A sophora alopecuroides RING-type E3 ubiquitin ligase SaRCHY1 and its application

Abstract

The present invention is applicable to the field of genetic engineering technology and provides a sophora alopecuroides RING type E3 ubiquitin ligase. SaRCHY1 , characterized in that the sophora alopecuroides RING-type E3 ubiquitin ligase SaRCHY1 The nucleotide sequence of the gene is shown in SEQ ID NO. 1, and the amino acid sequence is shown in SEQ ID NO. 2. The present invention constructed a plant overexpression vector using this gene and successfully introduced it into wild-type Arabidopsis thaliana for preliminary functional verification. The results showed that overexpression of this gene in Arabidopsis thaliana significantly improved the alkali tolerance of Arabidopsis thaliana, providing a new resource for improving the stress resistance of crops through genetic engineering technology.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a sophora alopecuroides RING-type E3 ubiquitin ligase SaRCHY1 and an application thereof. Background Art

[0002] Soil salinization is one of the most costly challenges facing sustainable development worldwide. Land salinization is a major obstacle to land resource utilization in many arid and semi-arid regions of the world. In nature, salinization and alkalization coexist, and studies have shown that alkaline stress causes far greater damage to plants than salt stress. However, compared to salt stress, our understanding of plant adaptation to alkaline stress remains relatively superficial, and research on its mechanisms is insufficient. Therefore, as the impacts of stress on plants continue to increase, research on abiotic stress in plants is gaining increasing attention.

[0003] Sophora alopecuroides (L.), a member of the genus Sophora in the Leguminosae family, also known as bitter bean grass and bitter liquorice, is a perennial herbaceous plant with rhizomes and underground shoots. It exhibits remarkable drought and salinity tolerance, representing a rich reservoir of resistance genes. Therefore, screening and cloning alkaline stress-related genes from Sophora alopecuroides, analyzing their alkaline tolerance, and elucidating their related functions will facilitate the further utilization of alkaline-tolerance genes.

[0004] All physiological processes in plants are carried out through the normal functioning of proteins, including stress resistance. Proteins play a vital role in processes such as material metabolism, energy metabolism, cellular signaling, developmental regulation, and body defense. Two pathways of protein degradation exist in organisms. One, such as the degradation that occurs in the digestive tract, does not require energy, where proteases break down dietary proteins into amino acids for utilization. The other pathway requires energy and is characterized by high efficiency, specificity, and selectivity. The ubiquitin-proteasome pathway (UPP) represents the second pathway and is highly conserved across eukaryotes. The UPP pathway consists of four main components: ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), ubiquitin ligase (E3), and the 26S proteasome. E1 covalently binds to and activates ubiquitin (Ub) in an ATP-dependent manner, and then ligates to E2 to form the E2-Ub complex. E3 is responsible for recognizing substrate proteins, promoting E2 to transfer Ub to target proteins, and ultimately attaching one or more Ubs to target proteins. Ubiquitinated proteins can be recognized and degraded by the 26S proteasome.

[0005] Compared to E1 and E2 ligases, E3 ligases are by far the most diverse. This diversity directly determines the agility with which plants respond to environmental changes and the precision with which they regulate developmental steps and growth timing. E3 ligases are classified into two major categories: single-subunit and multi-subunit. Single-subunit ligases include the RING-finger, HECT (homologous to the E6-AP carboxyterminus), and the U-box 3 group, while multi-subunit ligases contain a group of enzymes with F-box sequence signatures. The RING-finger domain refers to eight of the 70 amino acids (cysteine and histidine) that chelate zinc ions to form the C3H2C3 (RING-H2) or C3H1C4 (RING-HC) configurations. Due to the large number of RING-type E3 ubiquitin ligases and their specialized functions in plant development and stress responses, research on these E3 ubiquitin ligases has become a hot topic.

[0006] By using the cDNA yeast expression library of Sophora alopecuroides seedlings to screen for genes related to alkali tolerance in Sophora alopecuroides, a RING-type E3 ubiquitin ligase gene was obtained and named SaRCHY1.

[0007] In Sophora alopecuroides, so far, there is no report on the role of the SaRCHY1 gene. Summary of the Invention

[0008] The purpose of the embodiments of the present invention is to provide a sophora alopecuroides RING-type E3 ubiquitin ligase SaRCHY1 and its application, aiming to solve the problems raised in the above background technology.

[0009] The embodiment of the present invention is achieved by providing a Sophora alopecuroides RING-type E3 ubiquitin ligase SaRCHY1, wherein the nucleotide sequence of the Sophora alopecuroides RING-type E3 ubiquitin ligase SaRCHY1 is shown in SEQ ID NO.1, and the amino acid sequence thereof is shown in SEQ ID NO.2.

[0010] Another object of the embodiments of the present invention is to use a sophora alopecuroides RING-type E3 ubiquitin ligase SaRCHY1 in improving plant alkali tolerance.

[0011] A further technical solution is that the specific steps of the application include:

[0012] Step 1: Tissue-specific expression of the SaRCHY1 gene in Sophora alopecuroides;

[0013] Step 2: Cloning of the SaRCHY1 gene and construction of a plant expression vector;

[0014] Step 3: Expression of SaRCHY1 in plants and analysis of alkali resistance.

[0015] The present invention provides a RING-type E3 ubiquitin ligase SaRCHY1 from Sophora alopecuroides and its applications. By sequencing transcriptomes of Sophora alopecuroides seedlings treated with simulated stress (NaCl, Na2CO3, and NaHCO3), and analyzing the differentially expressed genes, Sophora alopecuroides genes associated with alkaline stress were screened. Bioinformatics analysis of the nucleic acid sequence obtained by sequencing revealed that the gene belongs to the RING-type E3 ubiquitin ligase family and was named SaRCHY1. Cloning and quantitative primers were designed based on the sequence obtained. RT-PCR was used to detect changes in the expression of the gene in Sophora alopecuroides under alkaline stress (NaHCO3) to preliminarily investigate its role in the response to alkaline stress. A plant overexpression vector was constructed for the gene and successfully transformed into wild-type Arabidopsis thaliana for preliminary functional verification. The results showed that overexpression of the gene in Arabidopsis significantly improved alkaline tolerance, providing a new resource for improving crop stress resistance through genetic engineering. In the present invention, Arabidopsis thaliana transformed with pCHF3300-SaRCHY1 showed significant resistance to alkali stress compared to the wild type. Furthermore, in the native plant Sophora alopecuroides, expression of the SaRCHY1 gene also showed a significant response to alkali stress, indicating that overexpression of the SaRCHY1 gene can improve plant alkali tolerance. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The expression level of SaRCHY1 in the root transcriptome results of Sophora alopecuroides under control and adverse stress (salt, alkali and drought) treatments (CK: control; ST: 1.2% NaCl treatment; 1.2% NaHCO3 treatment; 8% PEG6000 treatment);

[0017] Figure 2 is the expression level of SaRCHY1 gene in different tissues of Sophora alopecuroides;

[0018] Figure 3 is the relative expression level of SaRCHY1 gene in leaves of Sophora alopecuroides L. in the control and 1.2% NaHCO3 treated condition;

[0019] Figure 4 is the relative expression level of SaRCHY1 gene in roots of Sophora alopecuroides L. in the control and 1.2% NaHCO3 treated condition;

[0020] Figure 5 This is the gel image of SaRCHY1 gene cloning ("M1" is 2000bp Marker);

[0021] Figure 6This is the electrophoresis diagram of double enzyme digestion and recovery of the pCHF3300-SaRCHY1 vector ("M" is a 2000 bp marker; "H2O" is negative);

[0022] Figure 7 PCR results for the construction of pCHF3300-SaRCHY1 vector in Agrobacterium EHA105 (Note: “M” is a 2000bp marker; “-” is negative; “+” is positive);

[0023] Figure 8 The following are the detection results of the T1 generation of Arabidopsis thaliana transgenic with the SaRCHY1 gene (M: 2000bp marker; L1-L30: Arabidopsis thaliana transgenic lines; the gel image below shows the corresponding bar gene detection results);

[0024] Figure 9 Relative quantitative analysis of each line of Arabidopsis thaliana transgenic with SaRCHY1 gene (WT: wild type; L5, L11, L16, and L21 are SaRCHY1 transgenic lines);

[0025] Figure 10 This is the test result of T3 generation of Arabidopsis thaliana transformed with SaRCHY1 gene;

[0026] Figure 11 The germination rate statistics of wild-type Arabidopsis and transgenic SaRCHY1 Arabidopsis under the control and different concentrations of NaHCO3 treatment conditions (Note: WT is wild type; #5 and #21 are transgenic SaRCHY1 Arabidopsis);

[0027] Figure 12 The growth of wild-type Arabidopsis and transgenic SaRCHY1 Arabidopsis seedlings on 1 / 2MS solid medium under control and different concentrations of NaHCO3 treatment (Note: WT is wild type; #5 and #21 are transgenic SaRCHY1 Arabidopsis);

[0028] Figure 13 The statistics of cotyledon green percentage of wild-type Arabidopsis and transgenic SaRCHY1 Arabidopsis seedlings under control and different concentrations of NaHCO3 treatment conditions (Note: WT is wild type; #5 and #21 are transgenic SaRCHY1 Arabidopsis);

[0029] Figure 14 Seedling growth of wild-type Arabidopsis and transgenic SaRCHY1 Arabidopsis under control and 300 mM NaHCO3 treatment conditions (Note: WT is wild type; #5 and #21 are transgenic SaRCHY1 Arabidopsis);

[0030] Figure 15Survival statistics of wild-type Arabidopsis and SaRCHY1-transgenic Arabidopsis after 8 days of treatment with 300 mM NaHCO3 (Note: WT is wild type; #5 and #21 are SaRCHY1-transgenic Arabidopsis);

[0031] Figure 16 Fresh weight statistics of wild-type Arabidopsis and SaRCHY1 transgenic Arabidopsis under control and 300mM NaHCO3 treatment conditions (Note: WT is wild type; #5 and #21 are SaRCHY1 transgenic Arabidopsis). DETAILED DESCRIPTION

[0032] 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.

[0033] The specific implementation of the present invention is described in detail below with reference to specific embodiments.

[0034] One embodiment of the present invention provides a Sophora alopecuroides RING-type E3 ubiquitin ligase SaRCHY1, wherein the nucleotide sequence of the Sophora alopecuroides RING-type E3 ubiquitin ligase SaRCHY1 is shown in SEQ ID NO.1, and the amino acid sequence thereof is shown in SEQ ID NO.2.

[0035] The acquisition of the sophora alopecuroides RING-type E3 ubiquitin ligase SaRCHY1 comprises the following steps:

[0036] Step 1: Cultivation of Sophora alopecuroides seedlings:

[0037] 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 1.2%, a NaHCO3 concentration of 1.2%, and a PEG6000 concentration of 8% for 72 hours respectively. The roots of bitter beans under each treatment were taken, and the total RNA of the bitter bean roots under different treatment conditions was extracted respectively. The RNA of the bitter bean roots of the four treatments extracted above was used for transcriptome sequencing.

[0038] Step 2: Extraction of total RNA:

[0039] (1) Take 50-100 mg of sample in a pre-cooled mortar, add liquid nitrogen and grind thoroughly into powder;

[0040] (2) Quickly add the ground powder to a 1.5 mL centrifuge tube containing 1 mL of TransZol Up, shake well, let stand at room temperature for 5 minutes, and centrifuge at 10,000 × g for 5 minutes at 4°C.

[0041] (3) Take the supernatant and place it in a new centrifuge tube. Add 200 μL of chloroform and shake vigorously for 30 seconds. Mix well and incubate at room temperature for 5 minutes. Centrifuge at 10,000 × g for 15 minutes at 4°C.

[0042] (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.

[0043] (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;

[0044] (6) Discard the supernatant and dry at room temperature for 5 min;

[0045] (7) Dissolve the sample in 50 µL of RNase-free water, incubate at 58°C for 10 min, and store at -80°C.

[0046] Step 3: Library construction:

[0047] The library was constructed using total RNA. Oligo (dT) magnetic beads were used to enrich all mRNAs with polyA tails. The resulting mRNA was then randomly fragmented in Fragmentation Buffer using divalent cations. The fragmented mRNA was 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 was then degraded with RNase H, and the second-strand cDNA was synthesized using the DNA polymerase I system. The purified, end-repaired double-stranded cDNA was then A-tailed and ligated to sequencing adapters. cDNAs between 370 and 420 bp were selected using AMPure XP beads for PCR amplification. The PCR products were then purified again using AMPure XP beads to obtain the final library.

[0048] After the library is constructed, a Qubit2.0 Fluorometer is used for preliminary quantification, and the library is diluted to 1.5 ng / uL. The insert size of the library is then detected using an Agilent 2100 bioanalyzer. When the insert size meets the expected value, the effective concentration of the library is quantified by qRT-PCR (the effective concentration of the library is required to be greater than 2 nM) to ensure that the library quality meets the requirements.

[0049] Table 1 Real-time fluorescence quantitative PCR system

[0050]

[0051] Table 2 Real-time fluorescence quantitative PCR program

[0052]

[0053] Step 4: Transcriptome sequencing analysis:

[0054] Sequencing: After preliminary library quality checks, the library is pooled according to the effective concentration and target data volume requirements before undergoing Illumina sequencing, generating 150 bp paired-end reads. The basic principle of sequencing is 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 strand. Each added fluorescently labeled dNTP emits a corresponding fluorescence. The sequencer captures the fluorescence signal and, using computer software, converts the light signal into sequencing peaks, thereby obtaining the sequence information of the fragment being tested.

[0055] Data quality control: Image data from sequencing fragments detected by high-throughput sequencers is converted into sequence data (reads) using CASAVA base calling. These files are in fastq format and primarily contain sequence information and quality information. The raw sequencing data may contain reads with sequencing adapters and poor sequencing quality. To ensure the quality and reliability of data analysis, raw data filtering is required. This primarily involves removing reads with uncertain base information, reads with adapters, and low-quality reads (e.g., reads with a Qphred value of 20 or less, representing at least 50% of the total read length). Furthermore, Q20, Q30, and GC content are calculated for the clean data. All subsequent analyses are based on high-quality clean data.

[0056] Transcript splicing: Trinity is an efficient and stable splicing software for RNA-seq data. It combines three independent software modules: Inchworm, Chrysalis, and Butterfly, to sequentially process and splice large amounts of RNA-seq data. The main steps are as follows: Inchworm: First, read the fq file of all reads, then convert the fq file into fa format. The 3' and 5' reads are merged to obtain 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 then sorted from high to low according to their frequency. The k-mer with the highest frequency is selected as the starting point and extended towards the 3' end, extending only one base at a time. The number of occurrences of each k-mer after extension is then counted, and the k-mer with the highest frequency is selected as the extension path. Finally, the k-mers are extended using the overlap relationship to form a contig sequence. Chrysalis: Clusters all contigs with similarity greater than k-1-mers to form components. De Bruijn graphs are constructed based on the components, and reads are aligned with the components for verification. Butterfly: Counts transcripts, splits the graph into linear sequences, eliminates any erroneous sequences using the read-pair relationship, simplifies the De Bruijn graph for each component, outputs the full-length transcript, and ultimately generates a splicing result file.

[0057] 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 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.

[0058] Gene Function Annotation: Gene function annotation is based on the following databases: Nr: NCBI's official protein sequence database, including protein sequences from the PDB (Protein DataBank) protein database, GenBank gene protein coding sequences, SwissProt protein sequences, PRF (Protein Research Foundation), and PIR (Protein Information esource) databases. Nt: NCBI's official nucleotide sequence database, including nucleotide sequences from GenBank, EMBL, and DDBJ, excluding EST, PAT, STS, WGS, GSS, TSA, and HTG sequences. Pfam: A classification system for protein domain annotation. Proteins are composed of domains, and the protein sequences of each specific domain are conserved to a certain extent. PFAM classifies protein domains into different protein families and establishes HMM statistical models for the 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 the metabolic pathways of gene products and compounds in cells, as well as the functions of these gene products. It integrates data on genomes, chemical molecules, and biochemical systems, including metabolic pathways, drugs, diseases, functional models, gene sequences, and genomes. The KO system links together various KEGG annotation systems. KEGG has established a complete KO annotation system that can be used for functional annotation of the genomes or transcriptomes of newly sequenced species. GO: An internationally standardized classification system for describing gene function. PFAM: Based on the credibility of the annotation results, it is divided into two categories: the highly reliable Pfam-A family and the program-generated Pfam-B family. The HMMER3 program can be used to search the established HMM model 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 4,852 classifications. Genes from the same ortholog have the same function.

[0059] 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 bowtie2 default parameters.

[0060] Step 5: Differential gene analysis:

[0061] 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 calculate the false discovery rate (FDR). Differentially expressed genes were screened under the criteria of |log2FoldChange| >= 1 and FDR < 0.05. These differentially expressed genes were then annotated using the KEGG and GO databases. KEGG pathway and GO term enrichment analyses were performed using GOseq and KOBAS software, respectively, based on the hypergeometric test. A corrected p-value of less than 0.05 was considered significant for enrichment analysis.

[0062] Among the differentially expressed genes obtained, we screened for a gene annotated as a homeodomain leucine zipper transcription factor. Sequence alignment analysis using the NCBI database Blast revealed that it consists of 738 base pairs, with a reading frame from base 1 to base 738 at the 5' end, encoding a protein composed of 245 amino acid residues. The SaRCHY1 protein contains a homeodomain tightly linked to the leucine zipper sequence, suggesting that the SaRCHY1 gene may have similar functions to genes in the homeodomain leucine zipper transcription factor family.

[0063] In a preferred embodiment of the present invention, the application of the sophora alopecuroides RING-type E3 ubiquitin ligase SaRCHY1 in improving plant alkali tolerance comprises the following specific steps:

[0064] Step 1: Tissue-specific expression of the SaRCHY1 gene in Sophora alopecuroides:

[0065] The bitter bean was subjected to stress treatment, and the treatment method was the same as the above embodiment. After 4 hours of treatment. The roots and leaves of the bitter bean were taken at the same time. 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 SaRCHY1 gene in different tissues of the bitter bean 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 SGExcel FastSYBR Mixture (With ROX) of Sangon Company. Sophora alopecuroides Lectin was used as the internal reference gene, and the primers are as follows:

[0066] Table 3 Quantitative primers for SaRCHY1

[0067]

[0068] The PCR reaction system and procedure are as follows:

[0069] Table 4 qRT-PCR reaction system

[0070]

[0071] Table 5 qRT-PCR reaction procedure

[0072]

[0073] 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.

[0074] The results showed that the SaRCHY1 gene was expressed in the roots, stems and leaves of Sophora alopecuroides, with the highest expression level in the leaves. Under alkali treatment conditions, the expression level in the roots and leaves was significantly increased, with the highest expression level in the leaves at 24 hours and in the roots at 12 hours.

[0075] Step 2: Cloning of the SaRCHY1 gene and construction of a plant expression vector:

[0076] Primer 5.0 was used to design primers for candidate genes (see Table 6 for specific primer sequences):

[0077] Table 6 SaRCHY1 cloning primer sequences

[0078]

[0079] The candidate gene was cloned using the cDNA of Sophora alopecuroides root as a template. The cloning system is as follows:

[0080] Table 7 Candidate gene cloning reaction system

[0081]

[0082] The procedure is as follows:

[0083] Table 8 PCR amplification program

[0084]

[0085] Agarose gel electrophoresis was used to verify the candidate gene bands. For target genes whose band sizes matched the expected values, gel excision and recovery were performed. The specific experimental steps for recovery were as follows:

[0086] 1. Cut the gel containing the target band on a blue light analyzer and place it into a 2 mL centrifuge tube;

[0087] 2. Add an equal volume of PG solution to the centrifuge tube and incubate in a 50°C water bath until the gel is completely dissolved.

[0088] 3. Transfer the liquid from the previous step into the adsorption column, let it stand at room temperature for 2 minutes, centrifuge at 12,000 rpm for 1 minute, and discard the waste liquid.

[0089] 4. Add 600 µL of PW to each tube and centrifuge at 12,000 rpm for 1 min. Discard the waste liquid and return the adsorption column to the centrifuge tube.

[0090] 5. Repeat the previous step;

[0091] 6. Centrifuge at 12,000 rpm for 12 minutes.

[0092] 7. Prepare a new 1.5 mL centrifuge tube, place the adsorption column into the tube, and dropwise add 50 µL of ddH2O to the middle of the adsorption column. Let it stand at room temperature for 2 minutes, then centrifuge at 12,000 rpm for 2 minutes to collect the DNA solution in the tube.

[0093] The recovered fragments are A-tailed, and the system is as follows:

[0094] Table 9 Recycling fragment plus A reaction system

[0095]

[0096] 72°C for 20 min, then on ice for 2 min.

[0097] The candidate gene was connected to the cloning vector pMD18-T. The connection system is as follows:

[0098] Table 10 Ligation reaction system

[0099]

[0100] Ligation at 16°C for 14 h or overnight.

[0101] Transformation of Escherichia coli DH5α

[0102] Preparation and transformation of Escherichia coli DH5α:

[0103] 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. Incubate at 37°C with shaking at 180 rpm for 10-12 hours.

[0104] 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.

[0105] 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.

[0106] 4. Add 5 mL of pre-chilled 0.1 M CaCl2 to resuspend the bacteria and place on ice for 30 min.

[0107] Centrifuge at 5000 rpm for 10 min at 5.4°C and discard the supernatant.

[0108] 6. Pipette 1 mL of pre-chilled 0.1 M CaCl2 and resuspend the cells.

[0109] 7. Add 200 µL of 80% glycerol, mix evenly, aliquot 100 µL into each tube, snap freeze in liquid nitrogen, and store at -80°C.

[0110] The extracted yeast plasmid was transformed into E. coli DH5α using the freeze-thaw method as follows:

[0111] 1. Thaw the competent cells in an ice box, add 10 µL of plasmid, and incubate on ice for 30 min.

[0112] 2. Heat shock at 42°C for 90 seconds, immediately place on ice for 4 minutes, then add 800 µL of LB medium and shake at 200 rpm at 37°C for 1-2 hours.

[0113] 3. Centrifuge at 8000 rpm for 5 min, discard the supernatant, and add appropriate amount of LB medium to resuspend the bacteria;

[0114] 4. Pipette 50 µL of bacterial solution and spread it on LB medium (containing Amp, add 1 µL Amp per ml of medium), and invert and culture at 37°C for 12-16 hours.

[0115] 5. Pick a single colony and add it to 1 mL of liquid LB medium (containing Amp). Incubate the culture at 37°C with shaking at 180 rpm for 8 h. Pipette 1 µL of the bacterial solution as a template for PCR. Perform PCR on the bacterial solution. The reaction system and conditions are shown in Tables 11 and 12.

[0116] Table 11 PCR amplification reaction system

[0117]

[0118] Table 12 PCR amplification conditions

[0119]

[0120] 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.

[0121] Plasmid extraction

[0122] The extraction method refers to the plasmid extraction kit method of Sangon Company.

[0123] 1. Add 5 mL of Buffer P1 to the precipitated cells, pipette, and vortex until the cells are completely suspended.

[0124] 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.

[0125] 3. Add 7 mL of Buffer P3, immediately invert the tube to mix thoroughly, and let it stand at room temperature for 5 minutes.

[0126] 4. Incubate at 90°C in a water bath for 10 min, then at -20°C for 10 min, and centrifuge at 12,000 rpm for 15 min.

[0127] 5. Transfer all supernatant to the adsorption column, let it sit for 5 minutes, centrifuge at 8000 rpm for 2 minutes, discard the waste liquid in the collection tube, and return it to the adsorption column.

[0128] 6. Add 5 mL of buffer DW1 and centrifuge at 8000 rpm for 2 min. Discard the waste liquid in the collection tube and return it to the adsorption column.

[0129] 7. Add 5 ml of Wash Solution, centrifuge at 8000 rpm for 2 min, discard the waste liquid in the collection tube, return the column to the adsorption column, and repeat once;

[0130] 8. Centrifuge the empty adsorption column at 10,000 rpm for 2 min;

[0131] 9. Place the adsorption column in a 50 mL centrifuge tube, add 1 mL of Elution Buffer 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.

[0132] Plant expression vector construction

[0133] After the target gene recombinant cloning vector with correct sequencing is shaken to extract the plasmid, enzyme cutting sites are designed according to the target gene sequence and the multiple cloning site information of the plant expression vector pCHF3300, and primers with double enzyme cutting sites are designed. The target gene is cloned and recovered using the recloning vector as a template.

[0134] The recovered fragments of the target gene and the pCHF3300 vector were digested with double enzymes. The enzyme digestion system is shown in Table 13.

[0135] Table 13 Enzyme digestion reaction system

[0136]

[0137] 37°C, digest for 3 h.

[0138] Run electrophoresis, cut the gel, recover the fragments and connect them; connect the target gene fragment to the plant expression vector; after the enzyme digestion product is detected by agarose gel electrophoresis imaging, recover the fragments by gel according to the recovery method. The recovered product is connected. The connection system is shown in Table 14.

[0139] Table 14 Ligation reaction system

[0140]

[0141] Ligate overnight at 16°C for 14 h.

[0142] The preparation and transformation of Escherichia coli DH5α competent cells were carried out according to the above method.

[0143] Design universal vector primers for PCR detection of recombinant vectors, select single clones with correct bands, take 200 μL and send to biological companies for sequencing verification.

[0144] Shake the cells and extract the plasmid

[0145] Inoculate a correctly sequenced monoclonal clone into 50 mL of liquid LB medium (containing 50 μg / mL Kana). Incubate overnight at 37°C, 220 rpm, and incubate for 12 hours. Once the concentration reaches an OD600 of 1.0-1.5, proceed to plasmid extraction. Simultaneously, add 700 μL of the aliquot to 200 μL of 80% glycerol, mix well, and store at -80°C. Follow the same method for plasmid extraction as described above.

[0146] Recombinant plasmid was transformed into Agrobacterium EHA105 and verified

[0147] Preparation of competent Agrobacterium EHA105

[0148] 1. Streak and activate EHA105 bacterial culture on YEP solid plates and incubate at 28°C for 2 days.

[0149] 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.

[0150] 3. Add 2 mL of activated bacterial solution to 50 mL of YEP liquid medium and culture at 28°C with shaking at 200 rpm until the OD600 reaches approximately 0.5.

[0151] 4. Place the above culture on ice for 30 minutes;

[0152] 5. Centrifuge at 3000 rpm for 5 min at 4°C and discard the supernatant.

[0153] 6. Add 1 mL of pre-chilled 0.2 M CaCl2 to resuspend the cells. Add 200 µL of glycerol and aliquot. Quickly freeze in liquid nitrogen and store at -80°C.

[0154] Transformation of Agrobacterium with recombinant plasmid

[0155] 1. Thaw the competent Agrobacterium cells on ice;

[0156] 2. Add 3 µL of plasmid, freeze in liquid nitrogen for 5 min, and then incubate in a 37°C water bath for 5 min.

[0157] 3. Add 1 mL of antibiotic-free YEP liquid medium to (2) and culture at 200 rpm at 28°C for 2-4 h.

[0158] 4. Centrifuge at 10,000 rpm for 1 min, discard the supernatant, and resuspend the cells in 80 µL of liquid YEP.

[0159] 5. Spread 40 µL of the bacterial solution on a YEP plate (containing Rif and Kan) and incubate at 28°C for 2 days.

[0160] 6. Pick a single colony for PCR verification, add glycerol, and store at -80°C.

[0161] Step 3: Expression of SaRCHY1 in Arabidopsis and analysis of alkali tolerance:

[0162] The plant expression vector pCHF3300-SaRCHY1 was transformed into wild-type Arabidopsis thaliana using Agrobacterium-mediated transformation. The transgenic Arabidopsis thaliana was screened for basta, and the expression level of the target gene in the positive plants was detected. The alkaline tolerance of the transgenic Arabidopsis thaliana was also analyzed. The specific method is as follows:

[0163] Arabidopsis sowing and cultivation

[0164] Select dried Arabidopsis seeds and sterilize them with 1 mL of 10% NaClO for 5 minutes. Rinse with sterile distilled water several times until clear (typically 4-6 rinses). Use a sterile pipette tip or forceps to sow the sterilized seeds onto 1 / 2 MS solid medium. Incubate in the dark at 4°C for 72 hours for vernalization. Cultivate in an incubator until the four-leaf stage, then transplant to prepared potting soil (1:1 peat soil:vermiculite). Move to an Arabidopsis cultivation room (day / night cycle: 16 h / 8 h, 22°C / 20°C) for 3-4 weeks, watering with 1 / 2× Hoagland nutrient solution every 3 days. Once the Arabidopsis begins to shoot, proceed to the next step of infection and transformation.

[0165] Agrobacterium tumefaciens-mediated floral dip infection of Arabidopsis

[0166] 1. Activate Agrobacterium EHA105 transformed with the recombinant plant expression vector by streaking on YEP solid medium containing Kan and Rif antibiotics and culture at 28°C for 30-48 hours.

[0167] 2. Pick a single colony and inoculate it into YEP liquid medium containing Kan and Rif antibiotics. Incubate at 28°C, 200 rpm, and culture for 12-16 hours until the OD600 of the culture reaches 0.8-1.0.

[0168] 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 of Switt77 per 100 µL of medium) to an OD600 of 0.7-0.9.

[0169] 4. Select wild-type Arabidopsis buds that are only white. Gently dip the inflorescence into the infection solution for 60-90 seconds. Carefully remove any remaining infection solution with filter paper.

[0170] 5. After infection, culture the Arabidopsis plants in darkness for 24 hours, then continue culturing under normal conditions and replenish nutrient solution promptly.

[0171] 6. Observe the growth of the Arabidopsis thaliana. Re-infect after 12-15 days. Harvest the seeds when the Arabidopsis thaliana matures and dry them.

[0172] Transgenic Arabidopsis screening

[0173] After disinfection and vernalization, the T0 generation Arabidopsis seeds were 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 two screenings were considered preliminary positive.

[0174] CTAB method for DNA extraction from Arabidopsis thaliana

[0175] 1. Place an appropriate amount of Arabidopsis leaves in a 2 mL centrifuge tube (use two steel balls per tube), quickly freeze in liquid nitrogen, and grind into powder using a grinder.

[0176] 2. Add 600 µL of CTAB extract (preheated at 65°C) to the centrifuge tube, followed by 15 µL of β-mercaptoethanol. Mix thoroughly by inversion, then place in a 65°C water bath for 40 min, mixing every 10 min.

[0177] 3. Add 600 µL of chloroform, mix thoroughly by inversion, and centrifuge at 12,000 rpm for 15 min at 4°C.

[0178] 4. Transfer the supernatant after centrifugation to a new 1.5 mL centrifuge tube, add 400 µL of isopropanol, mix thoroughly, and allow to settle at -20°C for 30 min.

[0179] 5. Centrifuge at 12,000 rpm for 15 min at 4°C and discard the supernatant.

[0180] 6. Add 1 mL of pre-chilled 75% ethanol to wash the precipitate. Centrifuge at 12,000 rpm for 2 min at 4°C and discard the supernatant.

[0181] 7. Repeat step (6) and dry the precipitate at room temperature.

[0182] 8. Dissolve the DNA pellet in 30 µL of ddH2O and store at -20°C.

[0183] Propagation and testing of transgenic Arabidopsis

[0184] 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.

[0185] Real-time fluorescence quantitative PCR of overexpression lines

[0186] RNA was extracted from each transgenic Arabidopsis line screened and reverse transcribed into cDNA. The expression level was analyzed by real-time fluorescence 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 referred to the above method.

[0187] Alkali stress treatment of Arabidopsis thaliana at the germination stage and germination rate statistics

[0188] Dried T3 transgenic and wild-type Arabidopsis seeds were removed, disinfected, and sown on 1 / 2 MS solid medium containing CK, 7 mM NaHCO₃, 8 mM NaHCO₃, and 9 mM NaHCO₃. Vernalize at 4°C for 72 hours and incubate in a culture chamber for 3–15 days. Observe the growth of the Arabidopsis daily. Each experiment was repeated three times in triplicate. During germination, count the germination status of the Arabidopsis seeds.

[0189] Analysis of Alkali Tolerance in Arabidopsis thaliana at the Seedling Stage

[0190] For the alkaline stress experiment on Arabidopsis seedlings, when only four leaves have expanded, the plants are transferred to soil for cultivation (peat soil: vermiculite = 1:1). One month later, 300 mM NaHCO3 is poured into the tray. After about 7 days, when the overexpression lines show obvious differences from the wild type, the relevant indicators are counted.

[0191] 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 RING-type E3 ubiquitin ligase SaRCHY1 , characterized in that, Sophora alopecuroides RING type E3 ubiquitin ligase SaRCHY1 The nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.

2.

2. A sophora alopecuroides RING-type E3 ubiquitin ligase according to claim 1 SaRCHY1 Application in improving plant alkali resistance.

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