Protein elicitor SyCD1 and application thereof

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a protein elicitor SyCD1 and application thereof. According to the invention, a protein elicitor SyCD1 gene is obtained by cloning from Rabdolothrix poplar Hhs.015, the nucleotide sequence of the protein elicitor SyCD1 gene is as shown in SEQ ID No: 1, and the amino acid sequence of the protein coded by the protein elicitor SyCD1 gene is as shown in SEQ ID No: 2. The efficacy of the unknown functional protein-protein elicitor SyCD1 is discussed and studied for the first time, the unknown functional protein-protein elicitor SyCD1 is applied to improvement of plant disease resistance, and tests prove that the SyCD1 protein induces plant active oxygen accumulation and callose bursting, regulates plant defense gene expression and improves plant disease resistance. The invention provides a new biological material for improving the disease resistance of plants.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a protein elicitor SyCD1 and its applications. Background Technology

[0002] *Saccharomyces yanglingense* is a rare actinomycete. The *Saccharomyces yanglingense* strain Hhs.015 is an endophytic actinomycete isolated from cucumber roots in the inventor's laboratory. Both indoor and field trials have demonstrated that this strain is effective against *Tomato Leaf Mold* (*Saccharomyces yanglingense*). Fulvia fulva ), Apple black rot fungus ( Valsa mali ) and Sclerotinia sclerotiorum ( Sclerotinia sclerotiorum It exhibits significant inhibitory effects against various plant pathogens, including *H. 015*. Based on the 16S rRNA sequencing results of *H. 015* strain from Yangling, and through DNA-DNA hybridization, it was found that H. 015 is closely related to four members of the *H. 015* genus. Further research determined that H. 015 belongs to the phylum Actinobacteria (…). Actinobacteria Actinomycetes ( Actinobacteria ), Pseudococciales ( Pseudonocardiales ), Nocardiaceae ( Pseudonocardiaceae ), genus *Saccharomyces* ( Saccharothrix ).

[0003] Protein elicitors are a class of molecules of significant research value among numerous elicitors and have received widespread attention in recent years. Studies have shown that various protein elicitors have been successfully isolated and identified from pathogens, demonstrating their crucial role in regulating plant immune responses. Besides protein elicitors derived from pathogens, fungi with biocontrol functions have also become an important source of protein elicitors. Research has found that different strains of *Trichoderma* can produce a variety of protein elicitors and secondary metabolites, among which *Trichoderma harzianum* (…) is particularly valuable. Trichoderma harzianum Harzianolide, produced by *Fusarium oxysporum*, is a secondary metabolite with significant functions. It not only induces the expression of defense-related enzymes (such as peroxidase and phenylalanine ammonia-lyase) and related defense genes in plants, but also enhances systemic resistance and plays an important role in plant growth regulation. Furthermore, *Fusarium oxysporum* (…) Fusarium oxysporum The protein elicitors PeFOC1 and FocCP1, isolated from fermentation broth, have been shown to induce disease resistance responses in plants, such as enhancing the resistance of tobacco and banana seedlings to various pathogens. These elicitors have demonstrated good application potential in both field and laboratory trials. In recent years, protein elicitors derived from biocontrol bacteria have also attracted widespread attention from researchers. This is especially true for various bacteria of the genus Bacillus, such as Bacillus subtilis (…). Bacillus subtilis ), Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) and Bacillus laterosporus ( Brevibacillus laterosporusAll of these elicitors have been found to effectively suppress disease occurrence by stimulating plant hypersensitive responses (HR) and enhancing systemic resistance in plants. Furthermore, some studies have shown that these elicitors also exhibit significant effects in controlling plant pests.

[0004] Yangling sugar filament fungus has a significant inhibitory effect on a variety of plant pathogens. However, whether its protein elicitor is involved in the regulation of plant immune response or plant growth has not been reported in the literature. Studying the protein elicitor of Yangling sugar filament fungus is of great significance in improving plant disease resistance. Summary of the Invention

[0005] The primary objective of this invention is to provide a protein elicitor SyCD1 protein and its gene derived from *Saccharomyces yanglingensis* Hhs.015.

[0006] The second objective of this invention is to provide the application of the protein elicitor SyCD1 protein and its gene in improving plant disease resistance.

[0007] To achieve the above objectives, the present invention adopts the following technical measures: On one hand, the present invention provides an application of the protein elicitor SyCD1 in improving the disease resistance of plants, wherein the amino acid sequence of the protein elicitor SyCD1 is shown in SEQ ID No:2, the nucleotide sequence of the gene of the protein elicitor SyCD1 is shown in SEQ ID No:1, and the plant is tobacco or apple tree.

[0008] Furthermore, in the aforementioned application, enhancing plant disease resistance means improving plant resistance to pathogenic fungi and / or inducing plant defense responses.

[0009] Furthermore, in the aforementioned application, the enhancement of plant resistance to pathogenic fungi is achieved by inducing the accumulation of reactive oxygen species and the release of callose.

[0010] Furthermore, in the aforementioned application, the enhancement of plant resistance to pathogenic fungi is achieved by regulating the expression of plant defense genes.

[0011] Furthermore, in the application, the defense genes include genes related to the SA and JA signaling pathways.

[0012] Furthermore, in the application, the pathogenic fungus is *Sclerotinia sclerotiorum*.

[0013] Furthermore, in the application, the pathogenic fungus is *Penicillium aubergine*.

[0014] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages: This invention obtained the clone from *Saccharomyces yanglingensis* Hhs. O15. SyCD1 Gene ， The SyCD1 protein was obtained through genetic engineering, and its function was studied. Its application in improving plant disease resistance revealed that SyCD1 can enhance tobacco resistance to Sclerotinia sclerotiorum and improve the resistance of different apple varieties to black rot fungus, particularly in the Qin Cui apple, where SyCD1 showed the most significant improvement in resistance to black rot fungus.

[0015] The protein elicitor SyCD1 enhances tobacco resistance to *Sclerotinia sclerotiorum* and apple tree resistance to *Hemiberlesia oleracea* by inducing reactive oxygen species accumulation and callose bursting; furthermore, SyCD1 regulates apple tree defense genes. MdPR1 , MdPR2 , MdPR5 , MdEDS1 , MdNPR1 , MdPR4 and MdHAB1 The expression of [a specific gene] enhances the resistance of apple trees to apple black rot fungus; the protein elicitor SyCD1 regulates the expression of genes related to the SA and JA signaling pathways in tobacco, thereby improving tobacco's resistance to Sclerotinia sclerotiorum. This invention provides new biomaterials for improving plant disease resistance. Attached Figure Description

[0016] Figure 1 for SyCD1 Gene PCR electrophoresis image ( Figure 1 SDS-PAGE electrophoresis images of A and SyCD1 proteins (in the image) Figure 1 (B) in the middle; Figure 2 The SyCD1-induced improvement of resistance test results against *Nicotinus benthamiana* was achieved.

[0017] Figure 3 The results of tests on reactive oxygen species accumulation and callosity bursting induced by SyCD1 in Benedict's tobacco.

[0018] Figure 4 Expression map of SyCD1 activation of the tobacco defense gene.

[0019] Figure 5 To improve the resistance test results of Gala3 apple tissue culture seedlings induced by SyCD1 to apple black rot and peel mold.

[0020] Figure 6 The results of tests on reactive oxygen species accumulation and callose bursting induced by SyCD1 in Gala3 apple tissue culture seedlings.

[0021] Figure 7 Expression map of defense genes in SyCD1-activated Gala3 apple tissue culture seedlings.

[0022] Figure 8 The purpose of SyCD1 was to improve the resistance of apple leaves of different varieties to apple black rot fungus. Detailed Implementation

[0023] The technical solution of the present invention will be described below with reference to the embodiments. However, the present invention is not limited to the following embodiments.

[0024] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments and accompanying drawings. However, the embodiments described are not intended to limit the present invention.

[0025] Unless otherwise specified, the experimental and detection methods described in the following embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0026] Example 1 This embodiment provides a protein exciton. SyCD1 Gene sequence cloning and construction of pET28a-SyCD1-His vector.

[0027] (1) Genomic DNA extraction of *Saccharomyces yanglingensis* Hhs.015 The *Saccharomyces yanglingensis* strain Hhs.015, stored at -80℃, was streaked onto Gao's No. 1 medium using sterile bamboo sticks and incubated at 28℃ until white spores were produced. Five to seven mycelial discs were inoculated into 100 mL of tryptic soy broth (TSB) and incubated at 28℃ for 48 h on a shaker. The mycelial cells were then collected by centrifugation for DNA extraction. Genomic DNA was extracted using the CTAB method, and 3 µL of the sample was subjected to agarose gel electrophoresis to assess DNA quality.

[0028] (2) Cloning of SyCD1 gene and construction of prokaryotic expression vector Primers were designed using the adapter sequence of the prokaryotic expression vector pET28a. Genomic DNA from *Gymnophiopogon yanglingensis* Hhs.015 was used as a template for sequence amplification. The amplification system was 50 μL, specifically: 2×Phanta Max Buffer 25 μL, pET28a-SyCD1-F 2 μL, pET28a-SyCD1-R 2 μL, dNTPs 1 μL, genomic DNA 1 μL, Phanta® Max Super-Fidelity DNA Polymerase 1 μL, and ddH2O was added to bring the total volume to 50 μL. The PCR reaction program was: denaturation at 95℃ for 3 min, pre-denaturation at 95℃ for 15 s, annealing at 56-64℃ for 15 s, extension at 72℃ for 30 s / kb, for a total of 33 cycles, with a final extension at 72℃ for 5 min. PCR products were detected using a 1% agarose gel electrophoresis. The results are shown below. Figure 1 As shown in A, lane M is the marker, and lane 6 is the target band. SyCD1 Gene strips), will SyCD1 Gene strips were excised and recovered using a gel extraction kit. SyCD1 Take a portion of the PCR product of the gene. SyCD1 The PCR product of the gene was sent to a biotechnology company for sequencing. Sequencing yielded... SyCD1 Gene sequence, SyCD1 The gene sequence is shown in SEQ ID No:1. Primers used are detailed in Table 1.

[0029] Take part SyCD1 The PCR product of the gene was recovered and ligated into the linearized vector using the ClonExpress-II® one-step cloning kit. The ligation product was transformed into E. coli DH5α by heat shock and cultured upside down at 37°C for 14-18 h on a selection plate containing kanamycin. After single colonies were picked for colony PCR detection, they were inoculated into LB liquid medium containing antibiotics and cultured at 37°C and 200 rpm for 14-18 h. The plasmid was extracted and sent to Beijing Qingke Biotechnology Co., Ltd. for sequence detection. The sequencing results were compared with the sequence shown in SEQ ID No:1. The sequence was consistent, indicating that the pET28a-SyCD1-His prokaryotic expression vector was successfully constructed.

[0030] Table 1 Primer sequence listing

[0031] Example 2 This example demonstrates the fermentation process for obtaining the SyCD1 protein.

[0032] The prokaryotic expression vector pET28a-SyCD1-His was transformed into Escherichia coli BL21(DE3) competent cells and cultured at 37℃ and 220 rpm for 2-3 hours until OD. 600 The concentration was 0.6; IPTG was added to a final concentration of 0.3 mM for induction (16℃, 18h or more); the bacterial cells were collected by centrifugation at 8000 rpm at 4℃ for 3 min, and the bacterial suspension was washed 2-3 times with PBS buffer; lysozyme, 1 mM PMSF, and 1 mM β-mercaptoethanol were added to a final concentration, and the mixture was reacted at room temperature for 30 min; the bacterial suspension was then sonicated on ice (40% power, 3s on, 4s off, total sonication time 30 min); the mixture was centrifuged at 12000 rpm at 4℃ for 15 min, the bacterial cells were removed using a 0.22 μM bacterial filter, the supernatant was collected, and 10 μL of the supernatant was analyzed by SDS-PAGE electrophoresis. The results are as follows. Figure 1 As shown in Figure B, lane M is the marker, and lane SyCD1 is the target protein SyCD1. The measurement results show that the size of SyCD1 protein is between 10kD and 15kD, which is consistent with the expected size.

[0033] The collected supernatant was purified according to the Thermo HisPur Ni-NTA Resin instructions. A primary purified SyCD1 protein was obtained. Imidazole was removed from the primary purified SyCD1 protein by dialysis with PBS buffer, yielding the SyCD1 protein. The SyCD1 protein was then flash-frozen in liquid nitrogen and stored at -80°C for later use.

[0034] Example 3 This example demonstrates a test experiment demonstrating the effect of the protein elicitor SyCD1 on enhancing the resistance of Nicotiana benthamiana to Sclerotinia sclerotiorum.

[0035] The SyCD1 protein prepared in Example 2 was diluted to 1 μM with sterile ddH2O to prepare a SyCD1 protein dilution solution for later use. Using PBS, the final dialysis solution of SyCD1 protein purification, as a control, the PBS was diluted with sterile ddH2O at the same ratio as the SyCD1 protein dilution solution to prepare a PBS dialysis solution dilution solution for later use.

[0036] (1) SyCD1 protein induces increased resistance of Nicotiana benthamiana to Sclerotinia sclerotiorum. Nicotiana benthamiana plants with a growth period of 4-6 weeks were selected. The dorsal side of the plant was injected with either a diluted SyCD1 protein solution (test group, SyCD1) or a diluted PBS dialysis solution (control group), and incubated at 25℃ for 24 hours. Tobacco leaves were cut and placed in an iron tray lined with moist filter paper. Cotton was wrapped around the petiole to retain moisture, and a wound was created using a sterile syringe needle to facilitate mycelial infection. Mycelial cakes were inoculated on the upper side of the leaves and sprayed with water to maintain moisture. After 24 hours at room temperature, photographs were taken for observation, and lesions were statistically analyzed. Each experiment was repeated three times. Test results are as follows: Figure 2 As shown, compared with the control, SyCD1 protein significantly enhanced the resistance of tobacco leaves to Sclerotinia benthamiana.

[0037] (2) SyCD1 protein induces reactive oxygen species accumulation and callosin bursting in Nicotiana benthamiana. Nicotiana benthamiana plants with a growth period of 4-6 weeks were selected. SyCD1 protein dilution (test group, SyCD1) or PBS dialysis solution dilution (control group) were injected dorsally, and the plants were cultured at 25℃ for 24 hours. Tobacco leaves were cut into 1×1cm cubes for later use. An aqueous solution (pH 3.8) containing 1 mg / mL 3,3'-diaminobenzidine (DAB) was prepared and reacted under light for 8 hours. After the reaction, the leaves were placed in a decolorizing solution (95% ethanol) until complete decolorization, then observed and photographed for preservation. For aniline blue staining, the cut leaves were completely decolorized with the decolorizing solution, washed with ddH2O, and placed in aniline blue staining solution (0.01% aniline blue dissolved in 150mM K2HPO4·3H2O (pH 9.5)). Staining was performed in the dark for 12-14 hours, followed by observation and photographing under a fluorescence microscope. The test results are as follows: Figure 3 As shown, compared with the control, SyCD1 protein induced the accumulation of brown reactive oxygen species and callosin bursting.

[0038] (3) SyCD1 protein activates the expression of genes related to defense against Nicotiana benthamiana. Nicotiana benthamiana plants with a growth period of 4-6 weeks were selected. SyCD1 protein dilution (test group, SyCD1) or PBS dialysis solution dilution (control group) was injected into the dorsal side of the leaves, using whole tobacco leaves. Samples were collected at 0h, 6h, 12h, and 24h after injection, flash-frozen in liquid nitrogen, and thoroughly ground into powder. RNA extraction was performed using the Beijing Huayueyang Plant Tissue RNA Extraction Kit; detailed instructions are provided in the product manual. Reverse transcription was performed using the RevertAid™ First Strand cDNA Synthesis Kit, with a total RNA amount of 1 μg for cDNA synthesis. The total reaction volume was 20 μL: 1 μL Oligo dT, 11 μL total RNA, and ddH2O, incubated at 65℃ for 5 min; then 4 μL 5×Reaction buffer, 1 μL Inhibitor, 1 μL RevertAid Reversase Trascriptase, and 2 μL 10 mM dNTP mix were added, incubated at 42℃ for 60 min and 72℃ for 8 min. After reverse transcription, dilute 10-fold and store at -80℃ for later use.

[0039] Using reverse-transcribed cDNA as a template, real-time quantitative PCR was performed using RealStar Green Mixture. The total volume was 20 μL: 1 μL cDNA, 1 μL Primer F / R, 10 μL RealStar Green Mixture, and 7 μL ddH2O. The real-time quantitative PCR instrument was a Roche LightCycler 96, and the reaction program was: 95℃ for 10 min, 45 cycles (95℃ for 15 s, 60℃ for 30 s, and 72℃ for 30 s). Actin was used as an internal control primer; the primers used are detailed in Table 1.

[0040] Test results are as follows Figure 4 As shown, real-time quantitative PCR results indicate that SyCD1 treatment upregulates genes involved in the salicylic acid (SA) signaling pathway. NbPAL The gene reached its peak relative expression level 6 hours after SyCD1 treatment. NbPAL Gene expression levels were compared to the control group. NbPAL Approximately 15 times the gene expression level; NbPR1a The relative expression level of the gene was highest 12 hours after SyCD1 treatment. NbPR1a Gene expression levels were compared to the control group. NbPR1a The gene expression level was nearly 220 times higher; NbPR4 Gene expression levels did not reach peak 24 hours after SyCD1 treatment. At this time, the SyCD1-treated group... NbPR4 The gene expression level is close to that of the control group. NbPR4 118 times the gene expression level; NbNPR1 Gene expression reached its peak 12 hours after SyCD1 treatment, with the SyCD1-treated group showing an expression level 5 times that of the control group.

[0041] In the jasmonic acid (JA) signaling pathway, NbLOX1 Gene expression reached its peak 6 hours after SyCD1 treatment, with the SyCD1-treated group showing approximately 6 times the expression level of the control group. NbCOI1 and NbPDF1.2 The gene was suppressed in the early stage, and the suppression was relieved 24 hours after SyCD1 treatment; NbERF1 Gene expression reached its peak 6 hours after SyCD1 treatment, with the SyCD1-treated group showing an expression level 3.5 times that of the control group.

[0042] In summary, this embodiment demonstrates that SyCD1 protein significantly enhances the resistance of *Nicotiana benthamiana* leaves to *Sclerotinia sclerotiorum*; SyCD1 protein can induce the accumulation of reactive oxygen species and callose release in *Nicotiana benthamiana*, thereby improving the plant's resistance to pathogenic fungi; and SyCD1 protein treatment disrupts the salicylic acid signal transduction pathway. NbPAL Gene, NbPR1a Gene, NbPR4 Genes and NbNPR1 Gene expression was upregulated, suggesting that SyCD1 protein enhances resistance to pathogenic fungi by upregulating genes related to the salicylic acid signaling pathway; jasmonic acid signaling pathway activity was also increased after SyCD1 protein treatment. NbLOX1 Upregulation of gene and NbERF1 gene expression NbCOI1 Genes and NbPDF1.2 The expression of the SyCD1 gene protein was suppressed in the early stage of treatment, and the inhibition was relieved 24 hours after treatment, suggesting that SyCD1 regulates plant resistance to pathogenic fungi by regulating gene expression in the jasmonic acid signaling pathway.

[0043] Example 4 This embodiment is a test experiment on the effect of the protein elicitor SyCD1 on the resistance of apple trees to apple black rot fungus.

[0044] (1) The protein elicitor SyCD1 enhances the growth of apple tissue culture seedlings Gala 3. Valsa mali Resistance and its mechanism of action.

[0045] Apple tissue culture seedlings Gala 3: Apple tissue culture seedlings Gala 3.

[0046] The SyCD1 protein prepared in Example 2 was diluted to 15 μM with PBS and sprayed onto one-month-old Gala 3 apple tissue culture seedlings as a control. The seedlings were cultured at 25°C for 16 h / h light / dark for 24 h. Leaves of similar growth were placed in 0.7% water agar medium, and wounds were created using a sterile syringe needle to pick out apple black rot fungus (…). V.mali The fungal cake was applied to the wound, and the area of ​​the lesion was counted and photographed after 24 hours.

[0047] Meanwhile, apple leaves of the same size after the above treatment were used to detect the expression of reactive oxygen species, callosity and defense-related genes. The experimental method is the same as in Example 3, and the PCR primers are detailed in Table 1.

[0048] Test results are as follows Figure 5 As shown, the leaves of Gala3 treated with SyCD1 had smaller lesion areas and weaker disease severity compared to the control group. Results of reactive oxygen species and callose tests are as follows... Figure 6 As shown, SyCD1 increased the accumulation of reactive oxygen species and callose production in the leaves of apple tissue culture seedling Gala 3. The results of defense-related gene testing are as follows... Figure 7 As shown, the SyCD1 treatment group is compared with the control group. MdPR1 , MdPR2 , MdPR5 , MdEDS1 , MdNPR1 , MdPR4 and MdHAB1 The expression levels all increased.

[0049] (2) SyCD1 improves the resistance of different apple varieties to the elements. V.mali resistance Cut leaves from Fuji, Red Sinike, Ruixue, and Qincui apple trees and place them in an iron tray lined with moist filter paper. Wrap cotton around the petioles to retain moisture. Dilute SyCD1 protein to 20 μM and spray with PBS as a control. Create wounds using a sterile syringe needle to facilitate fungal infection. Inoculate the upper surface of the leaves with... V. mali The fungal cake was sprayed with water to keep it moist. After being placed at room temperature for 3 days, photos were taken and the lesions were counted. Each group of experiments was repeated three times.

[0050] The results are as follows Figure 8 As shown, different varieties of apple leaves were sprayed with SyCD1 protein and then inoculated. V.mali The SyCD1 protein enhanced resistance to apple tree rot pathogens in different apple varieties. Compared with the control group, the leaf area of ​​Fuji, Red Sinike, Ruixue and Qincui apples showed a reduction in diseased area to varying degrees, with Qincui apples showing the most significant reduction in leaf area.

[0051] As described above, the basic principles, main features, and advantages of the present invention have been well described. The above embodiments and specifications are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the protection scope defined by the present invention.

Claims

1. The application of a protein elicitor SyCD1 in improving plant disease resistance, characterized in that, The amino acid sequence of the protein elicitor SyCD1 is shown in SEQ ID No:2, and the nucleotide sequence of the gene for the protein elicitor SyCD1 is shown in SEQ ID No:

1. The plant is tobacco or apple tree.

2. The application according to claim 1, characterized in that, The improvement of plant disease resistance refers to enhancing plant resistance to pathogenic fungi and / or inducing plant defense responses.

3. The application according to claim 2, characterized in that, The improvement of plant resistance to pathogenic fungi is achieved by inducing the accumulation of reactive oxygen species and the release of callose.

4. The application according to claim 2, characterized in that, The improvement of plant resistance to pathogenic fungi is achieved by regulating the expression of plant defense genes.

5. The application according to claim 4, characterized in that, The defense genes include genes related to the SA and JA signaling pathways.

6. The application according to claim 2, characterized in that, The pathogenic fungus is Sclerotinia sclerotiorum.

7. The application according to claim 2, characterized in that, The pathogenic fungus is *Pseudomonas aeruginosa*.

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