Application of sorghum SbPGK1 gene in improvement of plant disease resistance

Abstract

The invention discloses application of a sorghum SbPGK1 gene in improving plant disease resistance, and belongs to the technical field of biology. The nucleotide sequence of the sorghum SbPGK1 gene is as shown in SEQ ID NO.9. By analyzing phenotypes and related physiological indexes of SbPGK1 rice transgenic lines before and after inoculation of different pathogenic bacteria for infection and sorghum, the results show that compared with non-SbPGK1 gene rice lines, the SbPGK1 transgenic lines have obviously better resistance to magnaporthe oryzae and bacterial blight germs than wild types, silence SbPGK1 in sorghum has better resistance to rice blast germs and bacterial blight germs, and the SbPGK1 gene has better resistance to rice blast germs and bacterial blight germs. The SbPGK1 gene has the advantages that the SbPGK1 silent strain has reduced disease resistance to colletotrichum gloeosporioides, the SbPGK1 is overexpressed in sorghum, and the SbPGK1 overexpressed strain has enhanced disease resistance to colletotrichum gloeosporioides, so that the SbPGK1 gene is a potential disease-resistant breeding gene and can be used for plant disease-resistant genetic improvement.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to application of sorghum SbPGK1 gene in improving plant disease resistance. Background Art

[0002] Phosphoglycerate kinase (PGK) is a key enzyme in glycolysis, primarily composed of an N-terminal nucleotide-binding domain (NBD) and a C-terminal catalytic domain (CD) (Zheng et al., 2012). It catalyzes the conversion of 1,3-bisphosphoglycerate (1,3-BPG) into 3-phosphoglycerate (3-PG) and ATP, providing energy for life activities (Chen et al., 2022). It plays a crucial role in energy metabolism, biosynthesis, and redox homeostasis. PGK deficiency in organisms can lead to metabolic disorders, which in turn affect normal life activities.

[0003] In plants, PGK is not only involved in growth and development and carbon metabolism (Li, et al., 2019), but also in plant resistance to abiotic stresses. For example, expressing OsPGK2 and AtPGK2 in tobacco and Arabidopsis, respectively, can improve plant salt tolerance (Joshi, et al., 2015; Liu, et al., 2014); the loss of cPGK1 and cPGK2 in Arabidopsis will cause the plants to turn albinic (Watson, et al., 1982); and the silencing of PGK1 in tomato will inhibit plant growth (Yin Xiaowei, 2022).

[0004] PGKs are also involved in plant disease resistance. PGK genes play a crucial role in wheat resistance to stripe rust infection (Yang Donghe, 2017); PGKs regulate the accumulation of bamboo mosaic virus within chloroplasts (Cheng et al., 2013); silencing the PGK2 gene reduces Arabidopsis resistance to plum pox virus (Poque et al., 2015); PGK2 is essential for infection with watermelon mosaic virus (Ouibrahim et al., 2014); PGK1 promotes the assembly of replication components essential for tomato bushy stunt virus (Dinesh-Kumar et al., 2017); and NbPGK gene expression is significantly downregulated during the interaction between tobacco and rice stripe virus (Bi Ji'an, 2019). PGKs have been shown to be essential for the pathogenicity of single-stranded positive RNA (ssRNA+) viruses (Dinesh-Kumar, et al., 2017; Nagy & Lin, 2020).

[0005] As a key enzyme in the glycolysis pathway, the biological function of PGK is dynamically regulated by a variety of post-translational modifications of proteins. Studies have shown that modifications such as acetylation (Hu et al., 2017), phosphorylation (Zhang, et al., 2018b), ubiquitination (Dong, et al., 2019), succinylation (Zhang, et al., 2018a), and glycosylation (Nie, et al., 2020) jointly regulate the enzymatic activity, subcellular localization, and biological function of PGK, thereby affecting its role in processes such as anti-disease response. For example, during tumorigenesis, PGK1 glycosylation is regulated by O-GlcNAc glycosylation to promote tumor growth (Nie et al., 2020); phosphorylation of the PGK1 S203 site can promote cell metabolism and tumorigenesis. Acetylation at K323 significantly activates PGK1, a post-translational modification that plays a key regulatory role in the metabolic processes of liver cancer cells (Hu et al., 2017). Acetylation of PGK1 at K338 is crucial for hypoxia-induced autophagy and brain tumorigenesis (Qian et al., 2017). Insulin can induce the histone deacetylase HDAC3 to reduce PGK1 acetylation levels, thereby activating PGK1 and regulating the rapamycin (mTOR)-mediated signaling pathway (Wang et al., 2015). These studies indicate that PGK1 activity is regulated by acetylation and participates in anti-disease responses.

[0006] With the booming development of industries like winemaking and animal feed, the sorghum planting area is showing a growing trend. At the same time, various diseases frequently occur during sorghum growth, seriously threatening its yield and quality. Currently, there are over 60 common sorghum diseases. The wide variety of diseases that occur during cultivation makes the molecular mechanisms of plant disease resistance quite complex. Summary of the Invention

[0007] In this study, we used SbPGK transgenic rice and sorghum VIGS and VOX systems to detect SbPGK1-mediated plant disease resistance, preliminarily exploring the molecular mechanism of SbPGK1's involvement in sorghum disease resistance, and providing a basis for sorghum disease resistance breeding.

[0008] The present invention provides an application of a sorghum SbPGK1 gene in regulating disease resistance of rice and / or sorghum. The nucleotide sequence of the sorghum SbPGK1 gene is shown in SEQ ID NO.9.

[0009] In one embodiment of the present invention, the regulation is overexpression of the sorghum SbPGK1 gene in rice and / or sorghum.

[0010] The present invention also provides the use of a protein encoded by the sorghum SbPGK1 gene in regulating disease resistance of rice and / or sorghum. The nucleotide sequence of the sorghum SbPGK1 gene is shown in SEQ ID NO.9.

[0011] The present invention also provides a method for improving the disease resistance of rice and / or sorghum, which involves overexpressing the SbPGK1 gene in rice and / or sorghum; the nucleotide sequence of the SbPGK1 gene is shown in SEQ ID NO.9.

[0012] In one embodiment of the present invention, a vector containing the above-mentioned sorghum SbPGK1 gene is introduced into a plant.

[0013] In one embodiment of the present invention, the sorghum SbPGK1 gene is introduced into plants using Agrobacterium-mediated genetic transformation.

[0014] In one embodiment of the present invention, the disease resistance refers to resistance to rice blast, bacterial blight or anthracnose.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The present invention analyzes the phenotypes and related physiological indicators of SbPGK1 transgenic rice lines and sorghum before and after inoculation with different pathogens. The results show that compared with non-SbPGK1 transgenic rice lines, the SbPGK1 transgenic lines have significantly better resistance to rice blast and bacterial blight than the wild type. When SbPGK1 is silenced in sorghum, the SbPGK1-silenced lines have reduced resistance to anthracnose, while when SbPGK1 is overexpressed in sorghum, the SbPGK1-overexpressing lines have enhanced resistance to anthracnose. This indicates that the SbPGK1 gene is a potential disease-resistance breeding gene and can be used for genetic improvement of plant disease resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 PCR screening and detection of SbPGK1 transgenic rice in Example 1, including: A. PCR screening of SbPGK1#1 F1 generation plants; B. PCR screening of SbPGK1#1 F2 generation plants; C. PCR verification of homozygous SbPGK1 F3 generation plants. M represents DNA marker.

[0018] Figure 2 Results of the analysis of bacterial blight resistance of SbPGK1 transgenic rice in Example 1. SbPGK1 transgenic rice was inoculated with bacterial blight pathogens using the leaf clipping method, and lesion length was measured 20 days later. (A) Lesion phenotype of transgenic rice 20 days after inoculation with Xoo PX099A. (B) Lesion area. Bars represent 20 biological replicates. Significance was analyzed using the Student's t-test (***, p < 0.001).

[0019] Figure 3 Results of the analysis of blast resistance in SbPGK1 transgenic rice from Example 1. SbPGK1 transgenic rice was inoculated with a spore suspension of the blast fungus, and lesion area was counted 8 days later. (A) A partial image of the rice blast inoculation site (dpi = 8); (B) Lesion area statistics. Bars represent 20 biological replicates. Significance was analyzed using the Student's t-test (****, p < 0.0001).

[0020] Figure 4Phenotypic analysis of anthracnose resistance in sorghum silencing and overexpressing SbPGK1 in Example 1. A. Phenotypes of sorghum silencing FoMV-EV, SbPDS silencing (positive control), SbPGK1 silencing, and SbPGK1 overexpression 28 days after inoculation. Expression levels in sorghum silencing (SbPDS, SbPGK1) and overexpression (SbPGK1) were analyzed using RT-qPCR. Bar values ​​represent three technical replicates, and significance was analyzed using Student's t-test (*, p<0.05; **, p<0.001; ****, p<0.0001). B. Phenotypes of sorghum silencing and overexpressing SbPGK1 after inoculation with anthracnose (dpi = 5). Anthracnose lesion area was calculated based on a 10 cm radius of the inoculated leaf. Bar values ​​represent the standard error of 10 replicates (dpi = 5). Relative fungal biomass was also measured. Bar values ​​represent the standard error of three technical replicates, and significance was analyzed using Student's t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). DETAILED DESCRIPTION

[0021] Example 1

[0022] 1. Test materials

[0023] Plant material

[0024] The seeds of Nicotiana benthamiana and Sorghum bicolor (red tassel) are stored in our laboratory.

[0025] 1.2 Strains, vectors, and primers

[0026] Strain: Agrobacterium tumefaciens: GV1301, P19.

[0027] Escherichia coli strain: DH5α.

[0028] Rice blast fungus (Magnaporthe oryzae): RB22 (common strain).

[0029] Xanthomonas oryzae pv. oryzae: PXO99A.

[0030] Sorghum anthracnose: Colletotrichum sublineola.

[0031] Vector: Rice transgenic overexpression vector: pRHVcGFP (He F, Zhang F, Sun W, et al. AVersatile Vector Toolkit for Functional Analysis of Rice Genes[J]. Rice, 2018, 11(1): 27. DOI: 10.1186 / s12284-018-0220-7.) is preserved in our laboratory.

[0032] FoMV vector (foxtail mosaic virus): kindly provided by Professor Liu Wende from the Institute of Plant Protection, Chinese Academy of Agricultural Sciences.

[0033] Table 1 Primer sequences

[0034] 2 SbPGK1 gene cloning and expression vector construction

[0035] The nucleotide sequence of sorghum SbPGK1 is as follows:

[0036] SbPGK1 (SEQ ID NO. 9):

[0037]

[0038] 2.1 RNA extraction

[0039] Total RNA was extracted from plant leaves using the Trizol method. The steps are as follows:

[0040] (1) Take 0.1 g of plant leaves, place them in a sterilized centrifuge tube, quickly freeze them, and grind them into powder.

[0041] (2) Add 1 mL of Trizol extract and shake thoroughly. After standing for 2 minutes, add 200 μL of RNA extract, shake immediately and let stand for 5 minutes.

[0042] (3) Centrifuge at 12,000 rpm for 10 min at 4°C and transfer the supernatant to an RNase-free centrifuge tube.

[0043] (4) Immediately add an equal volume of pre-cooled isopropanol, gently invert the mixture and place in a -20°C refrigerator to settle for 30 minutes or at room temperature for 10 minutes.

[0044] (5) After standing, centrifuge at 2000 rpm for 10 min at 4°C. Carefully discard the supernatant and immediately add 1 mL of 70% alcohol to the precipitate to wash it. Repeat this step twice.

[0045] (6) Centrifuge at 4°C and 12,000 rpm for 5 min to carefully remove the alcohol and air dry. Dissolve the solution in 30 μL of DEPC water and store in a -80°C refrigerator until ready for use.

[0046] cDNA was synthesized using RNA as a template according to the instructions of the HiScript® III 1st Strand cDNA Synthesis Kit and stored in a -20°C refrigerator until use.

[0047] 2.2 Gene amplification

[0048] Primers were designed based on the full-length CDS sequence (excluding the stop codon) and restriction site sequences on the Novozyme online website (https: / / crm.vazyme.com / cetool / singlefragment.html). Primers were synthesized by Qingke Biotechnology (Table 1). High-fidelity Taq enzyme (TransStart FastPfu DNA Polymerase, Beijing Quanshijin Biotechnology Co., Ltd.) was used to amplify the target fragments using primers listed in Table 1, SEQ ID NOs. 1 and 2, and SEQ ID NOs. 3 and 4, respectively. The amplification protocol was as follows: 95°C denaturation for 5 min, 94°C denaturation for 30 s, 58°C annealing for 30 s, and 72°C extension for 1 min for 40 cycles, followed by an additional extension at 72°C for 5 min and storage at 4°C. After PCR, PCR product size was determined by 1% agarose gel electrophoresis at 120 V for 20 min, using a DL2,000 DNA marker (Takara) as a control.

[0049] The PCR product of the correct size was purified using the SanPrep column-based DNA gel recovery kit (Sangon Biotech (Shanghai) Co., Ltd.). The experimental method was carried out according to the instructions, and the recovered product was stored at -20°C until use.

[0050] 2.3 Expression vector construction

[0051] Construction of silencing and overexpression vectors in sorghum

[0052] (1) Target gene amplification and identification: Target gene fragments were obtained by PCR amplification, identified by 1% agarose gel electrophoresis, and recovered by gel excision;

[0053] (2) Enzyme digestion of vector: establish a 50 μL enzyme digestion system, including 5 μL 10× Buffer, 2 μL restriction endonuclease, 2 ng plasmid, and make up the volume with ddH2O. React at 37℃ for 2-4 h, inactivate at 80℃ for 5 min, and detect and recover by 1% agarose gel electrophoresis;

[0054] (3) Recombination ligation and transformation: The purified PCR product and the enzyme-cut vector were mixed in an appropriate ratio and ligated using T4 ligase (5 μL, 22°C, 1 h). The ligation product was immediately transformed into Escherichia coli competent cells.

[0055] Construction of rice overexpression vector

[0056] 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°C for 3 h, and recover the digestion products after identification by 1.2% agarose gel electrophoresis.

[0057] The PCR product amplified from SEQ ID NOs. 7 and 8 and the double-digested product (BamHI and HindIII) were ligated using homologous recombinases. The ligation system (10 μL) consisted of 1 μL of double-digested product, 2 μL of target gene fragment, and 5 μL of ligase. Add ddH2O to 10 μL and ligate at 50°C for 15 min to generate the SbPGK1-RHV-GFP overexpression vector. The ligation product was immediately transformed into E. coli.

[0058] 2.4 Cultivation and screening of SbPGK1 transgenic rice

[0059] The SbPGK1-RHV-GFP overexpression vector was transformed into Nipponbare (Oryza sativa L. spp. japonica). The F0 generation was harvested (SbPGK1: P#1), resulting in a heterozygous F1 generation. Twenty-four transgenic progeny of P#1 were selected and tested for SbPGK1 gene expression using specific primers, yielding 12 P#1-positive samples. The corresponding SbPGK1-positive heterozygous F1 plants were cultivated and harvested to yield the F2 generation. DNA from 240 F2 rice plants was extracted and sequenced for PCR, yielding 41 P#1-positive samples. Positive plants were further cultivated to the F3 generation, and random F3 transgenic samples were tested using specific PCR primers, yielding positive results. The SbPGK1 F3 transgenic rice plants were designated P#1-3.

[0060] The results are as follows Figure 1 shown.

[0061] 3. Inoculation of transgenic rice with bacterial blight and rice blast

[0062] 3.1 Inoculation of bacterial blight pathogen

[0063] Rice cultures that have been incubated for approximately one month can be used to test the pathogenicity of bacterial blight. Activate the pathogen in NB liquid medium and shake at 28°C and 200 rpm until the OD600 reaches 1.0. Using sterilized scissors, apply an appropriate amount of bacterial suspension and cut off the tip 2-3 cm of a well-growing rice leaf. After inoculation, maintain moisture and maintain heat under the following conditions: 12 hours of light / 12 hours of dark, a temperature of 30°C, and a relative humidity of at least 70%. Measure leaf lesion length 20 days after inoculation. Set up 20 replicates for each sample.

[0064] The results are as follows Figure 2 shown.

[0065] 3.2 Rice blast inoculation

[0066] Use oatmeal medium to activate the strain and culture at 28°C under continuous light for 5-10 days. The specific inoculation steps are as follows:

[0067] (1) Elute spores with 0.5% Tween-20 and filter;

[0068] (2) Use a hemocytometer to measure the concentration of the spore suspension under a microscope and adjust it to 1×10 5 / mL;

[0069] (3) Use a small spray bottle to evenly spray the spore suspension onto the surface of rice leaves (at least 20 seedlings in each experimental group). After culturing in the dark for 2 days under a warm and moisturizing condition, continue the 12 h light / 12 h dark culture.

[0070] (4) Observe the results after 7 days and take photos and samples.

[0071] The results are as follows Figure 3 shown.

[0072] 4. Sorghum VIGS, VOX, and Anthrax Inoculation

[0073] 4.1 Sorghum VIGS, VOX

[0074] (1) The constructed SbPDS / SbPGK1-FoMV-VIGS and SbPGK1-FoMV-VOX vectors were transformed into Agrobacterium, and Nicotiana benthamiana was infiltrated and injected according to the tobacco transient expression method;

[0075] (2) Take 1 g of infected Nicotiana benthamiana leaves, add 10 mL of PBS buffer, grind, filter and collect the filtrate for later use;

[0076] (3) When the sorghum grows to the 2-3 leaf stage, sprinkle a little corundum evenly on the leaf surface, add the filtrate, and gently rub with your fingers to inoculate;

[0077] (4) After 28 days, take photos and observe, and extract RNA from sorghum leaf tissue. After reverse transcription into cDNA, store it in a -80℃ refrigerator for later use.

[0078] The results are as follows Figure 4 As shown in A.

[0079] 4.2 Anthrax inoculation

[0080] (1) After culturing Colletotrichum sorghum in PDA at 28°C for 3-5 days, spores were eluted and filtered using sterile water containing 0.5% Tween-20.

[0081] (2) Observe and record the number of spores under a microscope using a hemocytometer. Adjust the concentration to 10 5 / mL, place the spore suspension in a sterilized small spray bottle and spray it on the treated sorghum leaves;

[0082] (3) The culture was kept moist and dark for 2 days. At the same time, the sorghum was photographed and sampled at 0, 12, 24, 48, 60, 72, 84, 96, 108 and 120 hours after inoculation.

[0083] (4) DNA and RNA were extracted from the above sorghum leaves. DNA was used for subsequent fungal biomass statistics, and RNA was reverse transcribed into cDNA for RT-qPCR detection of SbPGK1 and SbHDA3 expression levels at different time periods.

[0084] The results are as follows Figure 4 As shown in Figure B, silencing SbPGK1 increased the number of anthracnose lesions and the biomass of anthracnose fungi in sorghum. Overexpression of SbPGK1 reduced the number of anthracnose lesions and the biomass of anthracnose fungi in sorghum. SbPGK1 positively regulates sorghum resistance to anthracnose.

[0085] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Application of the sorghum SbPGK1 gene in regulating disease resistance in rice and / or sorghum, characterized in that: The nucleotide sequence of the sorghum SbPGK1 gene is shown in SEQ ID NO.

9.

2. The use according to claim 1, characterized in that The regulation is to overexpress the sorghum SbPGK1 gene according to claim 1 in rice and / or sorghum.

3. Use of a protein encoded by the sorghum SbPGK1 gene in regulating disease resistance in rice and / or sorghum, characterized in that: The nucleotide sequence of the sorghum SbPGK1 gene is shown in SEQ ID NO.

9.

4. A method for improving disease resistance of rice and / or sorghum, characterized in that: The SbPGK1 gene is overexpressed in rice and / or sorghum; the nucleotide sequence of the SbPGK1 gene is shown in SEQ ID NO.

9.

5. The method according to claim 4, characterized in that The invention comprises transferring a vector containing the sorghum SbPGK1 gene according to claim 1 into a plant.

6. The method according to claim 5, characterized in that The sorghum SbPGK1 gene was introduced into plants using Agrobacterium-mediated genetic transformation.

7. The method according to any one of claims 4 to 6, characterized in that: The disease resistance refers to resistance to rice blast, bacterial blight or anthracnose.

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