Use of SbJMJ02 gene or its expression promoter to improve drought and / or salt tolerance of plants

Abstract

This invention belongs to the field of molecular biology, specifically involving SbJMJ02 The application of genes or their expression promoters in improving plant drought and / or salt tolerance. SbJMJ02 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2. This invention has experimentally demonstrated that... SbJMJ02 The gene's expression was significantly upregulated under drought and salt stress conditions, and its encoded protein, when heterologously expressed in yeast and Arabidopsis, significantly enhanced the organisms' tolerance to high salt, high osmotic pressure, and drought stress. Based on this, the present invention provides... SbJMJ02 The application of genes, their encoded proteins, or expression promoters in improving plant drought and salt tolerance provides new genetic resources and molecular breeding strategies for the genetic improvement of crop stress resistance.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, specifically to... SbJMJ02 Application of genes or their expression promoters in improving plant drought and / or salt tolerance. Background Technology

[0002] Under natural conditions, plants, as sessile organisms, face various influences during their growth and development, such as abiotic stresses like drought, salt, heat, and cold. Among these, drought and soil salinity, which affect the osmotic pressure balance within the plant, can cause irreversible damage and are major factors limiting crop growth. Plants may experience multiple abiotic stresses throughout their life cycle, rather than a single stress event. Research has shown that prior exposure to mild abiotic stress can prepare plants for subsequent exposure to more severe stress environments. This phenomenon, known as "plant priming," is considered a potential mechanism for plant adaptation to abiotic stress and is related to "plant stress memory." Existing results indicate that this stress memory participates in epigenetic modifications.

[0003] Histone methylation primarily occurs at the N-terminal lysine and arginine residues of histones H3 and H4. Methylation at different lysine sites on histones exerts different effects on gene expression regulation. Methylation at H3K4, H3K36, and H3K79, located in regions of active transcription in chromatin, is generally considered a promoter marker; methylation at H3K9, H3K27, and H4K20, on the other hand, acts as a repressor marker, associated with gene silencing and chromatin aggregation. Histone methylation is reversible and is regulated by both histone methyltransferases and histone demethylases to maintain a dynamic equilibrium.

[0004] Histone demethylases containing the JmjC domain (JHDM) use Fe(II) and α-ketoglutarate (α-KG) as cofactors to catalyze the removal of mono / di / trimethylmethyl groups from lysine residues via hydroxylation. Currently, based on sequence similarity and catalytic specificity, JHDM proteins in plants can be divided into five subfamilies: KDM5 / JARID subfamily members can remove methyl groups from H3K4me1 / 2 / 3; KDM4 / JHDM3 subfamily members clear H3K9me2 / 3 and H3K36me2 / 3; KDM3 / JHDM2 subfamily members can remove methyl groups from H3K9me1 and H3K9me2; JMJD6 subfamily proteins have H3K27me2 / 3 demethylation activity; and JmjC domain-only subfamily proteins can remove methyl groups from H3K27me3. JHDM proteins regulate the initiation of transcription or the repression of silencing of downstream genes by removing these types of histone methylation modifications.

[0005] In the model plant Arabidopsis thaliana, AtJMJ27 can reduce the level of H3K9me2 in ultraviolet-induced staining centers and form a complex with the photodamage recognition factor DNA damage binding protein 2 (DDB2) to promote photodamage repair. The AtJMJ15 loss-of-function mutant shows increased sensitivity to salt stress. Furthermore, AtJMJ15 directly binds to and demethylates the H3K4me3 markers in the promoters and coding regions of two transcription factors negatively correlated with abiotic stress tolerance, WRKY46 and WRKY70, thereby inhibiting the expression of these two WRKY genes under salt stress and directly leading to a significant increase in Arabidopsis salt tolerance. In dehydrated environments, AtJMJ17 can directly bind to OST1 (OPENSTOMATA1) and maintain normal plant condition by removing H3K4me3 to regulate OST1 expression levels. AtJMJ27 binds to the drought stress positive regulators GOLS2 (GALACTINOL SYNTHASE 2) and RD20 (RESPONSE TO DESICCATION). The removal of the inhibitory marker H3K9me2 from the chromatin of 20) promotes its expression, thereby positively regulating the drought stress response. Histone demethylases such as AtJMJ30, AtJMJ32, AtJMJ11, REF6, and AtJMJ13, which are involved in H3K27 methylation clearance, can alter the expression of HSP22 and HSP17.6C by clearing histone markers generated by the transient binding of the heat transcription factor HSFA2 (HEATSHOCK FACTOR) and heat shock proteins HSP (HSU). This allows Arabidopsis to develop heat memory to adapt to repeated high-temperature stress. Therefore, JHDM proteins are always in an "upstream" position, regulating the expression of downstream target genes by removing histone lysine methylation modifications on the chromatin of downstream target genes, playing an important role in the flexible response of plants to external abiotic stresses.

[0006] Sorghum is the world's fifth largest cereal crop after wheat, corn, rice, and barley, renowned for its versatility and widely cultivated in arid and semi-arid regions globally. Sorghum possesses numerous excellent traits, including high photosynthetic efficiency, drought and waterlogging resistance, and salt tolerance. Elucidating the molecular mechanisms underlying sorghum's drought and salt tolerance could potentially provide a new avenue for genetic improvement breeding of other crops sensitive to abiotic stresses. Summary of the Invention

[0007] Technical issue: The applicant previously used RT-qPCR technology to detect changes in the expression levels of all JmjC gene family members in sorghum under drought and salt stress, and found that the gene encoding the SbJMJ02 protein... SbJMJ02It showed a rapid response rate and a significant increase in expression level after both stress treatments, but whether this gene can improve the plant's drought and / or salt tolerance remains unknown.

[0008] The present invention specifically adopts the following technical solution: SbJMJ02 The application of a gene or its expression promoter in improving the drought and / or salt tolerance of plants, characterized in that... SbJMJ02 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0009] Furthermore, the expression promoter is selected from recombinant expression vectors containing the SbJMJ02 gene, expression cassettes, transgenic cell lines, transgenic plant tissues, or recombinant microorganisms.

[0010] Furthermore, the plants include sorghum and Arabidopsis thaliana.

[0011] Furthermore, improving plant drought tolerance manifests as an increased survival rate of plants after rehydration following drought stress.

[0012] Furthermore, improving plant drought resistance manifests as an enhanced water-holding capacity in plant leaves after drought stress.

[0013] Furthermore, improving plant drought tolerance manifests as improved stomatal regulation function after drought stress.

[0014] Furthermore, improved salt tolerance in plants manifests as improved growth in high-salt environments.

[0015] Furthermore, improving plant salt tolerance manifests as a reduction in the negative impact of high-salt environments on photochemical efficiency.

[0016] Furthermore, improved salt tolerance in plants manifests as enhanced ability to regulate ion balance in high-salt environments.

[0017] The present invention has the following beneficial effects: This invention is the first to discover in sorghum SbJMJ02 The genes can respond rapidly to drought and salt stress, and exhibit positive regulation. This discovery not only provides a scientific basis for elucidating the drought and salt tolerance mechanisms of sorghum, but also provides theoretical guidance for genetic improvement breeding of crops to enhance drought and salt tolerance. Attached Figure Description

[0018] Figure 1 For drought stress treatment, drought stress treatment and SbJMJs The expression pattern of the response. A: Growth status of sorghum seedlings under different drought stress treatment durations; B: Growth status of sorghum seedlings under different drought stress treatment durations. SbJMJsC: Changes in expression levels; D: Growth status of sorghum seedlings under different salt treatment times; E: Changes in expression levels under different salt stress treatment times. SbJMJs Changes in expression levels.

[0019] Figure 2 To obtain by quantitative fluorescence experiment SbJMJ02 Gene expression profile.

[0020] Figure 3 To determine the location of SbJMJ02 in cells through subcellular localization experiments. A: Structure of the subcellular localization expression vector; B: Results of subcellular localization experiments in onion epidermal cells; C: Results of subcellular localization experiments in sorghum leaf protoplasts.

[0021] Figure 4 The growth status of yeast with SbJMJ02 protein induced by heterologous expression in a medium containing high concentrations of NaCl and mannitol.

[0022] Figure 5 The growth rates of yeast strains induced with heterologous expression of SbJMJ02 protein in normal, high-concentration NaCl, and mannitol media are shown. A: Growth curves (OD600 vs. time) of the two yeast strains in normal medium (no stress factors); B: Statistical analysis of relative optical density values ​​(ΔΔOD600) corresponding to data in A; C: Growth curves of the two yeast strains under NaCl stress (300 mM); D: Statistical analysis of ΔΔOD600 corresponding to data in C; E: Growth curves of the two yeast strains under mannitol stress (500 mM, simulating osmotic / drought stress); F: Statistical analysis of ΔΔOD600 corresponding to data in E. P1, P2, and P3 represent comparisons of proliferation rates at different time points.

[0023] Figure 6 To identify Arabidopsis thaliana genetically transformed positive plants and conduct drought stress experiments. A: Structure of the overexpression vector; B: Screening of transgenic positive plants with hygromycin tags; C: Identification of the genotype of transgenic plants; D: Phenotypic characteristics of plants in drought stress-rehydration experiments.

[0024] Figure 7 This study investigated salt stress in genetically transformed Arabidopsis plants. A: Phenotypic identification of salt-stressed plants; B: Maximum photochemical efficiency of plants at 0, 3, 12, and 24 hours after salt stress treatment. Detailed Implementation

[0025] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0026] Example 1: In sorghum SbJMJ02 The discovery of genes Gene family bioinformatics analysis revealed that sorghum contains... JmjC Gene family members were identified by RT-qPCR after sorghum was subjected to drought and salt stress treatments. SbJMJ02 Changes in expression level ( Figure 1 ). SbJMJ02 The nucleotide sequence of the gene is shown in SEQ ID NO.1. SbJMJ02 The amino acid sequence of the encoded protein SbJMJ02 is shown in SEQ ID NO.2.

[0027]

[0028] SEQ ID NO.2: MRLPMPQVEGRSYLPAEVRNGLETLKRRRLERMRLSAQNEVGDNPAVAARSGGDALRSPANCGVRLHSNNSTGLPGNVQTKDPFAKRNVEKFDMSNLEWIGKIPECPVYCPTKEEFEDPIAYIQKISPEAAKYGICKIVSPVCASVPAGVVLMKEQPSFKFMTRVQPLRLAEWAEDDTVTFFMSGRKYTFRDYEKMANKVFSKKYSSSSCLPARYVEEEFWREIAFGKMDFVEYACDVDGSAFSSSPHDQLGKSNWNLKNFSRLPNSVLRLLQTPIPGVTDPMLYIGMLFSMFAWHVEDHYLYSINYHHCGAFKTWYGIPGDAAPGFERVASQYVYNKDILVGDGEDAAFDVLLGKTTMFPPNVLLDHNVPVYKAVQRPGEFVITFPRSYHAGFSHGFNCGEAVNFAIGDWFPLGSLASKRYALLNRTPLLAHEELLCRSAVLLSQKLLNCDPRSLDKLDHPYSQYCVKSCFVRLIRFQRRARGLLAKMGSQICYLPKTFPNLSCSMCRRDCYITHVLCGCNLDPVCLHHEQELRSCPCESNRVVYVREDILELEALSRKFEEDVCLSKERSCIGSCKEAEISDTNVERVPNLGITVDFGNSKAGSSGFMTVDGGNSSAAVSILTSSAHHKAPKHSEARAINTSMTKGTYTVDESSSGMDDACNEHGSCNASAMECSDNSDSESEIFRVKRRSTSFDKPTSETKTSTLSEQQVLRRLKKVHPEVQQASKRPEEYDNGSVHSARMSQKSSNPASSDDEREDKVPISWRIKRRQLETQHNVTSPGVRLQSNLASSGGSREETAERTRDAAAELRPKRVKIRLPSSASRQIEQQGSSGQRFAREDKLSLGFPRTF。

[0029] Example 2: Subcellular Localization Experiment of SbJMJ02 Protein in Cells I. Construction of GFP Green Fluorescent Protein Fusion Vector of SbJMJ02 Protein 1. Retrieved from the Phytozome plant genome database SbJMJ02 The nucleotide sequence of the gene, as shown in SEQ ID NO.1, can be amplified by Shanghai Sangon Biotech Co., Ltd. SbJMJ02 Primers for the CDS region sequence of the gene (SEQ ID NO.3 and SEQ ID NO.4), based on the expression profile ( Figure 2 )Sure SbJMJ02 By extracting RNA from the corresponding tissue or organ at the location of gene-specific expression and reverse transcribing it into cDNA as a template, the gene can be amplified. SbJMJ02 The CDS region was identified and the amplified sequence was confirmed to be correct before being ligated into the intermediate vector.

[0030] 2. Double digestion of GFP vector: The circular GFP vector was digested with restriction endonucleases KpnI and XbaI to obtain the linearized GFP vector.

[0031] 3. Recombinant vector construction: Based on the sequence of the GPF multiple cloning site, design... SbJMJ02 Primers (SEQ ID NO. 5 and SEQ ID NO. 6) were used to ligate the gene and the GFP homologous arm of the linearized vector, and the recombinant vector ( Figure 3 A) The bacteria were transformed into DH5α Escherichia coli strains and inoculated onto solid LB medium supplemented with kanamycin to screen for positive strains.

[0032] 4. Obtaining the SbJMJ02-GFP recombinant vector: [The following steps were performed using...] SbJMJ02 The specific sequences of the gene and the GFP vector were used as primers (SEQ ID NO.5 and SEQ ID NO.7) for bacterial testing. After confirming that the strain was positive, the strain was propagated, plasmids were extracted, and the SbJMJ02-GFP recombinant vector was obtained.

[0033] 5. In onion epidermal cells and sorghum protoplasts, the SbJMJ02 protein is fused with nuclear localization signals and is a type of nuclear protein. Figure 3 B).

[0034] SEQ ID NO. 3: 5'-AGCATCTCGTACTGCTATGGA-3'.

[0035] SEQ ID NO. 4: 5'-ATTTTCTGCCACTGACAGCC-3'.

[0036] SEQ ID NO. 5: 5'-ACGGGGGACGAGCTCGGTACATGCGTTTACCTATGCC-3'.

[0037] SEQ ID NO. 6: 5'-TTGCTCACCATGTCGACTCTAAATGTACGTGGAAAA-3'.

[0038] SEQ ID NO. 7: 5'-GCCGTCGTCCTTGAAGAAGAT-3'.

[0039] Example 3: SbJMJ02 Heterologous expression of genes in yeast I. Construction of the SbJMJ02-pYES2 recombinant vector 1. Double digestion of pYES2 vector: The circular pYES2 vector was digested with restriction endonucleases KpnI and SacI to obtain the linearized pYES2 vector.

[0040] 2. Homologous recombination: Based on the sequence of the pYES2 restriction site, design... SbJMJ02 The recombinant vector was ligated to the homologous arms (SEQ ID NO.8 and SEQ ID NO.9) of the linearized vector pYES2, and then transformed into DH5α Escherichia coli strains. Positive strains were screened by inoculating the recombinant vector into solid LB medium supplemented with ampicillin.

[0041] 3. Obtaining the SbJMJ02-pYES2 recombinant vector: [The following text appears to be incomplete and requires further context: "using..."] SbJMJ02 After confirming the positive strain with specific sequences and pYES2 vector specific sequences as primers (SEQ ID NO.8 and SEQ ID NO.10), the strain was propagated, plasmids were extracted, and the SbJMJ02-pYES2 recombinant vector was obtained.

[0042] II. Induction of heterologous protein expression 1. Select positive clones with a diameter of 2-3 mm transformed with SbJMJ02-pYES2 and pYES2 vectors respectively, and inoculate them into liquid yeast medium with glucose as the carbon source of SD-Ura. Incubate on a shaker at 220 rpm and 30°C for 16-20 h until OD is reached. 600 =0.8~1.2; 2. Centrifuge 20 ml of bacterial culture at 10000 rpm for 10 seconds to collect the bacteria. Add 20 ml of yeast liquid culture medium containing 300 mM NaCl with galactose as the carbon source, mix well, and divide into 4 portions. Adjust OD. 600 Similarly, the bacterial culture was placed on a shaker at 200 rpm and incubated at 30°C. Oddi concentrations (OD) were measured at 0, 3, 6, and 12 hours. 600 The experiment was repeated three times and the results were recorded. The proliferation rates of yeast strains containing the SbJMJ02-pYES2 and pYES2 vectors were compared.

[0043] 3. The mannitol treatment method is the same as above, and the concentration of mannitol in the liquid culture medium is 500mM. 4. Take the two bacterial cultures from step 1, centrifuge at 10,000 rpm for 10 seconds to collect the cells, resuspend the cells in liquid yeast culture medium with SD-Ura carbon source and galactose, induce overnight, and adjust OD. 600 To be consistent.

[0044] 5. According to 10 0 10 -1 10 -2 10 -3 Yeast colony growth was recorded by inoculating yeast cells with different concentration gradients onto solid culture medium without stress factors (glucose as carbon source), solid culture medium containing NaCl yeast (glucose as carbon source), and solid culture medium containing mannitol yeast (glucose as carbon source).

[0045] 6. The results showed that... Figure 4 and Figure 5 As shown, in a culture medium containing stress factors, the expression of SbJMJ02 protein enhanced the yeast's tolerance to stress, and its proliferation rate was significantly greater than that of yeast strains without SbJMJ02 protein expression.

[0046] SEQ ID NO. 8: 5'-GGGAATATTAAGCTTGGTACATGCGTTTACCTATGCC-3'.

[0047] SEQ ID NO. 9: 5'-GGCCGTTACTAGTGGATCCGCTAAAATGTACGTGGA-3'.

[0048] SEQ ID NO. 10: 5'-TTCTAAAATGTACGTGGAAA-3'.

[0049] Example 4: Overexpression SbJMJ02 Construction and identification of Arabidopsis thaliana lines with specific genes and phenotypic analysis after drought and salt stress treatment. one, SbJMJ02 Construction of gene overexpression vectors 1. Linearize the pCambia1300 vector using restriction enzymes KpnI and XbaI.

[0050] 2. Based on the sequence of the pCambia1300 restriction site, design... SbJMJ02 The recombinant vector (pCambia1300) was linked to its homologous arms (SEQ ID NO.11 and SEQ ID NO.12) and then the recombinant vector (pCambia1300) was attached. Figure 6A) The bacteria were transformed into DH5α Escherichia coli strains and inoculated onto solid LB medium supplemented with kanamycin to screen for positive strains.

[0051] 3. After selecting single colonies, they were tested using pCambia1300-SbJMJ02 vector-specific primers (SEQ ID NO.13 and SEQ ID NO.14) and then sent to the company for sequencing.

[0052] 4. Take the strain with correct sequencing results, extract the plasmid according to the steps shown in the plasmid rapid extraction kit (Genstar), and store it in a -20℃ freezer for later use.

[0053] II. Arabidopsis SbJMJ02 Identification of overexpression lines 1. Sow the sterilized T0 generation Arabidopsis thaliana seeds on solid 1 / 2 MS medium (containing hygromycin 25 μg / ml) and seal with sealing film.

[0054] 2. Place the petri dishes at 4 ℃ in the dark for vernalization for 2-3 days, then transfer them to an incubator at 22 ℃ / 25 ℃ (night / day), with a photoperiod of 8 h / 16 h (dark / light). Select normally growing Arabidopsis plants ( Figure 6 B) Transplanted into vermiculite nutrient soil (1:1) for continued cultivation.

[0055] 3. After cultivation to the 6-leaf stage, a small number of leaves were cut off, and DNA was extracted. The authenticity of positive plants was further detected using pCambia1300-SbJMJ02 vector-specific primers (SEQ ID NO.11 and SEQ ID NO.12). Figure 6 (C).

[0056] 4. Continue to cultivate genuine positive plants until they self-pollinate, produce seeds, and are harvested.

[0057] III. Arabidopsis SbJMJ02 Drought stress treatment and phenotypic analysis of overexpression lines 1. Obtain the Arabidopsis thaliana SbJMJ02 Overexpression lines and untransformed Arabidopsis lines were evenly sown in breeding pots and then transferred to normal temperature and light incubators after vernalization.

[0058] 2. When most Arabidopsis thaliana plants reached the 6-leaf stage, watering was stopped for 7 days, followed by rehydration for 3 days, during which the growth of the Arabidopsis thaliana was observed. The results showed that... Figure 6 As shown in D, SbJMJ02 After rehydration, a large number of transgenic Arabidopsis plants overexpressing the protein recovered their phenotype, while all plants in the control group died. This indicates that the presence of the SbJMJ02 protein in Arabidopsis can enhance its drought tolerance.

[0059] IV. Arabidopsis SbJMJ02 Salt stress treatment and phenotypic analysis of overexpression lines 1. Obtain the Arabidopsis thaliana SbJMJ02 Overexpression lines and untransformed Arabidopsis lines were evenly sown in breeding pots and then transferred to normal temperature and light incubators after vernalization.

[0060] 2. When most Arabidopsis plants reach the early bolting stage, stop normal watering and replace it with a 100mM NaCl solution for irrigation. Observe the growth of the Arabidopsis plants. Figure 7 (A). And the values ​​of Arabidopsis thaliana (A) were measured at 0, 4, 12, and 24 hours. Figure 7 (B). The results showed that... Figure 7 As shown, wild-type and wild-type after salt treatment SbJMJ02 The transgenic Arabidopsis thaliana overexpressing the protein showed varying degrees of wilting, but the overexpressing plants were more robust. Moreover, the maximum photochemical efficiency of the overexpressing plants was significantly higher than that of the wild type at 12 h and 24 h after treatment, indicating that the SbJMJ02 protein in Arabidopsis thaliana can enhance its salt tolerance.

[0061] SEQ ID NO. 11: 5'-ACGGGGGACGAGCTCGGTACATGCGTTTACCTAT-3'.

[0062] SEQ ID NO. 12: 5'-TTGCTCACCATGTCGACTCTAAATGTACGTGGAA-3'.

[0063] SEQ ID NO. 13: 5'-ACGGGGGACGAGCTCGGTACATGCGTTTACCTAT-3'.

[0064] SEQ ID NO. 14: 5'-GCTTCATGTGGTCGGGGTAGC-3'.

[0065] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0066] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. SbJMJ02 The application of genes or their expression promoters in improving the drought and / or salt tolerance of plants, characterized in that, The SbJMJ02 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The expression promoter is selected from the group containing SbJMJ02 Recombinant gene expression vectors, expression cassettes, transgenic cell lines, transgenic plant tissues, or recombinant microorganisms.

3. The application according to claim 2, characterized in that, The plants mentioned include sorghum and Arabidopsis thaliana.

4. The application according to claim 3, characterized in that, Improving plant drought tolerance is manifested in an increased survival rate of plants after rehydration following drought stress.

5. The application according to claim 4, characterized in that, Improving plant drought tolerance is manifested in the enhanced water-holding capacity of plant leaves after drought stress.

6. The application according to claim 5, characterized in that, Improving plant drought tolerance manifests as an improvement in stomatal regulation function after drought stress.

7. The application according to claim 3, characterized in that, Improving plant salt tolerance manifests as an improvement in plant growth in high-salt environments.

8. The application according to claim 7, characterized in that, Improving plant salt tolerance manifests as a reduction in the negative impact of high-salt environments on photochemical efficiency.

9. The application according to claim 8, characterized in that, Improving a plant's salt tolerance manifests as an enhanced ability to regulate ion balance in high-salt environments.

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