Disease resistance related protease GmRD21A gene and application of encoding protein thereof in plant epidemic disease resistance

Abstract

The invention belongs to the technical field of plant molecular biology, and discloses a soybean disease resistance-related protease GmRD21A gene, a coding protein thereof, and an application of the gene in plant disease resistance. The nucleotide sequence of the protease GmRD21A gene is as shown in SEQ ID NO. 1, and the amino acid sequence of the polypeptide coded by the protease GmRD21A gene is as shown in SEQ ID NO. 2. Protein spectrum analysis of phytophthora sojae pectate lyase PsPL1 proves that the protease GmRD21A degrades pectate lyase secreted by phytophthora and can resist attack of pathogenic bacteria pectate lyase on plant cell walls, and meanwhile, overexpression of the GmRD21A gene in soybeans can remarkably improve the resistance of plants to phytophthora. The invention can be applied to crop breeding anti-epidemic disease improvement, and is hopeful to improve the disease resistance of plants to epidemic diseases, thereby achieving the purposes of increasing yield and reducing pesticide consumption.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular biology technology, and specifically discloses a disease resistance-related protease. GmRD21A Application of genes and their encoded proteins in plant disease resistance. Background Technology

[0002] Phytophthora infestans severely damages crops such as soybeans and potatoes, causing significant economic losses. Belonging to the phylum Oomycetes, Phytophthora infestans comprises over 160 known species and is a major culprit in many crop diseases, such as soybean Phytophthora (…). Phytophthora sojae Root rot caused by Phytophthora infestans is a devastating disease in soybean production; Potato late blight caused the Irish famine in the 19th century and now causes significant damage to potatoes and other crops annually. Phytophthora causes economic losses of up to 16 billion yuan annually to crops in my country, severely impacting crop production. Currently, disease control in production faces challenges such as pathogen resistance and the easy loss of crop resistance. Therefore, research on plant disease resistance mechanisms is of great significance for improving crop resistance and designing plant disease control strategies.

[0003] The apoplast space is the main battlefield for plants to defend against pathogens, and cysteine ​​proteases secreted by plants play a key role in plant disease resistance. For example, papain-like cysteine ​​proteases (PLCPs) in maize can cleave the Prozip1 precursor, and the released Zip1 peptide can activate the salicylic acid signaling pathway.

[0004] Therefore, utilizing and modifying plant proteases is a new approach to the long-term and safe control of plant diseases, which is more in line with the requirements of today's green and sustainable agricultural development. Summary of the Invention

[0005] The main objective of this invention is to provide a disease-resistant protease capable of degrading pathogenic pectin lyase. GmRD21A Application of genes and their encoded proteins in plant disease resistance.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solutions.

[0007] On the one hand, the present invention provides a disease-resistant protease. GmRD21A Genes, the disease-resistant proteases GmRD21A The nucleotide sequence of the gene is shown in any of the following examples. 1) The nucleotide sequence shown in SEQ ID NO.1 and its complementary sequence; or 2) A nucleotide sequence, such as SEQ ID NO.1, with substitution, deletion, and / or addition of one or more nucleotides to express a protein with the same function; or 3) A nucleotide sequence that hybridizes to the sequence shown in SEQ ID NO.1 under stringent conditions; or 4) A sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the nucleotide sequence shown in SEQ ID NO.1, the encoded polypeptide having the function of improving plant disease resistance.

[0008] On the other hand, the present invention also provides a disease-related protease. GmRD21A A gene-encoded polypeptide, wherein the amino acid sequence of the polypeptide is any of the following. 1) An amino acid sequence as shown in SEQ ID NO.2; or 2) An amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO. 2, wherein the polypeptide has the function of enhancing plant disease resistance; or 3) An amino acid sequence obtained by substituting and / or deleting and / or adding one or more amino acid residues in the sequence shown in SEQ ID NO.2, and the amino acid sequence has the function of regulating plant disease resistance.

[0009] On the other hand, the present invention also provides a recombinant expression vector comprising the aforementioned disease-associated protease. GmRD21A Gene.

[0010] On the other hand, the present invention also provides a host cell containing the aforementioned disease-related protease. GmRD21A Genes, or recombinant vectors containing the aforementioned gene.

[0011] On the other hand, the present invention also provides the aforementioned disease-related protease. GmRD21A The application of the gene, or the polypeptide, or the recombinant expression vector, or the host cell, wherein the application is any of the following: 1) Improve the basic disease resistance of plants; 2) Improve the ability of plants to degrade pectin lyase secreted by pathogens; 3) Select and breed disease-resistant plant varieties.

[0012] Furthermore, the pathogens that cause disease resistance or susceptibility include, but are not limited to, *Phytophthora indicum* ... and *Phytophthora indicum*. Furthermore, the pathogens that are resistant to or susceptible to disease include, but are not limited to, Phytophthora soybeani, Phytophthora tobaccoi, Phytophthora pathogenica, and Phytophthora capsici.

[0013] Furthermore, the plant is a legume or solanaceous plant, preferably soybean, tomato, potato or tobacco.

[0014] On the other hand, the present invention also provides a method for improving the basic disease resistance of plants, by using the aforementioned disease resistance-related protease. GmRD21A The gene, or the polypeptide, or the recombinant expression vector, or the host cell is transferred into the plant tissue.

[0015] On the other hand, the present invention also provides a method for breeding disease-resistant or disease-susceptible plant varieties, and for detecting the disease resistance-related proteases in the plant varieties to be tested. GmRD21A The expression level of the gene, if the disease-resistant protease... GmRD21A If the expression level of the gene is elevated, the plant variety being tested is a disease-resistant variety; if the disease resistance-related protease is elevated... GmRD21A If the expression level of a gene is reduced, the plant variety being tested is a susceptible variety.

[0016] Furthermore, the detection primer sequences used in the detection are shown in SEQ ID NO.3 and SEQ ID NO.4, wherein the upstream primer is shown in SEQ ID NO.3 and the downstream primer is shown in SEQ ID NO.4.

[0017] Furthermore, the pathogens that are resistant to or susceptible to disease include, but are not limited to, Phytophthora soybeani, Phytophthora tobaccoi, Phytophthora pathogenica, and Phytophthora capsici.

[0018] Furthermore, the plant is a legume or solanaceous plant, preferably soybean, tomato, potato or tobacco.

[0019] Compared with the prior art, the present invention has the following technical effects: 1) This invention, through proteomic analysis of the soybean Phytophthora pectin lyase PsPL1, discovered that the cysteine ​​protease GmRD21A interacts with PsPL1, and that GmRD21A degrades the pectin lyase secreted by Phytophthora, thus inhibiting the pathogen's pectin lyase from attacking the plant cell wall. This process silences the pectin lyase in soybeans. GmRD21A Genes that significantly weaken plant resistance to Phytophthora will have a significant impact on plant resistance to Phytophthora. This invention can be applied to crop breeding to improve disease resistance, and is expected to enhance plant resistance to Phytophthora, thereby achieving the goal of increasing yield and reducing pesticide use.

[0020] 2) As an important oilseed crop in my country, research on soybean GmRD21A can enrich the disease-resistant germplasm resources of soybean in my country. In addition, pectin lyases are conserved in different oomycetes and fungi, therefore GmRD21A has the ability to degrade pectin lyases of different pathogenic microorganisms. Attached Figure Description

[0021] Picture 1 This is the phylogenetic tree of GmRD21A.

[0022] Picture 2 This is a schematic diagram of the GmRD21A protein structure.

[0023] Picture 3 The co-localization results for PsPL1 and GmRD21A.

[0024] Picture 4 Results of Co-IP for the in vivo interaction between PsPL1 and GmRD21A proteins.

[0025] Picture 5 The results show the degradation of PsPL1 protein by GmRD21A.

[0026] Picture 6 This is a Western blot image showing the overexpression of GmRD21A and GmRD21AEM proteins in soybean root hairs.

[0027] Picture 7 Silence for soybean root hairs GmRD21A and GmRD21A-like The efficiency of silence.

[0028] Picture 8 For the silence of soybean root hairs GmRD21A and GmRD21A-like Phenotypic (left) and biomass (right) of plants subsequently inoculated with Phytophthora soybeanis.

[0029] Picture 9 To overexpress GmRD21A and GmRD21A in soybean root hairs EM Phenotypic (left) and biomass (right) of plants subsequently inoculated with Phytophthora soybeanis. Detailed Implementation

[0030] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions to the details and form of the present invention can be made without departing from the spirit and scope of the present invention, but such modifications and substitutions all fall within the protection scope of the present invention. The primers involved in the embodiments of the present invention were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The Phytophthora tobaccosa used in the following embodiments are strains preserved in our laboratory.

[0031] Example 1

[0032] Based on previous research, the pectin lyase PsPL1 is conserved in various pathogens and is a key virulence effector of Phytophthora sojae (Sun et al., (2024). Dual activation of soybean resistance against Phytophthora sojae by pectin lyase and degraded pectinoligosaccharides. Sci. China Life Sci. 67, 2746–2760.). Therefore, using LC-MS / MS technology, PsPL1 protein was incubated with soybean apoplastic fluid, and the interacting protein soybean GmRD21A was identified. The amino acid sequence of this protein is shown in SEQ ID NO.2. Mass spectrometry identification results showed that GmRD21A and its homolog GmRD21A-like were identified as interacting with PsPL1. GmRD21A is a type of PLCP belonging to the RD21 subfamily, with a size of 52 kDa. Phylogenetic tree construction and analysis showed that GmRD21A belongs to the RD21 subfamily, as shown in the following results. Picture 1 The protein structure includes a signal peptide, a pre-inhibitory domain, a peptidase C domain, a proline-rich sequence, and a granulin domain, as shown below. Picture 2 .

[0033] Example 2

[0034] Will PsPL1 and GmRD21A The gene was ligated into plasmids pBin-eGFP and pBin-RFP using homologous recombinase. The ligated pBin::PsPL1-eGFP and pBin::GmRD21A genes were then ligated into these plasmids. ΔGranulim The -RFP (Granulim domain removed) plasmid was transformed into E. coli strain JM109. E. coli at 37°C... o After 16 hours of growth in a C-type incubator, single-clone strains were picked and detected using PCR amplification primers. GmRD21AThe gene was extracted using the following primers: upstream primer 5'-CTTCGCAAGACCCTTCCTCTA-3' (SEQ ID NO.3), and downstream primer 5'-GGACAGGATGTCCCAGGCG-3' (SEQ ID NO.4). The PCR product was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were compared with SEQ ID NO.1 to confirm the positive vector. Recombinant plasmids were then extracted using a plasmid miniprep kit. pBin::PsPL1-eGFP and pBin::GmRD21A were then electroporated. ΔGranulim -RFP plasmids were transferred into Agrobacterium GV3101 strain. The transformed bacterial culture was evenly spread on LB solid medium containing 50 µg / mL kanamycin and 50 µg / mL rifampin, and incubated for 28 hours. o Incubate in the dark for 2 days. Select monoclonal Agrobacterium strains and detect them using PCR amplification primers. GmRD21A The gene was obtained with the upstream primer being 5'-CTTCGCAAGACCCTTCCTCTA-3' (SEQ ID NO.3) and the downstream primer being 5'-GGACAGGATGTCCCAGGCG-3' (SEQ ID NO.4).

[0035] Positive monoclonal Agrobacterium strains were picked and cultured in liquid LB medium (kanamycin 50 µg / mL, rifampin 50 µg / mL) for 28 hours. o Incubate overnight at 220 rpm at C. Wash the Agrobacterium three times by centrifugation at 5000 rpm using a washing buffer (10 mM pH 5.7 MES, 10 mM MgCl2, 200 μM acetylsuccinyl ketone). OD values ​​of all Agrobacterium species were determined. 600 Then pBin::PsPL1-eGFP+pBin::GmRD21A ΔGranulim -RFP is mixed in a 1:1 ratio, OD 600 The solution was adjusted to 0.6, allowed to stand at room temperature for 60 min, and then injected into leaves of Nicotiana benthamiana that had grown for 4-6 weeks. After injection, the Nicotiana benthamiana was kept under 16 h light / 8 h dark conditions at 22°C. o C. Greenhouse growth for 2 days.

[0036] The results are as follows Picture 3 Plasmolysis and confocal microscopy confirmed that GmRD21A is located in the apoplastic body and co-localizes with the apoplastic effector PsPL1, indicating that GmRD21A functions in the apoplastic body.

[0037] Example 3

[0038] The protein interaction between GmRD21A and PsPL1 was detected by Co-IP in plants. PsPL1 and GmRD21A The gene was ligated into plasmids pBin-eGFP and pBin-HA using homologous recombinase. The ligated plasmids pBin::PsPL1-eGFP, pBin::GmRD21A-FLAG-HA, pBin::SP+iGmRD21A-HA, and pBin::SP+mGmRD21A-HA were then transformed into *E. coli* strain JM109. *E. coli* was cultured at 37°C. o After 16 h of growth in a C-type incubator, single clones were selected and the GmRD21A gene was detected using PCR amplification primers. The upstream primer was 5'-CATTTCATTTGGAGAGGACC-3' (SEQ ID NO.5), and the downstream primer was 5'-TATCTGGGAACTACTCACAC-3' (SEQ ID NO.6). The PCR product was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were compared with SEQ ID NO.1 to confirm the positive vector. Recombinant plasmids were then extracted using a plasmid miniprep kit. The plasmids pBin::PsPL1-eGFP, pBin::GmRD21A-FLAG-HA, pBin::SP+iGmRD21A-HA, and pBin::SP+mGmRD21A-HA were transformed into Agrobacterium GV3101 strain by electroporation. The transformed bacterial culture was evenly spread on LB solid medium containing 50 µg / mL kanamycin and 50 µg / mL rifampicin and incubated for 28 hours. o Incubate in the dark for 2 days. Select monoclonal Agrobacterium strains and detect them using PCR amplification primers. GmRD21A The gene was obtained using the following primers: upstream primer 5'-CATTTCATTTGGAGAGGACC-3' (SEQ ID NO.5) and downstream primer 5'-TATCTGGGAACTACTCACAC-3' (SEQ ID NO.6).

[0039] Positive monoclonal Agrobacterium strains were picked and cultured in liquid LB medium (kanamycin 50 µg / mL, rifampin 50 µg / mL) for 28 hours. o Incubate overnight at 220 rpm at C. Wash the Agrobacterium three times by centrifugation at 5000 rpm using a washing buffer (10 mM pH 5.7 MES, 10 mM MgCl2, 200 μM acetylsuccinyl ketone). OD values ​​of all Agrobacterium species were determined. 600 Then, pBin::PsPL1-eGFP+pBin::GmRD21A-FLAG-HA, pBin::PsPL1-eGFP+pBin::SP+iGmRD21A-HA, and pBin::PsPL1-eGFP+pBin::SP+mGmRD21A-HA were mixed in a 1:1 ratio, and the OD...600 The solution was adjusted to 0.6, allowed to stand at room temperature for 60 minutes, and then injected into leaves of Nicotiana benthamiana that had grown for 4-6 weeks. After injection, the Nicotiana benthamiana was kept under 16 h light / 8 h dark conditions at 22°C. o C. Greenhouse growth for 2 days.

[0040] Tobacco leaves were collected for injection, ground with liquid nitrogen, and RIPA protein lysis buffer was added. 1 mL of RIPA buffer contained 35 μL of protease inhibitor (Sigma Cat No. P8340) and 10 μL of phosphatase inhibitor. 50 mL of RIPA buffer consisted of 1 mL of 2.5 M Tris-HCl (pH 7.5), 1.5 mL of 5 M NaCl, and 0.5 mL of NP-40, with water added to a final volume of 50 mL. The solution was incubated on ice for 30 min, centrifuged at 15000 rpm for 10 min, and the supernatant was collected. A portion of the supernatant was added to the protein loading buffer as input. The remainder was added to 15 µL of GFP-Trap gel beads (Chromotek, Planegg-Martinsried, Germany). o Incubate at C for 3 h. Wash the gel beads 4–6 times with RIPA buffer. Add 100 µL Lysis Buffer and 30 µL Protein Loading Buffer, mix well, and place the centrifuge tube in boiling water for 5 min. Load the sample onto an SDS-PAGE gel for electrophoresis separation, and then perform Western blot analysis.

[0041] The results are as follows Picture 4 PsPL1 can interact with the full-length GmRD21A protein, iGmRD21A, and mGmRD21A proteins, indicating that GmRD21A may exert its degradation function by binding to PsPL1.

[0042] Example 4

[0043] Agrobacterium, transformed into the pBin::GmRD21A-FLAG-HA vector and verified to be correct, was streaked onto LB agar plates and kept at 28°C. o Cultured at C for 24 h for later use. Positive monoclonal Agrobacterium strains were picked and cultured in liquid LB medium (kanamycin 50 µg / mL, rifampin 50 µg / mL) for 28 h. o Incubate overnight at 220 rpm at C. Wash the Agrobacterium three times by centrifugation at 5000 rpm using a washing buffer (10 mM pH 5.7 MES, 10 mM MgCl2, 200 μM acetylsuccinone). OD of Agrobacterium was determined. 600Then, the OD of pBin::GmRD21A-FLAG-HA 600 Adjust to 1.0, allow to stand at room temperature for 60 min, then inject into leaves of Nicotiana benthamiana that have grown for 4-6 weeks. After injection, the Nicotiana benthamiana is kept under 16 h light / 8 h dark conditions at 22°C. o C. Greenhouse growth for 2 days.

[0044] Tobacco leaves were collected for injection, ground with liquid nitrogen, and RIPA protein lysis buffer (without protease inhibitors) was added. A 50 mL RIPA buffer consisted of 1 mL of 2.5 M Tris-HCl (pH 7.5), 1.5 mL of 5 M NaCl, and 0.5 mL of NP-40, with water added to a final volume of 50 mL. The mixture was incubated on ice for 30 min, centrifuged at 15000 rpm for 10 min, and the supernatant was collected. This process was repeated with centrifugation at 15000 rpm for 10 min, collecting all supernatant. FLAG gel beads (Anti-DYKDDDDK Affinity Beads, Changzhou Tiandi Renhe Biotechnology Co., Ltd., catalog number: SA042005) were added to 1 mL of the supernatant at a ratio of 15 µL. The FLAG gel beads were resuspended and washed three times with RIPA buffer before being added to the supernatant. o Incubate at C for 3 h. Wash the gel beads 5 times with RIPA buffer, then wash again 5 times with TBS buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.5) without NP-40. For the final wash, discard all supernatant and elute with 100 mM Glycine (pH 3.0) at a 1:10 ratio for 10 min, repeating three times. Neutralize the eluted protein immediately with 1 M Tris-HCl (pH 8.0) at a 9:1 ratio. Finally, concentrate the GmRD21A-FLAG protein using ultrafiltration and aliquot for storage at -80°C. o C.

[0045] The yeast strain (KM71) transformed into the pPIC9K::PsPL1-His vector and verified to be correct was streaked onto YPD medium plates and incubated for 30 minutes. o Culture at C for 48 h for later use. Select positive monoclonal yeast cells and incubate in 50 mL of liquid BMGY medium for 30 h. o Incubate at 220 rpm for 48 h, allow to settle for 4-5 h, pour off the supernatant and replace with 50 mL of liquid BMMY medium, adding 1% methanol as a carbon source. o Cultured at C4 for 4 days, with 1% methanol added daily. Centrifuged at 8000 rpm for 30 min, the supernatant was collected and mixed with 50% ammonium sulfate. oAfter precipitating at C for 3 days, the precipitate was collected by centrifugation at 8000 rpm for 30 min and dissolved in 20 mL of deionized water. PsPL1-His protein was then purified by Ni-NTA column-mediated affinity chromatography, concentrated using ultrafiltration, aliquoted, and stored at -80°C. o C.

[0046] The purified mGmRD21A-FLAG protein and PsPL1-His protein were added in vitro to 100 µL of reaction buffer (100 mM NaAc, pH 6.0) in a specific ratio, and incubated for 25 minutes. o Incubate at C for 1 hour. After incubation, add loading buffer to terminate the reaction, 100 o Boil at C for 10 min, load the sample onto an SDS-PAGE gel for electrophoretic separation, and then analyze using Western blot. Results are as follows: Picture 5 As shown, GmRD21A degrades PsPL1, and the degradation effect weakens as the amount of protease used gradually decreases, indicating a clear dose-response effect.

[0047] Example 5

[0048] Construct overexpression of pBin::eGFP-EV, pBin::GmRD21A-eGFP, and pBin::GmRD21A EM -eGFP and silencing pFGC5941::RNAi-EV, pFGC5941::RNAi- GmRD21A / GmRD21A-like Vector. A common silencing fragment of GmRD21A and its homologous gene GmRD21A-like was inserted into two multiple cloning sites of the pFGC5941 vector in both forward and reverse orientations. Within plant cells, the vector transcribes into an RNA that folds into a "hairpin" structure. This structure is recognized and cleaved by the plant cell into small interfering RNAs (siRNAs). The siRNAs precisely locate and degrade target gene mRNAs that match their sequences, thus silencing the gene. pBin::eGFP-EV, pBin::GmRD21A::eGFP, and pBin::GmRD21A were then inserted into the vector. EM -eGFP, pFGC5941::RNAi-EV, pFGC5941::RNAi- GmRD21A / GmRD21A-like The vector was transformed into K599 Agrobacterium rhizogenes, and positive single colonies were picked and streaked onto YEP plates. 28 o Cultivate at C for 24 hours for later use. Select healthy soybean seeds, sterilize with chlorine for 14 hours, remove residual chlorine in a clean bench, and then place them on germination medium at 28°C. oC germinates for 2 days. Positive colonies are picked and incubated in 4 mL of liquid YEP medium (with kanamycin, streptomycin, and chloramphenicol added to 50 mg / L) for 12 hours, then transferred to 50 mL of liquid YEP medium for further culture for 12 hours. Agrobacterium cells are collected by centrifugation at 4000 rpm for 10 min, resuspended in liquid co-culture medium, and the OD of the bacterial culture is adjusted. 600 The value was 0.6. After germination, the soybean germ was removed, the seed coat was removed, and the mixture was soaked in bacterial solution for 30 min, then transferred to solid co-culture medium for 28 hours. o Cultured at C for 5 days. Once the soybeans turned green, they were transferred to liquid rooting medium and soaked for 1 hour (with 0.2 g / L cephalosporin added) to remove excess Agrobacterium, then transferred to solid rooting medium at 28°C. o Cultured in the dark for 14 days, after hairy roots have emerged, positive root hairs were selected using a fluorescence microscope and transferred to a new rooting medium (Ø 15 cm). o Cultured in the dark for 7 days.

[0049] Western blot analysis was performed to detect root hair overexpression of GmRD21A and its enzyme activity mutant GmRD21A. EM Protein, results as follows Picture 6 As shown, GmRD21A and GmRD21A EM Success is expressed in the root hair.

[0050] Gene silencing efficiency was detected by qRT-PCR. GmRD21A The primers for gene detection are: pre-quantification primer: 5'-ACGCTGGAACTTGTCTCAGG-3' (SEQ ID NO.7), and post-quantification primer: 5'-ACGTGCAGCATTATGTGAAGA-3' (SEQ ID NO.8). Homologous genes GmRD21A-like The primers for gene detection are: pre-quantification primer: 5'-AACAACGTCGTCCAAGACCAT-3' (SEQ ID NO.9), and post-quantification primer: 5'-GCCGTGCTTTACTAGCCACT-3' (SEQ ID NO.10).

[0051] The PCR reaction mixture contained 5 μL cDNA, 10 μL SYBR Premix Ex Taq II (Tli RNase H Plus), 0.4 μL each of the pre- and post-primer primers, 0.4 μL ROX Reference Dye II, and 13.8 μL water. The reaction procedure was: I: 95 o C 30s, II: 95 o C 5 s, 60 oC 34 s, step II reaction 40 cycles. The dissolution curve analysis procedure is: 95 o C 15 s, 60 o C1 min, 95 o C 15 s.

[0052] Data analysis uses 2 −ΔΔCT Methods and test results are as follows Picture 7 As shown, pFGC5941- GmRD21A / GmRD21A-like Dealing with Silent Roots GmRD21A and GmRD21A-like Gene expression levels were significantly lower compared to the control.

[0053] Example 6

[0054] Overexpression and silencing of GmRD21A GmRD21A / GmRD21A-like Soybean Phytophthora mycelial blocks were inoculated onto the root hairs. Two days after inoculation, if... Picture 8 As shown, compared with the control, silence GmRD21A / GmRD21A-like The number of oospores in *Phytophthora sojae* was significantly increased. For example... Picture 9 As shown, overexpression GmRD21A The number of Phytophthora oospores in root hairs of soybean was significantly reduced, and the resistance induced by GmRD21A depended on its enzyme activity.

[0055] The biomass of *Phytophthora sojae* was detected by qRT-PCR. The detection primers are shown in Table 1. Picture 8 As shown, this confirms silence. GmRD21A / GmRD21A-like Afterwards, the biomass of Phytophthora soybeanum increased significantly, such as Picture 9 As shown, in overexpression GmRD21A On the root hairs, the biomass of Phytophthora soybeanis was significantly reduced, indicating that GmRD21A positively regulates the resistance of soybean to Phytophthora soybeanis.

[0056] Table 1 Primer Sequences

[0057] Unless otherwise specifically stated, the numerical values ​​set forth in these embodiments do not limit the scope of the invention. In all examples shown and described herein, any specific value should be interpreted as merely exemplary and not as a limitation, unless otherwise specified; therefore, other examples of exemplary embodiments may have different values.

Claims

1. A disease-resistant protease GmRD21A The gene, characterized in that the disease-resistant protease... GmRD21A The nucleotide sequence of the gene is shown in any of the following examples. 1) The nucleotide sequence shown in SEQ ID NO.1 and its complementary sequence; or 2) A nucleotide sequence, such as SEQ ID NO.1, with substitution, deletion, and / or addition of one or more nucleotides to express a protein with the same function; or 3) A nucleotide sequence that hybridizes to the sequence shown in SEQ ID NO.1 under stringent conditions; or 4) A sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the nucleotide sequence shown in SEQ ID NO.1, the encoded polypeptide having the function of improving plant disease resistance.

2. A disease-resistant protease GmRD21A Gene-encoded polypeptides, characterized in that, The amino acid sequence of the polypeptide is any of the following: 1) An amino acid sequence as shown in SEQ ID NO.2; or 2) An amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO. 2, wherein the polypeptide has the function of enhancing plant disease resistance; or 3) An amino acid sequence obtained by substituting and / or deleting and / or adding one or more amino acid residues in the sequence shown in SEQ ID NO.2, and the amino acid sequence has the function of regulating plant disease resistance.

3. A recombinant expression vector, characterized in that, The recombinant expression vector includes the disease-related protease as described in claim 1. GmRD21A Gene.

4. A host cell, characterized in that, Contains the disease-associated protease as described in claim 1 GmRD21A Genes, or recombinant vectors as described in claim 3.

5. The disease-resistant protease according to claim 1 GmRD21A The application of the gene, or the polypeptide of claim 2, or the recombinant expression vector of claim 3, or the host cell of claim 4, is characterized in that... The application is any of the following: 1) Enhance the plant's ability to degrade pectin-lysing enzymes secreted by pathogens; 2) Improve plant resistance to Phytophthora; 3) Select and breed plant varieties that can fight diseases.

6. The application according to claim 5, characterized in that, The pathogens that are resistant to or susceptible to disease include, but are not limited to, Phytophthora soybeani, Phytophthora tobaccoi, Phytophthora pathogenica, and Phytophthora capsici.

7. The application according to claim 5, characterized in that, The plant is a legume or solanaceous plant, preferably soybean, tomato, potato or tobacco.

8. A method for improving the basic disease resistance of plants, comprising using the aforementioned disease resistance-related protease. GmRD21A The gene, or the polypeptide, or the recombinant expression vector, or the host cell is transferred into the plant tissue.

9. The method according to claim 8, characterized in that, The plant is a legume or solanaceous plant, preferably soybean, tomato, potato or tobacco.

10. A method for breeding disease-resistant plant varieties, comprising detecting the disease resistance-related proteases in the plant varieties to be tested. GmRD21A The expression level of the gene, if the disease-resistant protease... GmRD21A If the gene expression level is increased, the plant variety to be tested is a disease-resistant variety; preferably, the detection primer sequences used in the detection are shown in SEQ ID NO.3 and SEQ ID NO.4, wherein the upstream primer is shown in SEQ ID NO.3 and the downstream primer is shown in SEQ ID NO.4.

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