Protein agonist syCD1 and use thereof

Abstract

The application belongs to the technical field of bioengineering and particularly relates to a protein elicitor SyCD1 and application thereof. SyCD1 The nucleotide sequence of the gene is shown as SEQ ID No:1, and the amino acid sequence of the encoded protein is shown as SEQ ID No:2. The efficacy of the unknown function protein-protein elicitor SyCD1 is first explored in the application, and the protein is applied to improving the disease resistance of plants. It is verified through experiments that the SyCD1 protein induces the accumulation of active oxygen and the eruption of callose in plants, regulates the expression of plant defense genes, and improves the disease resistance of plants. The application 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 ), sacchariformis ( 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 laterosporus), all of which are found to be able to effectively inhibit the occurrence of diseases by stimulating the hypersensitive response (HR) of plants and enhancing the systemic resistance of plants. In addition, some studies have also shown that these elicitors also exhibit significant effects in preventing and controlling plant insect pests.

[0004] Yangling amylovorax has significant inhibitory effect on various plant pathogenic fungi, however, whether its protein elicitor is involved in the regulation of plant immune response and the regulation of plant growth has not been reported in the literature, and it is of great significance to study the protein elicitor of Yangling amylovorax in improving the disease resistance of plants. SUMMARY

[0005] The first object of the present application is to provide a protein elicitor SyCD1 protein and its gene derived from Yangling amylovorax Hhs.015.

[0006] The second object of the present application is to provide the application of the protein elicitor SyCD1 protein and its gene in improving the disease resistance of plants.

[0007] In order to achieve the above object, the present application adopts the following technical measures:

[0008] On the one hand, the present application provides the application of a protein elicitor SyCD1 in improving the disease resistance of plants, the amino acid sequence of the protein elicitor SyCD1 is shown as SEQ ID No: 2, the nucleotide sequence of the gene of the protein elicitor SyCD1 is shown as SEQ ID No: 1, and the plant is tobacco or apple tree.

[0009] Further, in the application, the improvement of the disease resistance of plants is to improve the resistance of plants to pathogenic fungi and / or to induce the defense response of plants.

[0010] Further, in the application, the improvement of the resistance of plants to pathogenic fungi is achieved by inducing the accumulation of active oxygen and the burst of callose.

[0011] Further, in the application, the improvement of the resistance of plants to pathogenic fungi is achieved by regulating the expression of plant defense genes.

[0012] Further, in the application, the defense genes include SA and JA signal pathway related genes.

[0013] Further, in the application, the pathogenic fungi are Sclerotinia sclerotiorum.

[0014] Further, in the application, the pathogenic fungi are Venturia pyrola.

[0015] Compared with the prior art, the technical scheme provided by the present application at least has the following beneficial effects or advantages:

[0016] 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.

[0017] 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

[0018] 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;

[0019] Figure 2 The SyCD1-induced improvement of resistance test results against *Nicotinus benthamiana* was achieved.

[0020] Figure 3 The results of tests on reactive oxygen species accumulation and callosity bursting induced by SyCD1 in Benedict's tobacco.

[0021] Figure 4 Expression map of SyCD1 activation of the tobacco defense gene.

[0022] Figure 5 To improve the resistance test results of SyCD1-induced Gala3 apple tissue culture seedlings to apple black rot and peel mold.

[0023] Figure 6 The results of tests on reactive oxygen species accumulation and callose bursting induced by SyCD1 in Gala3 apple tissue culture seedlings.

[0024] Figure 7SyCD1 activates the expression profile of defense genes of Gala3 apple tissue culture seedlings.

[0025] Figure 8 SyCD1 induces different varieties of apple leaves to improve resistance to Valsa mali. DETAILED DESCRIPTION

[0026] The technical solutions of the present application will be described below in conjunction with examples, but the present application is not limited to the following examples.

[0027] In order to enable those skilled in the art to better understand the technical solutions of the present application and to implement them, the present application will be further described below in conjunction with specific examples and drawings, but the examples are not limiting to the present application.

[0028] The experimental methods and detection methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified.

[0029] Example 1

[0030] This embodiment provides a protein elicitor SyCD1 Gene sequence cloning and pET28a-SyCD1-His vector construction.

[0031] (1) Extraction of Hhs.015 genome DNA of Yangling sugar silk bacteria

[0032] The Hhs.015 strain preserved in the-80℃ refrigerator was streaked on the Gao's first medium with a sterilized bamboo stick, and cultured in a 28℃ incubator until white spores were produced. 5-7 bacterial cakes were picked and inoculated in 100mL TSB liquid medium, and cultured at 28℃ for 48h. The bacterial bodies were collected by centrifugation for DNA extraction. CTAB method was used for genome DNA extraction, and 3µL sample was taken for agarose gel electrophoresis to detect the quality of DNA.

[0033] (2) SyCD1 gene cloning and prokaryotic expression vector construction

[0034] The primer was designed with the linker sequence of the prokaryotic expression vector pET28a, and the genomic DNA of Yangling Leuconostoc Hhs.015 was used as the template for sequence amplification. The amplification system was 50 μL, and the details were as follows: 2x 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 50 μL. The PCR reaction program was as follows: denaturation at 95°C for 3 min, pre-denaturation at 95°C for 15 s, annealing temperature at 56-64°C for 15 s, extension at 72°C for 30 s / kb, a total of 33 cycles, and finally extension at 72°C for 5 min. The PCR product was detected by 1% agarose gel, and the detection results are shown in FIG. 1A, wherein lane M is Marker, and lane 6 is the target band (gene band). The gene band was cut off, and the PCR product of the gene was recovered using a gel recovery kit. Figure 1 SyCD1 The recovered PCR product of the gene was sequenced by a biological company, and the sequence of the gene was obtained. SyCD1 SyCD1 The sequence of the gene is shown in SEQ ID No: 1. The primers used are shown in Table 1. SyCD1 SyCD1 SyCD1

[0035] The recovered PCR product of the gene was sequenced by a biological company, and the sequence of the gene was obtained. SyCD1

[0036] Table 1 Primer sequence table

[0037]

[0038] Example 2

[0039] In this example, the SyCD1 protein was obtained by fermentation.

[0040] ​​​​​​The prokaryotic expression vector pET28a-SyCD1-His was transformed into E. coli BL21 (DE3) competent cells, which were cultured at 37°C and 220 rpm for 2-3 h until the OD 600 was 0.6, and induction was performed by adding IPTG to a final concentration of 0.3 mM (at 16°C for more than 18 h); the bacterial cells were collected by centrifugation at 8000 rpm and 4°C for 3 min, and then suspended in PBS buffer after being washed 2-3 times; 0.2 mg / mL 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 solution after the reaction was ultrasonically broken on ice (power 40%, ultrasonic on for 3 s and pause for 4 s, for a total of 30 min); the mixture was centrifuged at 12000 rpm and 4°C for 15 min, and the bacterial cells were removed using a 0.22 μM bacterial filter; the supernatant was collected, and 10 μL of the supernatant was subjected to SDS-PAGE electrophoresis for detection, and the detection results are shown in FIG. 1B, where lane M is a marker and lane SyCD1 is the target protein SyCD1, and the detection results show that the size of the SyCD1 protein is between 10 kD and 15 kD, which is consistent with the expected size. Figure 1

[0041] The supernatant collected above was purified according to the instructions of Thermo HisPur Ni-NTA Resin. The initial SyCD1 protein product was obtained, and the imidazole in the initial SyCD1 protein product was removed by dialysis using PBS buffer to obtain SyCD1 protein, which was quickly frozen using liquid nitrogen and stored at -80°C for later use.

[0042] Example 3

[0043] This example is a test experiment for testing the resistance of SyCD1 protein elicitor to S. sclerotiorum in N. benthamiana.

[0044] The SyCD1 protein prepared in Example 2 was diluted with sterilized ddH2O to 1 μM to prepare a SyCD1 protein diluent for later use; the last dialysate PBS of the SyCD1 protein purification was used as a control, and the PBS dialysate was diluted with sterilized ddH2O and the SyCD1 protein diluent at the same dilution factor to prepare a PBS dialysate diluent for later use.

[0045] (1) SyCD1 protein induces N. benthamiana to improve resistance to S. sclerotiorum

[0046] ​N.benthamiana with 4-6 weeks growth period were injected with SyCD1 protein dilution (test group, SyCD1) or PBS dialysate dilution (control group) on the back, and cultured in 25℃ greenhouse for 24h. The tobacco leaves were cut into 1x1cm small pieces for use. The water solution containing 1mg / mL 3,3'-diaminobenzidine (DAB) (pH3.8) was prepared, and reacted under light for 8h. After reaction, the leaves were placed in decolorizing solution (95% ethanol) until complete decolorization, and then observed and photographed. The cut leaves were completely decolorized with decolorizing solution, washed with ddH2O, and placed in aniline blue staining solution (0.01% aniline blue in 150mM K2HPO4·3H2O (pH9.5)) for 12-14h of dark staining, and then observed and photographed under fluorescence microscope. The test results are shown in Figure 2 SyCD1 protein significantly enhanced the resistance of N.benthamiana leaves to S.sclerotiorum compared with the control.

[0047] (2) SyCD1 protein induces accumulation of reactive oxygen species and callose burst in N.benthamiana

[0048] N.benthamiana with 4-6 weeks growth period were injected with SyCD1 protein dilution (test group, SyCD1) or PBS dialysate dilution (control group) on the back, and cultured in 25℃ greenhouse for 24h. The tobacco leaves were cut into 1x1cm small pieces for use. The water solution containing 1mg / mL 3,3'-diaminobenzidine (DAB) (pH3.8) was prepared, and reacted under light for 8h. After reaction, the leaves were placed in decolorizing solution (95% ethanol) until complete decolorization, and then observed and photographed. The cut leaves were completely decolorized with decolorizing solution, washed with ddH2O, and placed in aniline blue staining solution (0.01% aniline blue in 150mM K2HPO4·3H2O (pH9.5)) for 12-14h of dark staining, and then observed and photographed under fluorescence microscope. The test results are shown in Figure 3 SyCD1 protein induced accumulation of brown reactive oxygen species and callose burst compared with the control.

[0049] (3) SyCD1 protein activates the expression of defense-related genes in N.benthamiana

[0050] 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.

[0051] Using the 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.

[0052] 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. NbPR4118-fold; NbNPR1 The expression of the gene reached a peak at 12h after SyCD1 treatment, and the expression of the SyCD1 treatment group was 5 times that of the control group.

[0053] In the jasmonic acid (JA) signal pathway, NbLOX1 The expression of the gene reached a peak at 6h after SyCD1 treatment, and the expression of the SyCD1 treatment group was about 6 times that of the control group. NbCOI1 and NbPDF1.2 The gene was inhibited at the early stage, and the inhibition was released at 24h after SyCD1 treatment. NbERF1 The expression of the gene reached a peak at 6h after SyCD1 treatment, and the expression of the SyCD1 treatment group was 3.5 times that of the control group.

[0054] In summary, this embodiment shows that SyCD1 protein significantly enhances the resistance of N. benthamiana leaves to S. sclerotiorum; SyCD1 protein can induce the accumulation of reactive oxygen species and the release of callose in N. benthamiana, thereby improving the resistance of plants to pathogenic fungi; after SyCD1 protein treatment, the expression of NbPAL gene, NbPR1a gene, NbPR4 gene and NbNPR1 gene are up-regulated, suggesting that SyCD1 protein enhances the resistance of SyCD1 protein to pathogenic fungi by up-regulating the expression of genes related to the salicylic acid signal transduction pathway; after SyCD1 protein treatment, the expression of NbLOX1 gene and NbERF1 gene is up-regulated, NbCOI1 gene and NbPDF1.2 gene SyCD1 protein expression is inhibited at the early stage, and the expression inhibition is released at 24h after treatment, suggesting that SyCD1 regulates the resistance of plants to pathogenic fungi by regulating the expression of genes in the jasmonic acid signal pathway.

[0055] Example 4

[0056] This embodiment is a test of the resistance of apple trees to V. mali improved by protein elicitor SyCD1.

[0057] (1) The resistance of apple tissue culture seedlings Gala 3 to V. mali improved by protein elicitor SyCD1 and its mechanism. Valsa mali

[0058] Apple tissue culture seedlings Gala 3: apple tissue culture seedlings Gala 3.

[0059] ​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.

[0060] 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.

[0061] 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.

[0062] (2) SyCD1 improves the resistance of different apple varieties to the elements. V.mali resistance

[0063] 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.

[0064] 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.

[0065] The basic principles, main features and advantages of the present application have been described above. The above examples and descriptions are only for the preferred embodiments of the present application, and the present application is not limited by the above examples. Any changes and improvements made by those skilled in the art to the technical solutions of the present application without departing from the spirit and scope of the present application shall fall within the scope of protection of the present application.

Claims

1. A protein SyCD1 for use in improving the disease resistance of apple trees, characterized in that, The amino acid sequence of the protein elicitor SyCD1 is shown as SEQ ID No: 2, the nucleotide sequence of the gene of the protein elicitor SyCD1 is shown as SEQ ID No: 1, the improvement of the disease resistance of the apple tree is the improvement of the resistance of the apple tree to the pathogenic fungus, and the pathogenic fungus is Valsa mali.

2. Use according to claim 1, characterized in that, The improvement of the resistance of the apple tree to the pathogenic fungus is achieved by inducing the accumulation of active oxygen and the burst of callose.

3. Use according to claim 1, characterized in that, The improvement of the resistance of the apple tree to the pathogenic fungus is achieved by regulating the expression of the defense genes of the apple tree.

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