencing. The expression levels of SaPEI7 in different tissues of sophora alopecuroides were obtained through transcriptome data, and it was found that the expression level of SaPEI7 in roots was higher than that in stems and leaves. RT-PCR was used to detect changes in the expression of this gene in Sophora alopecuroides under salt stress conditions, preliminarily investigating its role in salt stress in Sophora alopecuroides. It was found to be significantly upregulated 4 and 72 hours after salt stress. A plant overexpression vector for SaPEI7 was constructed and successfully transferred into wild-type Arabidopsis thaliana for preliminary functional verification. The results showed that overexpression of this gene in Arabidopsis thaliana improved salt tolerance during the germination stage.

[0008] The present invention provides a cloning and application of the Sophora alopecuroides (Solanum sophora) SaPEI7 gene. By screening differentially expressed genes from transcriptome sequencing of Sophora alopecuroides under simulated stress conditions, a Sophora alopecuroides gene related to salt tolerance was identified through bioinformatics analysis as the Sophora alopecuroides (Solanum sophora) pectin methylesterase inhibitor protein gene SaPEI7. Quantitative detection using RT-PCR revealed a significant increase in gene expression under salt stress conditions. The gene was functionally verified by constructing a plant expression vector and successfully transformed into Arabidopsis thaliana. The results showed that overexpression of the gene in Arabidopsis thaliana improved its salt tolerance, providing a new resource for improving crop stress resistance through genetic engineering. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 The expression level (FPKM) of SaPEI7 in the root transcriptome results of Sophora alopecuroides under control and abiotic stress (NaCl, Na2CO3 and PEG) treatments;

[0010] Figure 2 is the expression level of SaPEI7 gene in different tissues of Sophora alopecuroides L. (FPKM);

[0011] Figure 3 is the relative expression level of SaPEI7 gene in Sophora alopecuroides at 0 h, 4 h and 72 h after treatment with 1.2% NaCl;

[0012] Figure 4 This is the gel image of SaPEI7 gene cloning;

[0013] Figure 5 PCR results for the construction of pCAMBIA3301-SaPEI7 vector in Escherichia coli DH5α;

[0014] Figure 6 PCR results for the construction of pCAMBIA3301-SaPEI7 vector in Agrobacterium EHA105;

[0015] Figure 7 For relative quantitative analysis of various Arabidopsis lines transfected with SaPEI7 gene;

[0016] Figure 8 Root length of wild-type Arabidopsis and transgenic SaPEI7 Arabidopsis at the germination stage under control and 100 mM NaCl treatment conditions;

[0017] Figure 9 Root length statistics of wild-type Arabidopsis and SaPEI7-transgenic Arabidopsis at the germination stage under control and 100 mM NaCl treatment conditions. DETAILED DESCRIPTION

[0018] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present 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 only used to explain the present invention and are not intended to limit the present invention.

[0019] The specific implementation of the present invention is described in detail below with reference to specific embodiments.

[0020] One embodiment of the present invention provides a Sophora alopecuroides SaPEI7 gene, the nucleotide sequence of which is shown in SEQ ID NO.1, and the amino acid sequence of the plant stress resistance-related protein encoded thereby is shown in SEQ ID NO.2.

[0021] Example 1: Sophora alopecuroides seedling cultivation and transcriptome sequencing

[0022] 10 g of bitter bean seeds with full grains were selected and soaked in 5 ml of 98% concentrated sulfuric acid for 20 minutes. After washing the seeds, they were sown in potted flower soil. The culture conditions were: 16 h of light, temperature 26 ° C, humidity 65%, and light intensity 30,000 lux. After four weeks of germination, the seedlings were transferred to Hoagland nutrient solution with a NaCl concentration of 200 mmol, a NaHCO3 concentration of 200 mmol, and a PEG6000 concentration of 8% and treated for 72 hours respectively. The roots of bitter beans under each treatment were taken, and the total RNA of the roots of bitter beans under different treatment conditions was extracted respectively. RNA was extracted from the roots of bitter beans treated above and the roots, stems, and leaves of bitter beans under control conditions for transcriptome sequencing.

[0023] Extraction of total RNA

[0024] (1) Take 50-100 mg of sample in a pre-cooled mortar and add liquid nitrogen to grind thoroughly until powder;

[0025] (2) The ground powder was quickly added to a 1.5 mL centrifuge tube containing 1 mL of TransZol Up, shaken, and allowed to stand at room temperature for 5 min. Centrifuged at 10,000 × g for 5 min at 4°C.

[0026] (3) The supernatant was placed in a new centrifuge tube, 200 μL of chloroform was added, and the tube was shaken vigorously for 30 seconds. After mixing, the tube was incubated at room temperature for 5 minutes and centrifuged at 10,000 × g for 15 minutes at 4°C.

[0027] (4) Take the supernatant and place it in a new centrifuge tube. Add an equal volume of isopropanol, invert evenly, incubate at room temperature for 10 min, centrifuge at 10,000 × g for 10 min at 4°C, and discard the supernatant.

[0028] (5) Add 1 mL of pre-chilled 75% ethanol, vortex vigorously using a vortex shaker, and centrifuge at 7500 × g for 5 min at 4°C;

[0029] (6) Discard the supernatant and dry at room temperature for 5 minutes;

[0030] (7) Dissolve the cells in 50 μL of RNase-free water, incubate at 58°C for 10 min, and store at -80°C.

[0031] Library construction and quality control

[0032] The library is constructed using total RNA. Oligo(dT) magnetic beads are used to enrich all mRNAs with polyA tails. The resulting mRNA is then randomly fragmented in Fragmentation Buffer using divalent cations. The fragmented mRNA is then used as a template and random oligonucleotides as primers to synthesize the first-strand cDNA using the M-MuLV reverse transcriptase system. The RNA strand is then degraded with RNase H, and the second-strand cDNA is synthesized using the DNA polymerase I system. The purified, end-repaired double-stranded cDNA is then A-tailed and ligated to sequencing adapters. cDNAs between 370 and 420 bp in size are selected using AMPure XP beads, amplified by PCR, and the PCR products are further purified using AMPure XP beads to obtain the final library.

[0033] After library construction, initial quantification was performed using a Qubit 2.0 Fluorometer. The library was diluted to 1.5 ng / uL. The insert size was then measured using an Agilent 2100 Bioanalyzer. Once the insert size met expectations, the effective concentration of the library was quantified by qRT-PCR (required to be greater than 2 nM) to ensure that the library quality met the requirements. The qRT-PCR system is shown in Table 1, and the procedure is shown in Table 2.

[0034] Table 1 Real-time fluorescence quantitative PCR system

[0035]

[0036]

[0037] Table 2 Real-time fluorescence quantitative PCR program

[0038]

[0039] Transcriptome sequencing analysis

[0040] Sequencing: After the initial library quality check is qualified, the library is pooled according to the effective concentration and the target data volume required for the sequencing process, and then Illumina sequencing is performed to generate 150bp paired-end reads. The basic principle of sequencing is the simultaneous synthesis and sequencing (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 chain. Each added fluorescently labeled dNTP releases corresponding fluorescence. The sequencer captures the fluorescent signal and uses computer software to convert the optical signal into sequencing peaks, thereby obtaining the sequence information of the fragment to be tested.

[0041] Data quality control: The image data of sequencing fragments that can be detected by high-throughput sequencers are converted into sequence data (reads) through CASAVA base recognition. The file is in fastq format, which mainly contains the sequence information of the sequencing fragments and the corresponding quality information. The raw data obtained by sequencing contains some reads with sequencing adapters and poor sequencing quality. In order to ensure the quality and reliability of data analysis, the raw data needs to be filtered. This mainly includes removing reads whose base information cannot be determined, reads with adapters, and low-quality reads (such as the number of bases with Qphred <= 20 accounting for more than 50% of the entire read length). At the same time, Q20, Q30 and GC content are calculated for clean data. All subsequent analyses are based on high-quality analysis of clean data.

[0042] Transcript splicing: Trinity software was used to process and splice a large amount of RNA-seq data. The main steps are as follows:

[0043] Inchworm: First, read the fq file of all reads, convert it to fa format, and merge the 3' and 5' reads to create both.fa. The reads are then decomposed into k-mers, and the types and number of k-mers of each type are counted. The k-mers are sorted from highest to lowest based on their frequency. The k-mer with the highest frequency is selected as the starting point for extension toward the 3' end, extending one base at a time. The occurrence of each k-mer after extension is counted, and the path with the highest k-mer frequency is selected as the extension path. Finally, the k-mers are extended using the overlap relationship to form a contig.

[0044] 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 by aligning reads with the components.

[0045] Butterfly: Counts transcripts (butterfly), splits the graph into linear sequences, uses the relationship between reads and pairs to eliminate all erroneous sequences, simplifies the de Bruijn graph of each component, outputs full-length transcripts, and finally obtains the splicing result file.

[0046] Transcript quality assessment: The Benchmarking Universal Single-Copy Orthologs (BUSCO) assessment uses a single-copy orthologous gene database in conjunction with software such as Augustus, Tblastn, and Hmmer to assess transcript integrity. BUSCO software was used to assess the quality of the splicing of Trinity.fasta, cluster.fasta, and unigene.fa. The integrity and accuracy of the splicing results were evaluated based on the proportion and completeness of the alignment results.

[0047] Gene function annotation: Gene function annotation is based on the following databases: Nr: NCBI official protein sequence database; Nt: NCBI official nucleic acid sequence database.

[0048] Pfam: A classification system for protein domain annotation. Proteins are composed of domains, and the protein sequences within each specific domain are conserved to a certain extent. PFAM classifies protein domains into families and constructs HMM statistical models for the amino acid sequences of each family through protein sequence alignment. Swiss-Prot: A collection of protein sequences compiled and studied by experienced biologists. KEGG: A database used to analyze the metabolic pathways of gene products and compounds in cells, as well as the functions of these gene products. It integrates data on genomics, chemical molecules, and biochemical systems, including metabolic pathways, drugs, diseases, functional models, gene sequences, and genomes. The KO system connects various KEGG annotation systems. KEGG has established a comprehensive KO annotation system that can be used for functional annotation of newly sequenced genomes or transcriptomes. GO: An internationally standardized classification system for describing gene function. PFAM: Based on the confidence of annotation results, it is divided into two categories: the highly reliable Pfam-A family and the automatically generated Pfam-B family. The HMMER3 program can be used to search the constructed HMM models for gene annotation. KOG: KOG (euKaryotic Ortholog Groups) classifies homologous genes from different species into different ortholog clusters based on evolutionary relationships. Currently, KOG has 4852 classifications. Genes from the same ortholog have the same function.

[0049] Reference sequence alignment: The transcripts assembled using Trinity served as the reference sequence (Ref). Clean reads from each sample were mapped to the Ref. Reads with alignment quality values ​​less than 10 and reads with non-pairwise alignments were removed. RSEM software was used for alignment, using the default bowtie2 parameters.

[0050] Differential gene analysis

[0051] RSEM and bowtie2 software were used to calculate the FPKM (Fragments Per Kilobase of transcript per Million fragments mapped) of transcriptome unigenes assembled using Trinity. Differentially expressed genes between sample groups were analyzed using DESeq2 software. After differential analysis, the probability of hypothesis testing (P_value) was corrected for multiple hypothesis testing using the Benjamini-Hochberg method to obtain the false discovery rate (FDR). Differentially expressed genes were screened under the conditions of |log2FoldChange|>=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, KEGG pathway and GO term enrichment analysis were performed using GOseq and KOBAS software, respectively, using the KEGG pathway and GO term units in the GO database. A corrected p-value less than 0.05 was used as the critical value for significant enrichment analysis.

[0052] Among the differentially expressed genes obtained, we found a gene annotated as pectin methylesterase inhibitor protein that was significantly upregulated under salt, alkali and drought stress conditions (see attached Figure 1 As shown in the table, CK: control; ST: 1.2% NaCl treatment; A_ST: 1.2% NaHCO3 treatment; DT: 8% PEG6000 treatment), and transcriptome data analysis in different tissues showed that the expression level of this gene in roots was much higher than that in leaves and stems (see attached figure). Figure 2 Sequence alignment analysis using the NCBI database Blast (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome) revealed that the SaPEI7 protein consists of 1062 base pairs, with a reading frame from bases 180 to 806 at the 5' end (sequence shown in SEQ ID NO.1), encoding a protein composed of 208 amino acid residues (sequence shown in SEQ ID NO.2). The SaPEI7 protein contains a pectin methylesterase inhibitor homology domain, suggesting that the SaPEI7 gene may have similar functions to genes in the pectin methylesterase inhibitor protein family.

[0053] Example 2: Analysis of the expression level of SaPEI7 gene in Sophora alopecuroides

[0054] The bitter bean was treated with 200mM NaCl, and the treatment method was the same as in Example 1. After 4 hours of treatment, the root tissue of the bitter bean was taken, quick-frozen in liquid nitrogen, and then stored in a -80°C refrigerator. The total RNA of the treated material was extracted with reference to the column-type plant total RNA extraction and purification kit of Sangon Company, and the integrity of the RNA was detected by 1% agarose electrophoresis. The synthesis of cDNA was carried out according to the instructions of Reverse Transcriptase M-MLV (RNase H-). The expression of the SaPEI7 gene in the roots of bitter bean treated with salt at different times was detected by real-time fluorescence quantitative PCR. The experimental operation was carried out in a real-time fluorescence quantitative PCR instrument ABI 7500 according to the instructions of Sangon Company SGExcelFastSYBR Mixture (With ROX). Sophora alopecuroides Lectin was used as the internal reference gene, and the primers are shown in Table 3 below:

[0055] Table 3 Quantitative primers for SaPEI7

[0056]

[0057] The PCR reaction system and procedure are shown in Tables 4 and 5 below:

[0058] Table 4 qRT-PCR reaction system

[0059]

[0060] Table 5 qRT-PCR reaction procedure

[0061]

[0062] The 2-ΔΔCT method was used to analyze the data and determine the relative expression of genes. The experiment was repeated three times technically and three times biologically.

[0063] The results showed that the expression of SaPEI7 gene was significantly increased after 4 h of salt stress treatment (see attached figure). Figure 3 As shown in the figure, note: "**" indicates a very significant difference compared with the control (p < 0.01). It was relatively downregulated at 72 h, but the expression level was still higher than that at 0 h, indicating that the SaPEI7 gene actively participated in the response process of sophora flavescens to salt stress.

[0064] Example 2: Cloning of SaPEI7 gene and construction of plant expression vector

[0065] Primer 5.0 was used to design primers for candidate genes (see Table 6 for specific primer sequences).

[0066] Table 6 SaPEI7 cloning primer sequences

[0067]

[0068] The candidate genes were cloned using the cDNA of Sophora alopecuroides root as a template. The cloning system is shown in Table 7:

[0069] Table 7 Candidate gene cloning reaction system

[0070]

[0071] The procedure is as shown in Table 8:

[0072] Table 8 PCR amplification program

[0073]

[0074] Agarose gel electrophoresis was used to verify the candidate gene bands (see attached Figure 4 As shown in the figure, note: "M" is a 2000bp marker). For target genes whose band size meets the expected value, gel excision and recovery are performed. The specific experimental steps for recovery are as follows:

[0075] 1. Cut the gel containing the target band on a blue light analyzer and place it into a 2 mL centrifuge tube;

[0076] 2. Add an equal volume of PG solution to the centrifuge tube and place in a 50°C water bath until the gel is completely dissolved;

[0077] 3. Transfer the liquid in (2) into the adsorption column, place it at room temperature for 2 minutes, centrifuge it at 12000 rpm for 1 minute, and discard the waste liquid;

[0078] 4. Add 600 μL PW to each tube, centrifuge at 12,000 rpm for 1 min, discard the waste liquid, and return the adsorption column to the centrifuge tube;

[0079] 5. Repeat the previous step;

[0080] 6. Centrifuge at 12000 rpm for 12 min;

[0081] 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 it stand at room temperature for 2 minutes, centrifuge at 12000 rpm for 2 minutes, and collect the DNA solution in the centrifuge tube.

[0082] The recovered fragments were tailed with A, and the system is as shown in Table 9:

[0083] Table 9 Recycling fragment plus A reaction system

[0084]

[0085] 72°C, 20 min, then 2 min on ice.

[0086] The candidate gene was connected to the cloning vector pMD18-T. The connection system is shown in Table 10:

[0087] Table 10 Ligation reaction system

[0088]

[0089] 16°C, 14 h, overnight ligation.

[0090] Transformation of Escherichia coli DH5α

[0091] Preparation and transformation of E. coli DH5α: 1. Streak the E. coli culture onto LB solid medium and incubate at 37°C for 12-14 hours. Pick a single colony and transfer it to 10 mL of LB liquid medium. Shake and incubate at 37°C and 180 rpm for 10-12 hours.

[0092] 2. Add 1 mL of bacterial solution to 50 mL of LB liquid medium and culture at 37°C with shaking until the OD600 reaches 0.4-0.6;

[0093] 3. Place the bacterial solution in an ice bath for 10 minutes, then place it in a 50 mL centrifuge tube. Centrifuge at 5000 rpm for 10 minutes at 4°C and pour off the liquid.

[0094] 4. Add 5 mL of pre-chilled 0.1 M CaCl2 to resuspend the bacteria and place on ice for 30 minutes;

[0095] Centrifuge at 5000 rpm for 10 min at 5.4°C and discard the supernatant.

[0096] 6. Pipette 1 mL of pre-chilled 0.1 M CaCl2 and resuspend the cells;

[0097] 7. Add 200 μL of 80% glycerol, mix evenly, aliquot 100 μL into each tube, freeze quickly in liquid nitrogen, and store at -80°C.

[0098] The extracted yeast plasmid was transformed into E. coli DH5α using the freeze-thaw method as follows:

[0099] 1. Thaw the competent cells in an ice box, add 10 μL of plasmid, and place on ice for 30 minutes;

[0100] Heat shock at 42°C for 90 seconds, immediately place on ice for 4 minutes, then add 800 μL of LB medium and culture at 37°C with shaking at 200 rpm for 1-2 hours.

[0101] 3. Centrifuge at 8000 rpm for 5 min, discard the supernatant, and add appropriate amount of LB medium to resuspend the bacteria;

[0102] 4. Pipette 50 μL of bacterial solution, spread it on LB medium (containing Amp, add 1 μL Amp per ml of medium), and culture it upside down at 37°C for 12-16 hours.

[0103] 5. Pick a single colony and add it to 1 mL of liquid LB medium (containing Amp). Incubate with shaking at 180 rpm at 37°C for 8 h. Pipette 1 μL of the bacterial solution as a template for PCR. Perform bacterial solution PCR. The reaction system and conditions are shown in Tables 11 and 12 below.

[0104] Table 11 PCR amplification reaction system

[0105]

[0106] Table 12 PCR amplification conditions

[0107]

[0108]

[0109] Single clones were picked for PCR verification, and 200 μL of samples with expected target bands were sent to a biological company for sequencing to further confirm the sequence accuracy of the cloned target gene.

[0110] Plasmid extraction:

[0111] The extraction method refers to the plasmid extraction kit method of Sangon Company.

[0112] 1. Add 5 mL of Buffer P1 to the precipitated cells, pipette, and vortex until the cells are completely suspended.

[0113] 2. Add 5 mL of Buffer P2, immediately and gently invert the solution to mix, and let it stand at room temperature for 3-5 minutes;

[0114] 3. Add 7 mL of Buffer P3, immediately invert the tube to mix thoroughly, and let it stand at room temperature for 5 minutes.

[0115] 4. Incubate in a 90°C water bath for 10 minutes, then place at -20°C for 10 minutes, and centrifuge at 12,000 rpm for 15 minutes.

[0116] 5. Transfer all the supernatant to the adsorption column, let it stand for 5 minutes, centrifuge at 8000 rpm for 2 minutes, pour out the waste liquid in the collection tube, and return it to the adsorption column;

[0117] 6. Add 5 mL of buffer DW1, centrifuge at 8000 rpm for 2 min, discard the waste liquid in the collection tube, and return it to the adsorption column;

[0118] 7. Add 5ml Wash Solution, centrifuge at 8000rpm for 2min, discard the waste liquid in the collection tube, return the adsorption column, and repeat once;

[0119] 8. Centrifuge the empty adsorption column at 10,000 rpm for 2 minutes;

[0120] 9. Place the adsorption column in a 50 mL centrifuge tube, add 1 mL of Elution Buffe to the adsorption membrane, let it stand for 2 minutes, centrifuge at 10,000 rpm for 2 minutes, and store the collected plasmid DNA solution at -20°C.

[0121] Plant expression vector construction

[0122] After the target gene recombinant cloning vector with correct sequencing is shaken to extract the plasmid, restriction sites are designed according to the target gene sequence and the multiple cloning site information of the plant expression vector pCAMBIA3301, and primers with double restriction sites are designed. The target gene is cloned and recovered using the recloning vector as a template.

[0123] The recovered fragments of the target gene and the pCAMBIA3301 vector after double enzyme digestion, the enzyme digestion system is shown in Table 13:

[0124] Table 13 Enzyme digestion reaction system

[0125]

[0126] 37℃, enzyme digestion for 3h.

[0127] Run electrophoresis, cut the gel, recover the fragments and connect them.

[0128] Connect the target gene fragment to the plant expression vector:

[0129] After the digestion product is detected by agarose gel electrophoresis imaging, it is recovered by gel recovery according to the recovery method. The recovered product is ligated. The ligation system is shown in Table 14:

[0130] Table 14 Ligation reaction system

[0131]

[0132] 16°C, overnight ligation for 14 h.

[0133] The preparation and transformation of Escherichia coli DH5α competent cells were carried out according to the above method.

[0134] Design universal primers for vector PCR detection of recombinant vector (see attached Figure 5 As shown, note: "M" is a 2000bp marker; "-" is negative; "+" is positive), and 200 μL of a single clone with the correct band was selected and sent to a biological company for sequencing verification.

[0135] Shake the cells and extract the plasmid:

[0136] The monoclonal bacterial solution with correct sequencing was inoculated into 50 mL of liquid LB (containing 50 μg / mL Kana) medium and cultured overnight at 37°C, 220 rpm for 12 h until the bacterial solution concentration reached OD 600 =1.0-1.5, then proceed to plasmid extraction. Simultaneously, take 700 μL and add 200 μL of 80% glycerol, mix well, and store at -80°C. Follow the above method for plasmid extraction.

[0137] Recombinant plasmid was transformed into Agrobacterium EHA105 and verified

[0138] Preparation of competent culture of Agrobacterium EHA105:

[0139] 1. Streak and activate EHA105 bacterial culture on YEP solid plates and incubate at 28°C for 2 days;

[0140] 2. Pick a single colony and place it in 50 mL of YEP liquid medium. Culture overnight at 28°C with shaking at 200 rpm.

[0141] 3. Take 2 mL of activated bacterial solution and add it to 50 mL of YEP liquid medium. Cultivate with shaking at 200 rpm at 28°C until the OD600 reaches about 0.5.

[0142] 4. Place the above culture on ice for 30 minutes;

[0143] Centrifuge at 3000 rpm for 5 min at 5.4°C and discard the supernatant.

[0144] 6. Add 1 mL of pre-cooled 0.2 M CaCl2 to suspend the cells, add 200 μL of glycerol and aliquot, freeze in liquid nitrogen and store at -80°C.

[0145] Transformation of Agrobacterium with recombinant plasmid:

[0146] 1. Thaw the competent Agrobacterium cells on ice;

[0147] 2. Add 3 μL of plasmid, quick freeze in liquid nitrogen for 5 minutes, and incubate in a water bath at 37°C for 5 minutes;

[0148] 3. Add 1 mL of antibiotic-free YEP liquid medium to (2) and culture at 200 rpm with shaking at 28°C for 2-4 h;

[0149] 4. Centrifuge at 10,000 rpm for 1 minute, discard the supernatant, and resuspend the cells in 80 μL of liquid YEP;

[0150] 5. Take 40 μL of bacterial solution and spread it on YEP plate (containing Rif and Kan) and culture it at 28℃ for 2 days.

[0151] 6. Pick a single clone for PCR verification (see attached Figure 6 Note: “M” is a 2000 bp marker; “-” is negative; “+” is positive) and then add glycerol and store at -80°C.

[0152] Example 4: Expression of SaPEI7 in Arabidopsis and Analysis of Salt Tolerance

[0153] The plant expression vector pCAMBIA3301-SaPEI7 was transformed into wild-type Arabidopsis thaliana using Agrobacterium-mediated transformation. The transgenic Arabidopsis thaliana was screened by basta. The expression level of the target gene in the positive plants was detected, and the salt tolerance of the transgenic Arabidopsis thaliana was analyzed. The specific method is as follows:

[0154] Arabidopsis sowing and cultivation

[0155] Select dried Arabidopsis seeds and sterilize them with 1 mL of 10% NaClO for 5 minutes. Then rinse them with sterile distilled water several times until clear (usually 4-6 times). Use a sterile pipette tip or tweezers to sow the sterilized seeds on 1 / 2 MS solid culture medium. Incubate them in the dark at 4°C for 72 hours for vernalization. After incubation in an incubator until the four-leaf stage, transplant them into prepared potting soil (peat soil: vermiculite = 1:1). Move them to an Arabidopsis culture room (day / night, 16 hours / 8 hours, 22°C / 20°C) and culture for 3-4 weeks. During this period, water them with 1 / 2× Hoagland nutrient solution every 3 days. After the Arabidopsis begins to shoot, prepare for the next step of infection and transformation.

[0156] Agrobacterium tumefaciens-mediated floral dip infection of Arabidopsis

[0157] 1. Streak activation of Agrobacterium EHA105 transformed with the recombinant plant expression vector on YEP solid medium containing Kan and Rif antibiotics, and culture at 28°C for 30-48 hours;

[0158] 2. Pick a single colony and inoculate it into YEP liquid medium containing Kan and Rif antibiotics. Incubate at 28°C, 200 rpm for 12-16 hours until the OD600 of the bacterial solution reaches 0.8-1.0.

[0159] 3. Centrifuge at 5000 rpm for 10 min at 4°C to collect Agrobacterium cells and resuspend in 1 / 2 MS liquid medium (add 20 μL Switt77 per 100 μL medium) to an OD600 of 0.7-0.9;

[0160] 4. Select wild-type Arabidopsis buds with only white spots, gently dip the inflorescence into the infection solution for 60-90 seconds, and carefully remove the remaining infection solution with filter paper;

[0161] 5. After infection, culture the Arabidopsis in dark conditions for 24 hours, then cultivate under normal conditions and replenish nutrient solution in time;

[0162] 6. Observe the growth of Arabidopsis thaliana. Re-infect after 12-15 days. Harvest the seeds after the Arabidopsis thaliana matures and dry them.

[0163] Transgenic Arabidopsis screening

[0164] The T0 generation Arabidopsis seeds were sterilized and vernalized, then sown on 1 / 2 MS solid medium containing 4 mg / L Basta for screening. Arabidopsis seedlings that grew normally on the screening medium were transplanted and then sprayed with 200 mg / L Basta for a second screening three days later. Arabidopsis seedlings that grew normally after the second screening were considered preliminary positive.

[0165] CTAB method for DNA extraction from Arabidopsis thaliana

[0166] 1. Place an appropriate amount of Arabidopsis leaves in a 2 mL centrifuge tube (put 2 steel balls in each tube), quickly freeze in liquid nitrogen, and grind into powder using a grinder.

[0167] 2. Add 600 μL of CTAB extract (preheated at 65°C) to the centrifuge tube, then add 15 μL of β-mercaptoethanol, mix thoroughly, and place in a 65°C water bath for 40 minutes, mixing every 10 minutes.

[0168] 3. Add 600 μL of chloroform, mix thoroughly by inversion, and centrifuge at 12,000 rpm for 15 min at 4°C.

[0169] 4. Transfer the supernatant after centrifugation to a new 1.5 mL centrifuge tube, add 400 μL of isopropanol, mix well, and let it settle at -20°C for 30 minutes;

[0170] 5. Centrifuge at 12,000 rpm for 15 min at 4°C and discard the supernatant.

[0171] 6. Add 1 mL of pre-cooled 75% ethanol to wash the precipitate, centrifuge at 12,000 rpm for 2 min at 4°C, and discard the supernatant.

[0172] 7. Repeat step (6) and dry the precipitate at room temperature.

[0173] 8. Dissolve the DNA pellet in 30 μL ddH2O and store at -20°C.

[0174] Propagation and testing of transgenic Arabidopsis

[0175] PCR molecular testing was performed using the screened Arabidopsis DNA as a template. The harvested T1 generation Arabidopsis seeds were sown, screened, and tested according to the above method to obtain T2 generation transgenic Arabidopsis. The above method was then repeated to obtain T3 generation transgenic Arabidopsis, and the seeds were harvested for the next experiment.

[0176] Real-time fluorescence quantitative PCR of overexpression lines

[0177] RNA was extracted from each transgenic Arabidopsis strain screened, reverse transcribed into cDNA, and expression levels were analyzed using real-time fluorescence quantitative PCR using cDNA as a template (see attached). Figure 7 As shown in the figure, Note: WT: wild type; L1, L2, L3, L4, L5 and L6 are SaPEI7 transgenic lines). In Arabidopsis, the Arabidopsis gene Actin2 was used as the internal reference gene. The specific quantitative system and procedure refer to the above method.

[0178] Salt stress treatment of Arabidopsis thaliana at the germination stage and germination rate statistics

[0179] Take out the dried T3 transgenic and wild-type Arabidopsis seeds, sterilize them and sow them on 1 / 2MS solid medium containing CK and 100mM NaCl. Vernalize them at 4℃ for 72h and culture them in the culture room for 3-15 days. Observe the growth of Arabidopsis every day. The specific growth status is shown in the attached figure. Figure 8 As shown (Note: WT is wild type; pei7 is Arabidopsis mutant, OE4 and OE5 are transgenic Arabidopsis with SaPEI7 gene). Three parallel experiments were performed each time and repeated three times. During the germination period of Arabidopsis, the germination status was counted in time, photos were taken, and root length was measured and counted (see attached). Figure 9 Note: WT is wild type; pei7 is Arabidopsis mutant, OE4 and OE5 are Arabidopsis transgenic with SaPEI7 gene, "*" indicates that the transgenic lines have significant differences compared with the control (p < 0.05), and "**" indicates that the transgenic lines have extremely significant differences compared with the control (p < 0.01).

[0180] 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 in the scope of protection of the present invention.

Claims

1. A Sophora alopecuroides SaPEI7 gene, characterized in that: The nucleotide sequence of the SaPEI7 gene is shown in SEQ ID NO.1, and the amino acid sequence of the sophora flavescens pectin methylesterase inhibitor protein SaPEI7 is shown in SEQ ID NO.

2.

2. An application of the Sophora alopecuroides SaPEI7 gene, based on the Sophora alopecuroides SaPEI7 gene according to claim 1, characterized in that: The application is the application of overexpressing the Sophora alopecuroides SaPEI7 gene in improving the salt tolerance of Arabidopsis thaliana.

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