Application of SbHDA3 gene in regulation and control of plant disease resistance

Abstract

The invention discloses application of a SbHDA3 gene in regulation and control of plant disease resistance, and belongs to the technical field of gene engineering. The nucleotide sequence of the SbHDA3 gene is as shown in SEQ ID NO. 11. It is found that the SbHDA3 gene is remarkably related to plant disease resistance, the resistance of plants to xanthomonas oryzae, magnaporthe oryzae and colletotrichum gloeosporioides can be improved by overexpressing the gene, and the resistance is reduced if the gene is silent. The research result of the invention provides a new gene resource for plant disease-resistant breeding, and lays a theoretical foundation for researching a plant disease-resistant mechanism.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to the application of the SbHDA3 gene in regulating plant disease resistance. Background Technology

[0002] Histone acetylation is a crucial mechanism in epigenetic regulation, primarily occurring at the N-terminal lysine residues of histones and mediated by histone acetylases (HATs) and histone deacetylases (HDACs). As the earliest discovered type of histone modification, the dynamic balance of acetylation plays a vital role in gene expression regulation in eukaryotes. HATs mainly include families such as GNAT, MYST, p300 / CREB, and TAFII-250, which can acetylate various histones and exhibit certain specificity. They play a significant role in gene expression and genome integrity, making them one of the most studied modifications. HDACs, as key regulators of histone acetylation, play a crucial role in chromatin remodeling and gene expression regulation. The main sites of action for HDACs are histones H3 and H4, including families such as RPD3 / HDA1, SIR2, and HD2. Studies have reported that HATs and HDACs, in addition to participating in the regulation of plant growth, reproductive development, and diurnal rhythms, are also widely involved in plant stress and disease resistance responses. The sorghum HDAC family includes 19 HDACs members, belonging to the RPD3 / HDA1, SIR2, and HD2 families.

[0003] Studies have shown that histone deacetylation affects pathogenicity by regulating the expression of pathogenicity-related genes. For example, in *Magnaporthe oryzae*, the deacetylation transferase MoHOS2 is closely related to the growth and conidia production of the pathogen; the MoSnt2-dependent deacetylation of histone H3 can mediate the MoTor signaling pathway-dependent autophagy process in *Magnaporthe oryzae*, thereby affecting the pathogen's ability to infect plants; and the histone deacetylases UvHOS2 and UvHOS3 affect the growth, development, and pathogenicity of the pathogen by regulating histone modifications. The absence of UvHOS2 leads to stunted pathogen growth, reduced spore production and germination, and decreased pathogenicity. Simultaneously, MoHOS2 regulates the acetylation levels of histone H3 at multiple lysine sites. MoHOS3, by mediating the deacetylation of histones H3 and H4, not only affects pathogen growth, branching, spore formation, and pathogenicity but also negatively regulates the biosynthesis of secondary metabolites. The TIG1 protein possesses deacetylase activity; knocking out the TIG1 gene results in malformed spores, inhibited mycelial formation, and complete loss of pathogenicity in *Pseudomonas oryzae*. Furthermore, histone deacetylation plays a crucial regulatory role in plant-pathogen interactions. HDA705 negatively regulates rice resistance to pathogens such as *Ustilaginoideavirens*, *Pseudomonas oryzae*, and *Xanthomonas oryzae* pv. *oryzae*; similarly, HDT701 negatively regulates rice resistance to *Pseudomonas oryzae* and *Xanthomonas oryzae*. The silent genes SlHDA6, SlHDT1, SlHDT2, SlSRT1, and SlSRT2 in tomatoes can enhance the resistance of susceptible tomato varieties to Ralstonia solanacearum. However, the mechanism of action of the SbHDA3 gene in sorghum in plant disease resistance has not been reported. Summary of the Invention

[0004] The purpose of this invention is to provide the application of the SbHDA3 gene in regulating plant disease resistance, thereby addressing the problems existing in the prior art. This invention reveals that the SbHDA3 gene is significantly correlated with plant disease resistance; overexpression of this gene enhances plant resistance to bacterial blight, rice blast fungus, and anthracnose, while silencing it reduces resistance. The findings of this invention provide new gene resources for plant disease resistance breeding and lay a theoretical foundation for studying plant disease resistance mechanisms.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides the application of the SbHDA3 gene in regulating plant disease resistance, and the nucleotide sequence of the SbHDA3 gene is shown in SEQ ID NO.11;

[0007] The regulation of plant disease resistance refers to overexpressing the SbHDA3 gene to increase plant disease resistance, and silencing the SbHDA3 gene to decrease plant disease resistance.

[0008] The present invention also provides the application of recombinant vectors or recombinant microorganisms containing the SbHDA3 gene in improving plant disease resistance, wherein the nucleotide sequence of the SbHDA3 gene is shown in SEQ ID NO.11.

[0009] The present invention also provides the application of the SbHDA3 gene in the breeding of plant varieties with high disease resistance, wherein the nucleotide sequence of the SbHDA3 gene is shown in SEQ ID NO.11;

[0010] The method for cultivating highly disease-resistant plant varieties is to obtain highly disease-resistant plant varieties by stably overexpressing the SbHDA3 gene in plants.

[0011] The present invention also provides the application of recombinant vectors or recombinant microorganisms containing the SbHDA3 gene in the cultivation of plant varieties with high disease resistance, wherein the nucleotide sequence of the SbHDA3 gene is shown in SEQ ID NO.11.

[0012] Furthermore, the disease resistance refers to the plant's resistance to bacterial blight, rice blast fungus, and anthracnose fungus.

[0013] Furthermore, the plants include rice and sorghum.

[0014] The present invention also provides a method for improving plant disease resistance, comprising the step of transferring the SbHDA3 gene into a plant using genetic transformation technology to improve plant disease resistance; the nucleotide sequence of the SbHDA3 gene is shown in SEQ ID NO.11.

[0015] The present invention also provides a method for cultivating highly disease-resistant plant varieties, comprising the steps of using genetic transformation technology to transfer the SbHDA3 gene into plants, thereby achieving stable overexpression of the SbHDA3 gene and cultivating disease-resistant plant varieties; the nucleotide sequence of the SbHDA3 gene is shown in SEQ ID NO.11.

[0016] Furthermore, the disease resistance refers to the plant's resistance to bacterial blight, rice blast fungus, and anthracnose fungus.

[0017] Furthermore, the plants include rice and sorghum.

[0018] The present invention discloses the following technical effects:

[0019] This invention discovered a significant correlation between the SbHDA3 gene and plant disease resistance. Overexpression of this gene enhances plant resistance to bacterial blight, rice blast, and anthracnose, while silencing the gene reduces resistance. Stable overexpression of this gene in rice and sorghum through genetic transformation technology allows for the breeding of highly disease-resistant varieties. Experimental results show that transgenic rice exhibits enhanced resistance to bacterial blight and rice blast, while sorghum overexpressing SbHDA3 demonstrates improved resistance to anthracnose. Silencing the gene exacerbates disease incidence, providing effective gene resources and technical means for plant disease resistance breeding. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 The image shows the detection results of positive SbHDA3 transgenic rice plants.

[0022] Figure 2 The results show the resistance of SbHDA3 overexpressing transgenic rice to bacterial blight pathogens; A shows the leaf disease status of transgenic rice and wild-type rice; B shows the statistical results of leaf lesion length of transgenic rice and wild-type rice.

[0023] Figure 3 The results show the resistance of SbHDA3 transgenic rice to rice blast fungus; A shows the leaf disease status of transgenic rice and wild-type rice; B shows the statistical results of leaf lesion length of transgenic rice and wild-type rice.

[0024] Figure 4 The results show the resistance of sorghum with silenced and overexpressed SbHDA3 to anthracnose. A represents the phenotype of sorghum leaves and the expression level of SbHDA3 before anthracnose inoculation. B represents the disease incidence, lesion area, and anthracnose biomass of sorghum leaves 120 h after anthracnose inoculation. Detailed Implementation

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0027] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0028] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0029] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0030] Example 1

[0031] 1. Test materials

[0032] 1.1 Plant materials

[0033] Seeds of Nicotiana benthamiana and Sorghum bicolor (red tassel).

[0034] 1.2 Strains and Vectors

[0035] Strains:

[0036] Agrobacterium tumefaciens strain: GV1301, P19.

[0037] Escherichia coli strain: DH5α.

[0038] Rice blast fungus (Magnaporthe oryzae): RB22.

[0039] Rice bacterial blight pathogen (Xanthomonas oryzaepv.oryzae): PX099A.

[0040] Sorghum anthracnose fungus: Sorghum anthracnose fungus (Colletotrichum sublineola).

[0041] Carrier:

[0042] Overexpression vector: pRHVcGFP(He F,Zhang F,Sun W,et al.A Versatile VectorToolkit for Functional Analysis of Rice Genes[J].Rice,2018,11(1):27.DOI:10.1186 / s12284-018-0220-7.).

[0043] FoMV vector (foxtailmosaic virus) (Characterization of afoxtail mosaic virus vector for gene silencing and analysis of innate immune responses in Sorghumbicolor, DOI:10.1111 / mpp.13270).

[0044] Primer sequences are shown in Table 1.

[0045] Table 1 Primer sequences

[0046]

[0047]

[0048] 2. Cloning of the SbHDA3 gene and construction of its expression vector

[0049] The research group previously obtained the gene sequence shown in SEQ ID NO.11 through gene family identification and named it SbHDA3:

[0050]

[0051] 2.1 RNA extraction

[0052] Total RNA was extracted from plant leaves using the Trizol method, and the steps are as follows:

[0053] (1) Take 0.1g of red sorghum leaves, put them into a sterilized centrifuge tube, freeze them quickly, and then grind them into powder.

[0054] (2) Add 1 mL of Trizol extract and shake well to mix. After standing for 2 min, add 200 μL of RNA extract, shake well immediately and let stand for 5 min.

[0055] (3) Centrifuge at 12,000 rpm for 10 min at 4℃, and aspirate the supernatant into an RNase-free centrifuge tube.

[0056] (4) Immediately add an equal volume of pre-cooled isopropanol, gently invert and mix well, then place in a -20°C refrigerator for 30 minutes to precipitate or let stand at room temperature for 10 minutes.

[0057] (5) After standing, centrifuge at 4°C and 2000 rpm for 10 min. After centrifugation, carefully discard the supernatant and immediately add 1 mL of 70% alcohol to the precipitate to wash the precipitate. Repeat this step twice.

[0058] (6) Centrifuge at 4℃ and 12000rpm for 5min to remove alcohol and air dry. Dissolve in 30μL of DEPC water and store in a -80℃ refrigerator for later use.

[0059] cDNA synthesis follows III 1st Stand cDNA Synthesis Kit Instructions: Use RNA as a template to synthesize cDNA and store at -20°C for later use.

[0060] 2.2 Gene Amplification

[0061] Primers were designed based on the full-length CDS sequence (with stop codon removed) and restriction enzyme site sequence of the SbHDA3 gene, as shown in Table 1 (SEQ ID NO.1-4 and SEQ ID NO.7-8). The primers were synthesized by Qingke Biotechnology.

[0062] High-fidelity Taq polymerase (TransStart FastPfu DNA Polymerase, Beijing TransGen Biotech Co., Ltd.) was used for amplification of the target fragment. The amplification program was as follows: 95℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 1 min, for 40 cycles, followed by a final extension at 72℃ for 5 min, and storage at 4℃. After the PCR reaction, the fragment size of the PCR product was detected by 1% agarose gel electrophoresis at 120V for 20 min, with DL2,000 DNA Marker (Takara) as a control.

[0063] The PCR products of the correct size were purified using the SanPrep Column DNA Gel Extraction Kit (Sangon Biotech (Shanghai) Co., Ltd.). The experimental method was performed according to the instructions. The recovered products were stored at -20℃ for later use.

[0064] 2.3 Construction of Recombinant Vectors

[0065] 2.3.1 Construction of sorghum silencing and overexpression vectors

[0066] Enzyme digestion system (20 μL): 2 μL 10× Buffer, 1 μL restriction endonuclease, 2 μL FoMV plasmid, add ddH2O to 20 μL, react at 37℃ for 3 h, and recover the enzyme digestion product after identification by 1.2% agarose gel electrophoresis.

[0067] The PCR product and the double digestion product from section 2.2 were ligated using T4 ligase. The ligation system (10 μL) consisted of 1 μL of double digestion product, 2 μL of the target gene fragment, 5 μL of ligase, and ddH2O was added to bring the total volume to 10 μL. Ligation was carried out at 50°C for 15 min. The ligation product was immediately transformed into E. coli.

[0068] 2.3.2 Construction of rice overexpression vector

[0069] Enzyme digestion system (20 μL): 2 μL 10× Buffer, 1 μL restriction endonuclease, 2 μL pRHVcGFP plasmid, add ddH2O to 20 μL, react at 37℃ for 3 h, and recover the enzyme digestion product after identification by 1.2% agarose gel electrophoresis.

[0070] The PCR product and double digestion product from section 2.2 were ligated using homologous recombinase or T4 ligase. The ligation system (10 μL) consisted of 1 μL of double digestion product, 2 μL of the target gene fragment, 5 μL of ligase, and ddH2O was added to bring the total volume to 10 μL. Ligation was carried out at 50°C for 15 min. The ligation product was immediately transformed into E. coli.

[0071] 3. Cultivation and resistance verification of SbHDA3 transgenic rice

[0072] 3.1 Breeding and Screening of Transgenic Rice

[0073] A SbHDA3-RHV-GFP overexpression vector was constructed and transformed into Nipponbare (Oryza sativa L. spp. japonica). The F0 generation was harvested to obtain a heterozygous F1 generation (SbHDA3:H#1). Twenty-four transgenic progeny plants were selected, and their expression was detected using SbHDA3 gene-specific primers (SEQ ID NO. 5-6), yielding H#1 positive material. The corresponding positive SbHDA3 heterozygous F1 plants were cultivated and harvested to obtain the F2 generation. DNA was extracted from 120 F2 generation rice plants for PCR detection and sequencing, yielding H#1 positive material. Positive plants were continued to be cultivated into the F3 generation, and the F3 transgenic material was randomly detected using PCR-specific primers. The F3 transgenic rice plants with positive results were named H#1-3. The detection results are as follows: Figure 1 As shown.

[0074] 3.2 Artificial inoculation with bacterial blight pathogen

[0075] Transgenic rice was cultured for one month and then used for pathogenicity testing of rice bacterial blight pathogen. The activated pathogen was cultured in NB liquid medium at 28°C and 200 rpm until OD... 600 =1.0. Using sterilized scissors dipped in a suitable amount of bacterial solution, cut off 2-3 cm from the leaf tips of healthy rice leaves. After inoculation, maintain humidity and warmth, with the following conditions: 12h light / 12h dark, temperature 30℃, and relative humidity above 70%. Measure the length of leaf lesions 20 days after inoculation. Set up 20 replicates for each group.

[0076] The results are as follows Figure 2 As shown, the SbHDA3 transgenic rice exhibits resistance to bacterial blight.

[0077] 3.3 Artificial inoculation with rice blast fungus

[0078] (1) Activate the pathogen with oat medium, culture at 28℃ under continuous light for 5-10 days, wash off the spores with 0.5% Tween-20 and filter;

[0079] (2) The concentration of the spore suspension was determined under a microscope using a hemocytometer and adjusted to 1×10⁻⁶. 5 cells / mL;

[0080] (3) Use a small spray bottle to evenly spray the spore suspension onto the surface of rice leaves (at least 20 seedlings per group), keep warm and moist in the dark for 2 days, and then continue to culture under 12 hours of light / 12 hours of darkness.

[0081] (4) Observe the results after 7 days and take photos of the samples.

[0082] The results are as follows Figure 3 As shown, SbHDA3 transgenic rice exhibits resistance to rice blast.

[0083] 4. Cultivation and resistance verification of SbHDA3 transgenic sorghum

[0084] 4.1 Cultivation of SbHDA3 transgenic (VIGS and VOX) sorghum

[0085] (1) Transform the constructed SbPDS (positive control gene) / SbHDA3-FoMV-VIGS, SbHDA3-FoMV-VOX vectors and blank FoMV-EV vectors into Agrobacterium tumefaciens and infiltrate and inject them into Nicotiana benthamiana according to the transient expression method.

[0086] (2) Take 1g of infected tobacco leaves, add 10mL of PBS buffer and grind, filter and collect the filtrate for later use;

[0087] (3) When the sorghum grows to the 2-3 leaf stage, sprinkle a small amount of carborundum evenly on the leaf surface and add the filtrate. Gently rub the inoculation with your fingers to obtain sorghum that overexpresses the SbHDA3 gene (SbHDA3-VOX), sorghum that silences the SbHDA3 gene (SbHDA3-VIGS), and control sorghum (FoMV-EV).

[0088] (4) After 28 days, photographs were taken and RNA was extracted from sorghum leaf tissue. The RNA was reverse transcribed into cDNA and stored at -80℃ for later use. The expression levels of SbPDS and SbHDA3 were detected, and the results are shown below. Figure 4 As shown in Figure A, after silencing the positive control gene SbPDS, the plant leaves became lighter in color. Further RT-qPCR analysis revealed that the expression level of the SbPDS gene in the silenced plants was lower than that in the control sorghum (FoMV-EV) and normal sorghum (Mock), indicating that this method can silence genes. The expression level of the SbPDS gene in the control sorghum (FoMV-EV) was slightly higher than that in normal sorghum (Mock), but it did not reach a significant level. The same situation was observed in multiple gene detections, which is a normal phenomenon. RT-qPCR analysis revealed that the expression level of the SbHDA3 gene in sorghum plants with silenced SbHDA3 was lower than that in control sorghum (FoMV-EV), while the expression level of the SbHDA3 gene in sorghum plants with overexpression of the SbHDA3 gene was higher than that in control sorghum (FoMV-EV). This result indicates that the SbHDA3 transgenic (VIGS and VOX) sorghum has been successfully bred.

[0089] 4.2 Artificial inoculation of anthrax bacteria

[0090] (1) After culturing sorghum anthracnose bacteria in PDA medium at 28℃ for 3-5 days, use sterile water containing 0.5% Tween-20 to wash off spores and filter.

[0091] (2) The number of spores was observed and recorded under a microscope using a hemocytometer, and the concentration was adjusted to 10. 5 The spore suspension was placed in a sterilized small spray bottle and sprayed onto the treated sorghum leaves.

[0092] (3) The sorghum was kept moist and protected from light for 2 days. At the same time, photos were taken and samples were collected from the sorghum at 0 and 120 h after inoculation.

[0093] (4) DNA and RNA were extracted from the sorghum leaves. The DNA was used for subsequent fungal biomass statistics, and the RNA was reverse transcribed into cDNA for RT-qPCR detection of SbHDA3 expression levels at different time points.

[0094] The results are as follows Figure 4 As shown in Figure B, silencing SbHDA3 resulted in an increase in anthracnose lesions and an increase in anthracnose bacterial biomass in sorghum; overexpression of SbHDA3 resulted in a decrease in anthracnose lesions and a decrease in anthracnose bacterial biomass. SbHDA3 positively regulates the resistance of sorghum to anthracnose.

[0095] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of the SbHDA3 gene in regulating plant disease resistance, characterized in that, The nucleotide sequence of the SbHDA3 gene is shown in SEQ ID NO.11; The regulation of plant disease resistance refers to overexpressing the SbHDA3 gene to increase plant disease resistance, and silencing the SbHDA3 gene to decrease plant disease resistance.

2. The application of recombinant vectors or recombinant microorganisms containing the SbHDA3 gene in improving plant disease resistance, characterized in that, The nucleotide sequence of the SbHDA3 gene is shown in SEQ ID NO.

11.

3. The application of the SbHDA3 gene in breeding highly disease-resistant plant varieties, characterized in that, The nucleotide sequence of the SbHDA3 gene is shown in SEQ ID NO.11; The method for cultivating highly disease-resistant plant varieties is to obtain highly disease-resistant plant varieties by stably overexpressing the SbHDA3 gene in plants.

4. The application of recombinant vectors or recombinant microorganisms containing the SbHDA3 gene in the cultivation of highly disease-resistant plant varieties, characterized in that, The nucleotide sequence of the SbHDA3 gene is shown in SEQ ID NO.

11.

5. The application according to any one of claims 1-4, characterized in that, The disease resistance refers to the plant's resistance to bacterial blight, rice blast, and anthracnose.

6. The application according to any one of claims 1-4, characterized in that, The plants mentioned include rice and sorghum.

7. A method for improving plant disease resistance, characterized in that, The method includes the step of transferring the SbHDA3 gene into plants using genetic transformation technology to improve plant disease resistance; the nucleotide sequence of the SbHDA3 gene is shown in SEQ ID NO.

11.

8. A method for cultivating highly disease-resistant plant varieties, characterized in that, The method includes the steps of using genetic transformation technology to transfer the SbHDA3 gene into plants, thereby achieving stable overexpression of the SbHDA3 gene and cultivating disease-resistant plant varieties; the nucleotide sequence of the SbHDA3 gene is shown in SEQ ID NO.

11.

9. The method according to claim 7 or 8, characterized in that, The disease resistance refers to the plant's resistance to bacterial blight, rice blast, and anthracnose.

10. The method according to claim 7 or 8, characterized in that, The plants mentioned include rice and sorghum.

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