Sophora alopecuroides SaHPPD gene and application thereof in regulation and control of plant stress tolerance

Abstract

The invention is suitable for the technical field of gene engineering, and provides a sophora alopecuroides SaHPPD gene, the nucleotide sequence of which is shown as SEQ ID NO: 1. The invention further provides a sophora alopecuroides p-hydroxyphenylpyruvate dioxygenase SaHPPD which is encoded by the SaHPPD gene according to the claim 1, and the amino acid sequence of the SaHPPD gene is as shown in SEQ ID NO: 2. The invention also provides an application of the sophora alopecuroides SaHPPD gene in the aspects of salt resistance, alkali resistance and drought resistance of plants. The invention further discloses application of the p-hydroxyphenylpyruvate dioxygenase SaHPPD in the aspects of salt resistance, alkali resistance and drought resistance of plants. The invention provides a sophora alopecuroides p-hydroxyphenylpyruvate dioxygenase gene which is named SaHPPD, the nucleotide sequence of the sophora alopecuroides p-hydroxyphenylpyruvate dioxygenase gene is as shown in SEQ ID NO: 1, the gene is composed of 1512 basic groups, and a complete open reading frame contains 1140bp, 296bp, 296bp, 296bp, 296bp and 296bp. The gene also contains 5'and 3 'untranslated region sequences, an initiation codon of an open reading frame is ATG, a termination codon is TAA, and the gene participates in plant salt, alkali and drought stress resistance.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a sophora alopecuroides SaHPPD gene and an application thereof in regulating plant stress tolerance. Background Art

[0002] At present, with the intensification of soil salinization and the decreasing cultivated land area, the impact on the yield and quality of crops is also increasing. At the same time, people's living standards are gradually improving, and the pursuit of nutritional value and quality is more prominent, which constitutes a contradiction between the two. Salt, alkali and drought stress are the main abiotic stresses that restrict plant growth and development, and are also serious problems that restrict crop production and ecological environment construction. China's saline-alkali land, especially inland saline-alkali land, is mostly a mixture of salinization and alkalization, with complex components and varying degrees, making it easy for people to confuse salt and alkali and collectively call them saline-alkali land. In fact, soil salinization and alkalization are mainly characterized by increased salinity and pH value, respectively. They are not two identical abiotic stresses, and salinization and alkalization often occur at the same time. This phenomenon is common in many areas. Exploring salt-tolerant, alkali-tolerant and drought-tolerant gene resources and studying the stress tolerance mechanism of crops are of great practical significance for the development and effective utilization of saline-alkali land.

[0003] Plants have different tolerance and adaptability to adverse environments at different growth stages. The seed germination and seedling stages are the most sensitive periods for plants to salt stress. Studies have shown that 0.9% NaCl is the most suitable salt tolerance identification index for legumes. Sophora alopecuroides is a plant with strong salt tolerance among legumes. It grows in arid and desert areas, has strong salt and drought resistance, and is an ideal stress resistance gene resource library. Transcriptome sequencing and yeast library screening have become effective means to mine stress resistance genes. So far, there are limited reports on stress resistance genes in Sophora alopecuroides. Therefore, it is of great significance to mine stress resistance genes from Sophora alopecuroides and explore its stress resistance mechanism.

[0004] 4-hydroxyphenylpyruvate dioxygenase (HPPD) is an enzyme that plays an important regulatory role in the process of tyrosine metabolism. It exists in almost all aerobic organisms. Tyrosine (Tyrosine) generates p-hydroxyphenylpyruvate acid (HPP) under the action of tyrosine aminotransferase (TAT), which is catalyzed by HPPD to convert into homogentisic acid (HGA) under aerobic conditions. Homodisic acid is further converted into plastoquinone and tocopherol. Tocopherol is an important compound for plant antioxidant effects and plays an important role in plant stress resistance. Plastoquinone can promote the synthesis of carotenoids in plants. Carotenoids are photosynthetic auxiliary pigments and important components of chlorophyll binding proteins and antenna systems in the photosynthetic reaction center. They also play the role of electron transporters and play an important role in free radical scavenging. The main way in which the external environment can cause direct damage to plants is through oxidation, and HPPD is one of the enzyme genes involved in the plant antioxidant pathway. Summary of the invention

[0005] The purpose of the embodiment of the present invention is to provide a sophora alopecuroides SaHPPD gene, aiming to solve the problems raised in the above background technology.

[0006] The embodiment of the present invention is implemented as follows: a sophora alopecuroides SaHPPD gene, whose nucleotide sequence is shown in SEQ ID NO: 1.

[0007] Another object of the embodiment of the present invention is to provide a sophora alopecuroides para-hydroxyphenylpyruvate dioxygenase SaHPPD, which is encoded by the SaHPPD gene as claimed in claim 1, and its amino acid sequence is shown in SEQ ID NO:2.

[0008] Another object of the embodiment of the present invention is to provide an application of the Sophora alopecuroides SaHPPD gene in plant tolerance to salt, alkali and drought.

[0009] Another object of the embodiment of the present invention is to provide an application of sophora alopecuroides para-hydroxyphenylpyruvate dioxygenase SaHPPD in plant tolerance to salt, alkali and drought.

[0010] The embodiment of the present invention provides a sophora alopecuroides p-hydroxyphenylpyruvate dioxygenase gene, which is named SaHPPD, and the nucleotide sequence is shown in SEQ ID NO: 1. The gene consists of 1512 bases in total, and the complete open reading frame contains 1140bp, and also contains 5' and 3' untranslated region sequences. The start codon of the open reading frame is ATG, and the stop codon is TAA. The gene is involved in plant resistance to salt, alkali and drought stress; the plant stress resistance-related protein SaHPPD provided in the embodiment of the present invention has an amino acid sequence as shown in SEQ ID NO: 2. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 The expression level of SaHPPD in the root transcriptome results of Sophora alopecuroides under control and adverse stress (salt, alkali and drought) treatments provided in Example 1 of the present invention;

[0012] Figure 2 The expression levels of the SaHPPD gene in different tissues of Sophora alopecuroides provided in Example 1 of the present invention;

[0013] Figure 3 This is a gel image of the SaHPPD gene clone provided in Example 2 of the present invention;

[0014] Figure 4 The PCR results of constructing the pCAMBIA3301-SaHPPD vector in DH5α provided in Example 2 of the present invention;

[0015] Figure 5 The PCR results of constructing the pCAMBIA3301-SaHPPD vector in Agrobacterium EHA105 provided in Example 2 of the present invention;

[0016] Figure 6 Relative quantitative analysis of the Arabidopsis thaliana strains transgenic with the SaHPPD gene provided in Example 3 of the present invention;

[0017] Figure 7 The phenotypes of wild-type Arabidopsis and Arabidopsis transgenic with SaHPPD gene under the control and NaCl, NaHCO3, and mannitol treatment conditions provided in Example 3 of the present invention;

[0018] Figure 8 The survival rate statistics of wild-type Arabidopsis and Arabidopsis transgenic with SaHPPD gene under the control and NaCl, NaHCO3, and mannitol treatment conditions provided in Example 3 of the present invention. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with 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.

[0020] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0021] Example 1: Cultivation of Sophora alopecuroides seedlings and transcriptome sequencing:

[0022] 10 g of bitter bean seeds with full grains were selected, and 5 mL of 98% concentrated sulfuric acid was added and soaked 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 NaCl concentration of 1.2%, NaHCO3 concentration of 1.2% and PEG6000 concentration of 8% and treated for 72 hours respectively; the roots of bitter bean under each treatment were taken, and the total RNA of the roots of bitter bean under different treatment conditions was extracted respectively, and RNA was extracted from the bitter bean roots extracted above and the stems and leaves of bitter bean under the 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 it into 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, and centrifuged at 10,000 × g for 5 min at 4 °C;

[0026] (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.

[0027] (4) Take the supernatant and place it in a new centrifuge tube. Add an equal volume of isopropanol, invert and 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-cooled 75% ethanol, vortex vigorously using a vortex oscillator, and centrifuge at 7500 × g for 5 min at 4°C;

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

[0030] (7) Add 50 μL RNase free water to dissolve, incubate at 58°C for 10 min, and store at -80°C.

[0031] Library construction and quality control:

[0032] The RNA used for library construction is total RNA. All mRNAs with polyA tails are enriched by Oligo (dT) magnetic beads, and then the obtained mRNA is randomly fragmented in Fragmentation Buffer with divalent cations. The fragmented mRNA is then used as a template and random oligonucleotides as primers to synthesize the first cDNA chain by M-MuLV reverse transcriptase system, and then the RNA chain is degraded by RNaseH, and the second cDNA chain is synthesized under the DNA polymerase I system. The double-stranded cDNA that has been purified and repaired at the end is connected to the sequencing adapter after adding the A tail, and the cDNA with a size of about 370 to 420 bp is screened by AMPure XPbeads, and PCR amplification is performed. The PCR product is purified again by AMPure XPbeads to obtain the final library.

[0033] After the library is constructed, Qubit2.0 Fluorometer is first used for preliminary quantification, and the library is diluted to 1.5 ng / uL. Then, the insert size of the library is detected using Agilent 2100bioanalyzer. When the insert size meets expectations, the effective concentration of the library is quantified by qRT-PCR (the effective concentration of the library is required to be higher than 2 nM) to ensure that the quality of the library meets the requirements.

[0034] The real-time fluorescence quantitative PCR system is shown in Table 1:

[0035] Table 1

[0036]

[0037] The real-time fluorescence quantitative PCR program is shown in Table 2:

[0038] Table 2

[0039]

[0040]

[0041] Transcriptome sequencing analysis:

[0042] Sequencing on the machine: After the quality of the preliminary library inspection 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 pool for amplification. Each sequencing cluster extends the complementary chain. Each addition of a fluorescently labeled dNTP can release fluorescence. The sequencer can capture the fluorescent signal and use computer software to convert the optical signal into a sequencing peak, thereby obtaining the sequence information of the fragment to be tested;

[0043] Data quality control: The image data of the sequencing fragments that can be detected by the high-throughput sequencer 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 connectors and poor sequencing quality. In order to ensure the quality and reliability of data analysis, the raw data needs to be filtered, mainly including removing reads whose base information cannot be determined, reads with connectors, 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 the clean data. All subsequent analyses are based on high-quality analysis of clean data;

[0044] 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 process and splice a large amount of RNA-seq data in turn. The main processes are as follows: Inchworm: First read the fq file of all reads, then convert the fq file into fa format, and merge the reads at the 3' end and the reads at the 5' end to obtain both.fa. Then decompose the reads according to k-mers, count the types of k-mers and the number of k-mers of each type, and sort them from high to low according to the frequency of k-mers. Select the k-mer with the highest frequency as the starting point to extend to the 3' end, extending only one base at a time, then count the number of occurrences of each k-mer after extension, select the one with the highest k-mer frequency as the extension path, and finally use the Overlap relationship to extend the k-mers to form a contig sequence; Chrysalis: cluster all contigs with similar regions higher than k-1-mers to form components, build a de Bruijn graph based on different components, and compare reads with components for verification; Butterfly: count transcripts (butterfly), split the graph into linear sequences, use the reads and pairs relationship to eliminate all erroneous sequences, simplify the de Bruijn graph of each component, output the full-length transcript, and finally obtain the splicing result file;

[0045] Transcript quality assessment: Benchmarking Universal Single-Copy Orthologs (BUSCO) assessment uses a single-copy orthologous gene database combined with software such as augustus, tblastn and hmmer to evaluate transcripts and their integrity. BUSCO software is used to evaluate the splicing quality of Trinity.fasta, cluster.fasta and unigene.fa, ​​and the integrity and accuracy of the splicing results are evaluated based on the proportion and integrity of the alignment results.

[0046] 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 esource) database protein sequences. Nt: NCBI official nucleic acid sequence database, including GenBank, EMBL and DDBJ nucleic acid sequences, which do not include 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 comparison; Swiss-Prot: concentrates on 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, etc. 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 genome or transcriptome of newly sequenced species. GO: It is an internationally standardized classification system for describing gene functions. PFAM: It is divided into two categories according to the credibility of the annotation results: the highly reliable Pfam-A family and the Pfam-B family automatically generated by the program. Through the HMMER3 program, you can search for the established HMM model to annotate genes. KOG: KOG (euKaryotic Ortholog Groups) combines evolutionary relationships to subdivide homologous genes from different species into different ortholog clusters. Currently, KOG has 4852 classifications, and genes from the same ortholog have the same function.

[0047] Reference sequence alignment: The transcripts obtained by Trinity splicing were used as reference sequences (Ref). The clean reads of each sample were mapped to Ref. Reads with alignment quality values ​​less than 10 and reads with non-paired successful alignment were filtered out. RSEM software was used in the alignment process, and bowtie2 default parameters were used in RSEM.

[0048] Differential gene analysis:

[0049] 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. DESeq2 software was used to analyze differentially expressed genes between sample groups. After differential analysis, the Benjamini-Hochberg method was used to correct the probability of hypothesis testing (P_value) for multiple hypothesis testing to obtain the false discovery rate (FDR). Differential genes were screened under the conditions of |log2Fold Change|>=1 and FDR<0.05, and the obtained differential 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 analysis in the KEGG database and GO-Term in the GO database, respectively, and the corrected p value less than 0.05 was used as the critical value for significant enrichment analysis.

[0050] Among the differentially expressed genes obtained, a gene annotated as p-hydroxyphenylpyruvate dioxygenase was screened and the NCBI database Blast

[0051] (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastn&PAGE_TYPE=

[0052] BlastSearch & LINK_LOC = blasthome) sequence comparison analysis revealed that it consists of 1512 base pairs, with a reading frame from the 169th to the 1308th base at the 5' end, encoding a protein composed of 379 amino acid residues. The SaHPPD gene contains an HPPD domain that is identical to that of the p-hydroxyphenylpyruvate dioxygenase family genes, indicating that the SaHPPD gene may have similar functions to the p-hydroxyphenylpyruvate dioxygenase family genes.

[0053] Among them, the expression level of SaHPPD in the root transcriptome results of Sophora alopecuroides under control and adverse stress (salt, alkali and drought) treatments is as follows Figure 1 As shown in the figure, CK is the control group; ST is the 1.2% NaCl treatment group; A_ST is the 1.2% NaHCO3 treatment group; DT is the 8% PEG6000 treatment group. Figure 1 It can be seen that the expression level of the gene SaHPPD was significantly upregulated under adverse stress (salt, alkali and drought);

[0054] The expression levels of SaHPPD in different tissues of Sophora alopecuroides are shown in Figure 2. Figure 2 shown.

[0055] Example 2: Cloning of SaHPPD gene and construction of plant expression vector:

[0056] Primer 5.0 was used to design the primers for the candidate genes. The specific primer sequences are shown in Table 3:

[0057] Table 3 SaHPPD cloning primer sequences

[0058]

[0059] The cDNA of Sophora alopecuroides root was used as a template to clone the candidate gene. The cloning system is shown in Table 4:

[0060] Table 4 Candidate gene cloning reaction system

[0061]

[0062] The procedure is shown in Table 5:

[0063] Table 5 PCR amplification program

[0064]

[0065] Agarose gel electrophoresis was used to verify the candidate gene bands (such as Figure 3 As shown in the figure, where "M" is a 2000bp Marker), the target gene whose band size meets the expected size is cut and recovered. The specific experimental steps for recovery are as follows:

[0066] (1) Cut the gel containing the target band on a blue light analyzer and place it in a 2 mL centrifuge tube;

[0067] (2) Add an equal volume of PG sol into a centrifuge tube and place in a 50°C water bath until the gel is completely dissolved;

[0068] (3) Transfer the liquid in (2) into the adsorption column, place it at room temperature for 2 min, centrifuge it at 12000 rpm for 1 min, and discard the waste liquid;

[0069] (4) Add 600 μL PW to each tube, centrifuge at 12,000 rpm for 1 min, discard the waste liquid, and put the adsorption column back into the centrifuge tube;

[0070] (5) Repeat the previous step;

[0071] (6) Centrifugation at 12000 rpm for 12 min;

[0072] (7) Prepare a new 1.5 mL centrifuge tube, place the adsorption column in the centrifuge tube, and drop 50 μL of ddH2O in the middle of the adsorption column. Let it stand at room temperature for 2 min, centrifuge at 12000 rpm for 2 min, and collect the DNA solution in the centrifuge tube.

[0073] The recovered fragments were tailed with A, and the system is shown in Table 6:

[0074] Table 6 Recycling fragment plus A reaction system

[0075]

[0076] 72°C, 20 min, on ice for 2 min.

[0077] The candidate gene was connected to the cloning vector pMD18-T, and the connection system is shown in Table 7:

[0078] Table 7 Ligation reaction system

[0079]

[0080] 16°C, 14h, overnight connection.

[0081] Transformation of E. coli DH5α:

[0082] The preparation and transformation of E. coli DH5α are as follows:

[0083] (1) Streak the E. coli culture on LB solid medium and culture at 37°C for 12-14 h. Pick a single colony and place it in 10 mL LB liquid medium and culture it at 37°C with shaking at 180 rpm for 10-12 h.

[0084] (2) Take 1 mL of bacterial solution and add it to 50 mL of LB liquid medium. Cultivate at 37°C with shaking until the OD600 is 0.4-0.6.

[0085] (3) Place the bacterial solution in an ice bath for 10 min in a 50 mL centrifuge tube and centrifuge at 5000 rpm for 10 min at 4 °C. Pour off the liquid.

[0086] (4) Add 5 mL of pre-cooled 0.1 M CaCl2, resuspend the bacterial solution, and place on ice for 30 min;

[0087] (5) Centrifuge at 5000 rpm for 10 min at 4°C and discard the supernatant;

[0088] (6) Pipette 1 mL of pre-cooled 0.1 M CaCl2 and suspend the cells;

[0089] (7) Add 200 μL of 80% glycerol, mix evenly, dispense 100 μL into each tube, freeze quickly in liquid nitrogen, and store at -80°C.

[0090] The extracted yeast plasmid was transformed into E. coli DH5α by freeze-thaw method as follows:

[0091] (1) Thaw the competent cells in an ice box, add 10 μL of plasmid, and place on ice for 30 min;

[0092] (2) 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;

[0093] (3) Centrifuge at 8000 rpm for 5 min, discard the supernatant, and add an appropriate amount of LB medium to resuspend the cells;

[0094] (4) Take 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.

[0095] (5) Pick a single colony and add it to 1 mL of liquid LB medium (containing Amp), shake and culture at 180 rpm for 8 h at 37°C, and take 1 μL of the bacterial solution as a template for PCR reaction. The reaction system and conditions are shown in Tables 8 and 9:

[0096] Table 8 PCR amplification reaction system

[0097]

[0098]

[0099] Table 9 PCR amplification conditions

[0100]

[0101] Single clone colonies 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.

[0102] Plasmid extraction:

[0103] The extraction method refers to the plasmid extraction kit method of Sangon Company, as follows:

[0104] (1) Add 5 mL of Buffer P1 to the precipitated cells, pipette and vortex until the cells are completely suspended;

[0105] (2) Add 5 mL of Buffer P2, gently invert the solution immediately, and let it stand at room temperature for 3-5 minutes;

[0106] (3) Add 7 mL of Buffer P3, immediately invert the solution to mix, and let stand at room temperature for 5 min;

[0107] (4) 90°C water bath for 10 min, then -20°C for 10 min, and centrifuged at 12,000 rpm for 15 min;

[0108] (5) Transfer all the supernatant to the adsorption column, place it for 5 min, centrifuge it at 8000 rpm for 2 min, pour out the waste liquid in the collection tube, and return it to the adsorption column;

[0109] (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;

[0110] (7) Add 5 ml of Wash Solution, centrifuge at 8000 rpm for 2 min, discard the waste liquid in the collection tube, return it to the adsorption column, and repeat once;

[0111] (8) Centrifuge the empty adsorption column at 10,000 rpm for 2 min;

[0112] (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 min, centrifuge at 10,000 rpm for 2 min, and store the collected plasmid DNA solution at -20°C.

[0113] Plant expression vector construction:

[0114] After the recombinant cloning vector of the target gene 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 pCHF3300, and primers with double restriction sites are designed. The target gene is cloned and recovered using the re-cloning vector as a template;

[0115] The recovered fragments of the target gene and the pCHF3300 vector after double restriction digestion, the restriction digestion system is shown in Table 10:

[0116] Table 10 Enzyme digestion reaction system

[0117]

[0118] 37℃, digest for 3h.

[0119] Run electrophoresis, cut the gel, recover it and connect it;

[0120] The target gene fragment is connected to the plant expression vector;

[0121] After the digestion product was detected by agarose gel electrophoresis imaging, the gel was recovered according to the recovery method, and the recovered product was connected. The connection system is shown in Table 11:

[0122] Table 11 Ligation reaction system

[0123]

[0124]

[0125] 16°C, overnight connection for 14h.

[0126] The preparation and transformation of Escherichia coli DH5α competent cells were carried out according to the above method.

[0127] Design universal vector primers for PCR detection of recombinant vectors, select single clones with correct bands, take 200 μL and send to biological company for sequencing verification. The results are as follows Figure 4 As shown, “M” is a 2000bp marker; “-” is negative, and “+” is positive.

[0128] Shake the cells and extract the plasmid as follows:

[0129] The monoclonal bacterial solution with correct sequencing was inoculated into 50 mL of liquid LB medium (containing 50 ug / mL Kana) and cultured overnight at 37°C, 220 rpm for 12 h until the bacterial solution concentration reached OD 600 =1.0-1.5, then you can extract the plasmid. At the same time, take 700μL and add 200μL 80% glycerol. Mix well and store at -80℃. Refer to the above method for plasmid extraction.

[0130] The recombinant plasmid was transformed into Agrobacterium EHA105 and verified:

[0131] Agrobacterium EHA105 competent cells were prepared as follows:

[0132] (1) Streak out and activate EHA105 bacterial culture on YEP solid plates and culture at 28°C for 2 days;

[0133] (2) Pick a single colony and place it in 50 mL of YEP liquid medium and culture it at 28°C with shaking at 200 rpm overnight;

[0134] (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 is about 0.5.

[0135] (4) Place the above culture on ice for 30 min;

[0136] (5) Centrifuge at 3000 rpm for 5 min at 4°C and discard the supernatant;

[0137] (6) Add 1 mL of pre-cooled 0.2 M CaCl2 to suspend the cells, add 200 μL of glycerol and aliquot, snap-freeze in liquid nitrogen and store at -80°C.

[0138] The recombinant plasmid was transformed into Agrobacterium as follows:

[0139] (1) Thaw the Agrobacterium competent cells on ice;

[0140] (2) Add 3 μL of plasmid, freeze in liquid nitrogen for 5 min, and place in a water bath at 37°C for 5 min;

[0141] (3) Add 1 mL of antibiotic-free YEP liquid medium to (2) and culture at 28°C with shaking at 200 rpm for 2-4 h;

[0142] (4) Centrifuge at 10,000 rpm for 1 min, discard the supernatant, and resuspend the cells in 80 μL of liquid YEP;

[0143] (5) Apply 40 μL of bacterial solution on YEP plate (containing Rif and Kan) and culture at 28°C for 2 days;

[0144] (6) Pick a single clone for PCR verification, add glycerol, and store at -80°C. The results are as follows Figure 5 As shown, “M” is a 2000bp marker; “-” is negative; and “+” is positive.

[0145] Example 3: Expression of SaHPPD in Arabidopsis and analysis of stress tolerance:

[0146] The plant expression vector pCHF3300-SaHPPD was transformed into wild-type Arabidopsis thaliana by Agrobacterium-mediated transformation, and the transgenic Arabidopsis thaliana was screened by basta. The expression 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:

[0147] Arabidopsis sowing and cultivation:

[0148] Select dried Arabidopsis seeds, first disinfect them with 1 mL of 10% NaClO for 5 min, then wash them with sterilized distilled water several times until they are clear (generally rinse 4-6 times), and use a sterilized gun tip or tweezers to sow the disinfected seeds on 1 / 2MS solid culture medium, culture them in the dark at 4°C for 72 h for vernalization, place them in an incubator and culture them until the four-leaf stage, then transplant them to the prepared potting soil (peat soil: vermiculite = 1:1), and move them to the Arabidopsis culture room (day / night, 16h / 8h, 22°C / 20°C) for culture for 3-4 weeks, during which time 1 / 2×hoagland nutrient solution is poured every 3 days. After the Arabidopsis begins to shoot, prepare for the next step of infection and transformation.

[0149] Agrobacterium tumefaciens-mediated floral dip method to infect Arabidopsis thaliana is as follows:

[0150] (1) Streaking and activating Agrobacterium EHA105 transformed with the recombinant plant expression vector on YEP solid medium containing Kan and Rif antibiotics, and culturing at 28°C for 30 h to 48 h;

[0151] (2) Pick a single colony and inoculate it into YEP liquid medium containing Kan and Rif antibiotics at 28°C, 200 rpm, and culture for 12-16 h until the OD600 of the bacterial solution reaches 0.8-1.0;

[0152] (3) Collect Agrobacterium cells by centrifugation at 5000 rpm for 10 min at 4°C and resuspend them in 1 / 2MS liquid medium (20 μL Switt77 per 100 μL medium) to an OD600 of 0.7-0.9;

[0153] (4) Select wild-type Arabidopsis thaliana flower 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;

[0154] (5) After infection, the Arabidopsis thaliana was cultured in the dark for 24 h, cultured under normal conditions, and supplemented with nutrient solution in time;

[0155] (6) Observe the growth of Arabidopsis thaliana. Re-infection can be carried out after 12-15 days. When the Arabidopsis thaliana matures, the seeds are harvested and dried.

[0156] Transgenic Arabidopsis screening:

[0157] The T0 generation Arabidopsis seeds obtained were disinfected and vernalized, and then sown in 1 / 2MS solid medium containing Basta (4 mg / L) for screening. After transplanting Arabidopsis seedlings that grew normally on the screening medium, they were sprayed with Basta at a concentration of 200 mg / L for secondary screening three days later. Arabidopsis seedlings that grew normally after two screenings were considered preliminary positive seedlings.

[0158] The CTAB method was used to extract Arabidopsis DNA as follows:

[0159] (1) Take an appropriate amount of Arabidopsis leaves and place them in a 2 mL centrifuge tube (two steel balls are placed in each tube), quickly freeze them in liquid nitrogen, and grind them into powder using a grinder;

[0160] (2) Add 600 μL of CTAB extract (preheated at 65°C) to the centrifuge tube, then add 15 μL of β-mercaptoethanol, invert to mix, and place in a 65°C water bath for 40 min, mixing every 10 min;

[0161] (3) Add 600 μL of chloroform, mix by inversion, and centrifuge at 12,000 rpm for 15 min at 4 °C;

[0162] (4) Transfer the supernatant after centrifugation into a new 1.5 mL centrifuge tube, add 400 μL of isopropanol, mix well, and settle at -20 °C for 30 min;

[0163] (5) Centrifuge at 12,000 rpm for 15 min at 4°C and remove the supernatant;

[0164] (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 remove the supernatant.

[0165] (7) Repeat step (6) and dry the precipitate at room temperature.

[0166] (8) Dissolve the DNA precipitate in 30 μL ddH2O and store at -20°C.

[0167] Transgenic Arabidopsis thaliana propagation and testing:

[0168] The Arabidopsis DNA obtained by screening was used as a template for PCR molecular detection. 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 used to obtain T3 generation transgenic Arabidopsis, and the seeds were harvested for the next experiment.

[0169] Real-time fluorescence quantitative PCR of overexpression strains:

[0170] RNA was extracted from each transgenic Arabidopsis strain screened, reverse transcribed into cDNA, and expression analysis was performed using real-time fluorescence quantitative PCR using cDNA as a template. The results were as follows: Figure 6 As shown, WT: wild type; L2, L3, L4, L5 are SaHPPD transgenic lines, and the Arabidopsis gene Actin2 is used as the internal reference gene in Arabidopsis. The specific quantitative system and procedure refer to the above method.

[0171] Arabidopsis thaliana salt, alkali and drought stress treatment and survival rate statistics at the germination stage:

[0172] Take out the dried T3 transgenic and wild-type Arabidopsis seeds, sterilize them and sow them on 1 / 2MS solid medium containing CK, 50mM NaCl, 10mM NaHCO3, and 200mM Mannitol. 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 as follows: Figure 7As shown, WT is the wild type, OE2 and OE3 are transgenic Arabidopsis thaliana with SaHPPD gene, and three parallel experiments were performed each time, and the survival rate statistics were obtained as shown in Figure 8 As shown, WT is the wild type; OE2 and OE3 are Arabidopsis thaliana transgenic with SaHPPD gene.

[0173] In summary, RNA-seq was performed on the sophora alopecuroides seedlings treated with simulated stress (NaCl, NaHCO3 and PEG), and differential expression analysis was performed on the obtained stress differential genes, so as to screen out the sophora alopecuroides genes related to stress response, and the gene CDS sequence was obtained by transcriptome sequencing technology. The gene was compared and analyzed by Blast comparison on the NCBI website, and it was found that the gene was a hydroxyphenylpyruvate dioxygenase gene, and it was named SaHPPD; according to the results of RNA-seq, the expression level of SaHPPD gene changed significantly under salt, alkali and drought stress, it was speculated that SaHPPD gene played an important role in the response of sophora alopecuroides to salt, alkali and drought stress. In order to verify whether the SaHPPD gene can improve the stress tolerance of plants, Arabidopsis thaliana overexpressing the SaHPPD gene was constructed to verify its stress tolerance. The experimental results showed that the gene can significantly improve the stress tolerance of Arabidopsis thaliana. The verification of the stress tolerance function of the SaHPPD gene provides a stress-resistant gene resource for creating new stress-resistant germplasm resources. In the embodiment of the present invention, Arabidopsis thaliana overexpressing the SaHPPD gene has more significant stress tolerance than the wild type. At the same time, in the bitter bean itself, the SaHPPD gene also has a significant response to external abiotic stress. The above experimental results prove that the SaHPPD gene is directly helpful for enhancing the stress tolerance of plants.

[0174] 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 protection scope of the present invention.

Claims

1. A sophora alopecuroides SaHPPD gene, characterized in that: Its nucleotide sequence is shown in SEQ ID NO:

1.

2. A sophora alopecuroides para-hydroxyphenylpyruvate dioxygenase SaHPPD, characterized in that It is encoded by the SaHPPD gene as claimed in claim 1, and its amino acid sequence is shown in SEQ ID NO:

2.

3. Use of the Sophora alopecuroides SaHPPD gene as claimed in claim 1 in plant tolerance to salt, alkali and drought.

4. Use of the sophora alopecuroides p-hydroxyphenylpyruvate dioxygenase SaHPPD as claimed in claim 2 in plant tolerance to salt, alkali and drought.

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