Application of lily disease course related protein LhSorPR10-1 in improvement of plant disease resistance

Abstract

The invention belongs to the technical field of molecular biology, and relates to application of a lily disease course related protein LhSorPR10-1 in improvement of plant disease resistance. The technical problem to be solved by the invention is to provide a new choice for cultivating high-resistance lily varieties. The technical scheme of the invention is the application of the lily disease course related protein LhSorPR10-1 in improving the disease resistance of plants. The lily disease course related protein LhSorPR10-1 disclosed by the invention is helpful for improving the resistance of a plant to gray mold.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology and relates to application of lily pathogenesis-related protein LhSorPR10-1 in improving plant disease resistance. Background Art

[0002] Lily (Lilium spp.) belongs to the genus Lilium of the family Liliaceae. It is a perennial aromatic bulbous plant that is well-known worldwide. Lilies have a variety of flower shapes and colors, and their fragrance is refreshing, elegant and refined. They have very high ornamental value and have become the main cash crop in the global fresh cut flower market. In addition, lilies also have medicinal, industrial and edible values. my country is the country with the most lily species and is also the origin center of lilies in the world. There are 55 species and 18 varieties of lily in my country, including 35 endemic species and 1 introduced species.

[0003] my country's floriculture industry developed relatively late. Compared with the Netherlands, Japan and other countries, my country's lily cut flower production is still in its early stages of development, and the bulbs used are basically imported. With the continuous expansion of my country's lily planting area, the process from bulb storage to fresh cut flower production has been seriously threatened by various diseases, including viral diseases, penicillium, anthracnose, wilt and gray mold, among which gray mold is the most common disease.

[0004] Gray mold, also known as leaf blight, can be caused by two species of Botrytis fungi on lilies: one is Botrytis cinerea; the other is Botrytis elliptica. Botrytis elliptica has a narrow host range and specifically infects only lily plants, causing the plants to wilt and rot, and the quality of the bulbs to decline, affecting lily field production and causing an annual production loss of 20%-30%. Severe epidemics can result in crop failure, and it is a major obstacle to cut flower cultivation and bulb propagation.

[0005] At present, the prevention and control of gray mold in lily production mainly relies on chemical prevention, but it faces the problems of environmental pollution and increased resistance of pathogens. The breeding of disease-resistant lily varieties is currently the most economical and effective way to prevent and control gray mold.

[0006] Pathogenesis-related proteins (PR) are a class of defense-related proteins synthesized by plants when attacked by pathogens. Studies have shown that the level of PR in plants directly affects the plant's defense response and related resistance. Therefore, the use and development of PR has become one of the effective ways to improve plant resistance. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a new option for breeding highly resistant lily varieties.

[0008] The technical solution of the present invention is lily pathogenesis-related protein LhSorPR10-1, and its amino acid sequence is shown in SEQ ID No.2.

[0009] Specifically, the coding gene of the lily pathogenesis-related protein LhSorPR10-1 has a nucleotide sequence as shown in SEQ ID No.1.

[0010] The present invention also provides application of lily pathogenesis-related protein LhSorPR10-1 in improving plant disease resistance.

[0011] Specifically, the disease resistance is resistance to gray mold.

[0012] Particularly, the pathogen of gray mold is Botrytis cinerea and / or Botrytis cinerea.

[0013] Wherein, the plant is lily.

[0014] The present invention also provides the use of lily pathogenesis-related protein LhSorPR10-1 in improving the antioxidant enzyme level, lignin content and / or proline content of diseased plants.

[0015] Specifically, the disease is infection with gray mold.

[0016] Particularly, the pathogen of gray mold is Botrytis cinerea and / or Botrytis cinerea.

[0017] Wherein, the plant is lily.

[0018] Furthermore, the antioxidant enzymes are SOD, POD, and CAT.

[0019] The present invention also provides the use of lily pathogenesis-related protein LhSorPR10-1 in reducing the plasma membrane damage level of diseased plants.

[0020] Specifically, the disease is infection with gray mold.

[0021] Particularly, the pathogen of gray mold is Botrytis cinerea and / or Botrytis cinerea.

[0022] Wherein, the plant is lily.

[0023] The present invention also provides the use of lily pathogenesis-related protein LhSorPR10-1 in the preparation of plant gray mold inhibitor.

[0024] Particularly, the pathogen of gray mold is Botrytis cinerea and / or Botrytis cinerea.

[0025] Wherein, the plant is lily.

[0026] The present invention also provides a method for improving the disease resistance of plants, which is achieved by increasing the expression level of lily pathogenesis-related protein LhSorPR10-1 in the plants.

[0027] Specifically, the method for increasing the expression level of the lily pathogenesis-related protein LhSorPR10-1 in plants is: transferring the coding gene of LhSorPR10-1 into the plants, so that the plants express the LhSorPR10-1 protein.

[0028] Specifically, the disease resistance is resistance to gray mold.

[0029] Particularly, the pathogen of gray mold is Botrytis cinerea and / or Botrytis cinerea.

[0030] Furthermore, the plant is lily.

[0031] The present invention also provides a method for increasing the antioxidant enzyme level, lignin content and / or proline content of diseased plants, which is achieved by increasing the expression level of lily pathogenesis-related protein LhSorPR10-1 in the plants.

[0032] Specifically, the method for increasing the expression level of the lily pathogenesis-related protein LhSorPR10-1 in plants is: transferring the coding gene of LhSorPR10-1 into the plants, so that the plants express the LhSorPR10-1 protein.

[0033] Specifically, the disease is infection with gray mold.

[0034] Particularly, the pathogen of gray mold is Botrytis cinerea and / or Botrytis cinerea.

[0035] The present invention also provides a method for reducing the plasma membrane damage level of diseased plants, which is achieved by increasing the expression level of lily pathogenesis-related protein LhSorPR10-1 in the plants.

[0036] Specifically, the method for increasing the expression level of the lily pathogenesis-related protein LhSorPR10-1 in plants is: transferring the coding gene of LhSorPR10-1 into the plants, so that the plants express the LhSorPR10-1 protein.

[0037] Specifically, the disease is infection with gray mold.

[0038] Particularly, the pathogen of gray mold is Botrytis cinerea and / or Botrytis cinerea.

[0039] Furthermore, the plant is lily.

[0040] The present invention also provides a plant gray mold inhibitor, the main components of which are lily pathogenesis-related protein LhSorPR10-1, engineered bacteria expressing lily pathogenesis-related protein LhSorPR10-1, fermentation broth of engineered bacteria expressing lily pathogenesis-related protein LhSorPR10-1 and / or secretions of engineered bacteria expressing lily pathogenesis-related protein LhSorPR10-1.

[0041] Particularly, the pathogen of gray mold is Botrytis cinerea and / or Botrytis cinerea.

[0042] Furthermore, the plant is lily.

[0043] Beneficial effects of the present invention:

[0044] The present invention provides a lily pathogenesis-related protein LhSorPR10-1, and clones its coding gene LhSorPR10-1. The maximum ORF frame of the gene is 471 bp, encoding 157 amino acids; the molecular weight of LhSorPR10-1 is 17.7 kDa, and the theoretical isoelectric point is 5.36. The present invention clarifies the expression induction characteristics of the LhSorPR10-1 gene and its influence on the disease resistance and stress resistance of lily by cloning the LhSorPR10-1 gene, analyzing the prokaryotic expression recombinant protein, analyzing the induction characteristics, constructing the overexpression vector and the antisense expression vector, and analyzing the resistance of the transgenic plant, enriches the understanding of the molecular mechanism of the disease resistance and stress resistance of lily, and provides a molecular theoretical basis for breeding new lily varieties with strong resistance. After infection with Botrytis cinerea, the LhSorPR10-1 gene was significantly induced to express; in an in vitro antibacterial experiment, the LhSorPR10-1 recombinant protein had an inhibitory effect on the growth of Botrytis cinerea hyphae; and the transgenic lily plants overexpressing LhSorPR10-1 had resistance to Botrytis cinerea. The results showed that the LhSorPR10-1 gene of the present invention plays an important role in plant resistance to biological stress, and can provide a reference for breeding new lily varieties with strong disease resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is the plasmid map of the prokaryotic expression vector pET-32a(+) described in the examples of the present invention.

[0046] Figure 2 It is the bacterial liquid detection and plasmid double enzyme digestion identification described in the embodiment of the present invention;

[0047] A, M, molecular weight standard; 1-2, PCR detection of pET32a(+)-LhSorPR10-1 bacterial solution; CK, negative control.

[0048] B, M, molecular weight standard; 1-5, pET32a(+)-LhSorPR10-1 plasmid PCR identification; CK, negative control.

[0049] Figure 3 It is the detection of the engineering bacteria liquid described in the embodiment of the present invention; M, molecular weight standard; 1-15, pET32a(+)-LhSorPR10-1-BL21 bacterial liquid PCR detection, CK, negative control.

[0050] Figure 4 The induced expression of the LhSorPR10-1 fusion protein described in the embodiment of the present invention was detected by SDS-PAGE electrophoresis; M, protein molecular weight standard protein; 1, uninduced pET32a(+) empty vector; 2, induced pET32a(+) vector; 3, uninduced pET32a(+)-LhSorPR10-1 recombinant vector; 4, induced pET32a(+)-LhSorPR10-1 recombinant vector.

[0051] Figure 5 This is the SDS-PAGE electrophoresis detection for solubility analysis of the LhSorPR10-1 fusion protein described in the example of the present invention; M, protein molecular weight standard protein; 1, uninduced pET32a(+) empty vector; 2, induced pET32a(+) vector total protein; 3, induced pET32a(+) vector supernatant; 4, induced pET32a(+) vector precipitate; 5, uninduced pET32a(+)-LhSorPR10-1 recombinant vector; 6, induced pET32a(+)-LhSorPR10-1 recombinant vector total protein; 7, induced pET32a(+)-LhSorPR10-1 recombinant vector supernatant; 8, induced pET32a(+)-LhSorPR10-1 recombinant vector precipitate.

[0052] Figure 6 The LhSorPR10-1 fusion protein induced purification SDS-PAGE electrophoresis detection described in the embodiment of the present invention;

[0053] A, SDS-PAGE electrophoresis detection of pET32a(+) recombinant protein purification; M, protein molecular weight standard protein; 1, induced pET32a(+) supernatant; 2-4: induced pET32a(+) flow-through 1-3; 5-8, pET32a(+) washing solution 1-4 after induction; 9-12, pET32a(+) elution solution 1-4 after induction;

[0054] B, SDS-PAGE electrophoresis detection of purified pET32a(+)-LhSorPR10-1 recombinant protein.

[0055] Figure 7 These are activity analysis diagrams of the recombinant protein described in the examples of the present invention in inhibiting Botrytis cinerea; A, activity analysis of the LhSorPR10-1 recombinant protein in inhibiting Botrytis cinerea; B, activity analysis of the LhSorPR10-1 recombinant protein in inhibiting Botrytis cinerea.

[0056] Figure 8 The inhibition area of ​​the LhSorPR10-1 recombinant protein described in the example of the present invention on the hyphae growth of two pathogenic fungi; the data are mean ± standard error, multiple T test, different letters represent P < 0.05.

[0057] Fig. 9 This is the RNase activity analysis of the recombinant protein purified from LhSorPR10-1 described in the examples of the present invention;

[0058] A, RNase activity analysis of LhSorPR10-1 recombinant protein; M, protein molecular mass standard protein; 1, lily leaf RNA; 2, RNase activity of HIS-tagged protein purified by pET32a(+); 3, RNase activity of LhSorPR10-1 purified protein after boiling; 4, RNase activity of LhSorPR10-1 purified protein after boiling; 5, RNase activity of LhSorPR10-1 purified protein; 6, RNase activity of LhSorPR10-1 purified protein after boiling; 7, RNase activity of LhSorPR10-1 purified protein.

[0059] B, RNase activity analysis of LhSorPR10-1 recombinant protein at different temperatures.

[0060] Fig.10 This is the DNase activity analysis of the recombinant protein purified from LhSorPR10-1 described in the examples of the present invention;

[0061] A, DNase activity analysis of LhSorPR10-1 recombinant protein; M, protein molecular weight standard protein; 1: adding Mg 2+ After boiling, the DNase activity of LhSorPR10-1 purified protein was detected; 2: Mg was added 2+ After adding Mg 2+ After that, the DNase activity of LhSorPR10-1 purified protein was detected; 4: Mg was added 2+ After boiling, the DNase activity of LhSorPR10-1 protein was measured; 5: Mg was added 2+ DNase activity of HIS-tagged protein purified from pET32a(+); 6: Add Mg 2+After boiling, the DNase activity of the HIS-tagged protein purified from pET32a(+) was determined.

[0062] B, DNase activity analysis of LhSorPR10-1 recombinant protein at different temperatures.

[0063] Fig.11 This is the phosphorylation site analysis of the LhSorPR10-1 recombinant protein described in the examples of the present invention.

[0064] Fig.12 This is the quality assessment of the mass spectrometry analysis data of the post-translational modification sites of the LhSorPR10-1 protein described in the embodiment of the present invention; A, peptide length distribution; B, peptide number distribution; C, missed cleavage site distribution; D, mass deviation distribution; E, contaminant protein spectrum distribution.

[0065] Fig.13 This is an overview of the mass spectrometry analysis samples of the post-translational modification sites of the LhSorPR10-1 protein described in the examples of the present invention; Identified proteins, identified proteins; Quantified proteins, quantified proteins; Identified peptides, identified peptides; Unique peptides, unique peptides; MS / MS count, MS / MS (tandem mass spectrometry) count.

[0066] Fig.14 This is the plasmid map of the plant expression vector pVM01-GFP described in the examples of the present invention.

[0067] Fig.15 This is a detection diagram of the LhSorPR10-1 recombinant plasmid bacterial solution described in the embodiment of the present invention;

[0068] A, detection diagram of LhSorPR10-1 recombinant plasmid bacterial solution; M, molecular mass standard; 1-10, PCR detection of LhSorPR10-1 recombinant plasmid bacterial solution.

[0069] B, LhSorPR10-1 antisense recombinant plasmid bacterial solution detection diagram; M, molecular mass standard; 1-6, LhSorPR10-1 antisense recombinant plasmid bacterial solution PCR detection.

[0070] Fig.16 The electrophoresis diagram is the restriction enzyme verification of the recombinant plasmid; M, molecular weight standard; 1-2, restriction enzyme verification of LhSorPR10-1 recombinant plasmid; 3-4, restriction enzyme verification of LhSorPR10-1 antisense recombinant plasmid.

[0071] Fig.17 This is a detection diagram of the LhSorPR10-1 recombinant plasmid Agrobacterium bacterial solution described in the embodiment of the present invention;

[0072] A, LhSorPR10-1 overexpression vector Agrobacterium tumefaciens culture detection diagram; M, molecular weight standard; 1-9, LhSorPR10-1 overexpression vector Agrobacterium tumefaciens culture PCR detection;

[0073] B, LhSorPR10-1 antisense expression vector Agrobacterium culture detection diagram; M, molecular weight standard; 1-7, LhSorPR10-1 antisense expression vector Agrobacterium culture PCR detection.

[0074] Fig.18 This is the detection result of LhSorPR10-1 overexpression (OE) transgenic lily described in the examples of the present invention;

[0075] A, LhSorPR10-1 transgenic lily strain test; M, molecular mass standard; P, plasmid; 1-9, LhSorPR10-1 transgenic lily strains;

[0076] B, Analysis of gene expression in LhSorPR10-1 lily line; multiple T test, WT was the control group, *P<0.05, **P≤0.01; ***P≤0.001.

[0077] Fig.19 This is the detection result of LhSorPR10-1 antisense expression (AS) transgenic lily described in the examples of the present invention;

[0078] A, antisense expression transgenic lily strain inspection; M, molecular mass standard; P, plasmid; 1-5, LhSorPR10-1 transgenic lily strains;

[0079] B, Analysis of gene expression in antisense-expressing lily strains; multiple T test, WT as control group, *P<0.05, **P≤0.01; ***P≤0.001;

[0080] C, Analysis of the expression level of the marker gene in the antisense-expressing transgenic lily lines; multiple T test, WT as the control group, ***P≤0.001.

[0081] Fig. 20 Antibacterial analysis and ROS reactive oxygen species detection of wild-type lily and transgenic lily as described in the examples of the present invention; scale bar is 1 cm; A, antibacterial analysis of wild-type lily and transgenic lily; B, nitro blue tetrazolium chloride staining (DAB); C, diaminobenzidine staining (NBT).

[0082] Fig.21This is the disease resistance analysis of the transgenic lily described in the example of the present invention; the data are mean ± standard error, multiple T test, WT is the control group, different letters represent P < 0.05; WT, wild type; OE, overexpression transgenic lily strain; AS, antisense inhibition transgenic strain.

[0083] Fig. 22 It is the malondialdehyde (MDA) content of the wild type and transgenic strains described in the examples of the present invention; the data are mean ± standard error, multiple T test, WT is the control group, *P<0.05, **P≤0.01; ***P≤0.001; T, wild type; OE, overexpression transgenic lily strain; AS, antisense inhibition transgenic strain.

[0084] Fig.23 The effects of gray mold on the protective enzyme activities of wild-type and transgenic lilies described in the examples of the present invention; A, superoxide dismutase (SOD) activity of LhSorPR10-1 transgenic strain; B, peroxidase (POD) activity of LhSorPR10-1 transgenic strain; C: catalase (CAT) activity of LhSorPR10-1 transgenic strain; data are mean ± standard error, multiple T test, WT is the control group, *P<0.05, **P≤0.01; ***P≤0.001; WT, wild type; OE, overexpression transgenic lily strain; AS, antisense inhibition transgenic strain.

[0085] Fig.24 It is the proline (Pro) content of the wild type and transgenic strains described in the examples of the present invention; the data are mean ± standard error, multiple T test, WT is the control group, *P<0.05, **P≤0.01; ***P≤0.001; WT, wild type; OE, overexpression transgenic lily strain; AS, antisense inhibition transgenic strain.

[0086] Fig.25 The lignin content of wild-type and transgenic lily before and after treatment with Botrytis cinerea described in the examples of the present invention; data are mean ± standard error, multiple T test, WT is the control group, *P < 0.05, **P ≤ 0.01; ***P ≤ 0.001; WT, wild type; OE, mixed sample of overexpression transgenic lily strains; AS, mixed sample of antisense suppression transgenic strains. DETAILED DESCRIPTION

[0087] In order to more clearly illustrate the technical solution of the embodiment of the present invention, the present invention will be described in detail below with reference to the accompanying drawings.

[0088] Example 1 Prokaryotic expression analysis of the cloned LhSorPR10-1 gene

[0089] Using lily cDNA as a template, primers for amplification of the LhSorPR10-1 gene were designed. The primer sequences are as follows:

[0090] LhSorPR10-1-F: ATGCCTTTCACTGTAGTGGTCG (SEQ ID No. 17);

[0091] LhSorPR10-1-R: TCAGACATAGGCATCAGGGTTG (SEQ ID No. 18).

[0092] The target band was connected to the pM19-T vector overnight and then transferred into TOP10 E. coli, and the positive bacterial solution was sequenced. BLAST comparison analysis of the sequencing results showed that a 471bp LhSorPR10-1 base sequence was cloned, containing a 471bp ORF, encoding 157 amino acids.

[0093] The cloned LhSorPR10-1 gene was analyzed for prokaryotic expression, the induction conditions of the LhSorPR10-1 gene were explored and optimized, and the purification conditions of the recombinant protein were explored, and the in vitro induced recombinant protein was successfully purified. Its nuclease activity was measured in vitro to explore the antibacterial activity of the LhSorPR10-1 protein, in order to provide a reference for further analysis of its anti-disease function.

[0094] SEQ ID No.1LhSorPR10-1 gene

[0095] ATGCCTTTCACTGTAGTGGTCGAGTCACCCGTCTCCGCTGCCCGGATGTTCAAGGCCGCCCTCGTCGACTGGCACAACCTCGGCCCCAAGATCGCCCCCCATGTCTTGTCCAGTGCTTCAATCATAGAAGGAGATGGCAGTGTCGGCAGTATTAGACAGCTCAATTTCACCTCAGATATACCATTCAGCTACGTCAAGGAACGCTTGGACTTCGTCGATCATGAAAAGTTCGAGT GCAAGGTCACCGCTCTCGAAGGAGGTCATCTGGAGACCATACTGGAATCTGCATCAGTCCACTTCAAGATCGAGCCCACCAGCACTGGAGGGTGCATCTGCAAGGCGGTGACCGAGTCCAAATTCAAACCAGGAGCTGAAGCCGGAGACGAGGAAGCCAAGGCCAAAGAGTCCATTGTCAAACTCTTCAAAGGCACCGAAGCCTACCTTCTTGCCAACCCTGATGCCTATGTCTGA

[0096] SEQ ID No.2LhSorPR10-1 protein

[0097] MPFTVVVESPVSAARMFKAALVDWHNLGPKIAPHVLSSASIIEGDGSVGSIRQLNFTSDIPFSYVKERLDFVDHEKFECKVTAEGGHLETILESASVHFKIEPTSTGGCICKAVTESKFKPGAEAGDEEAKAKESIVKLFKGTEAYLLANPDAYV

[0098] 1. Construction of prokaryotic expression vector of LhSorPR10-1 gene

[0099] A. Primer Design

[0100] According to the ORF frame sequence of LhSorPR10-1 gene and the multiple cloning site on the prokaryotic expression vector, two qualified restriction sites were selected, namely BamHI and HindⅠⅠⅠ, to design primers. The primer sequences are as follows:

[0101] LhSorPR10-1-BamHI-F: aaggccatggctgatatcGGATCCatgcctttcactgtagtggtcg (SEQ ID No. 3);

[0102] LhSorPR10-1-HindIII-R: gtgctcgagtgcggccgcAAGCTTgacataggcatcagggttggca (SEQ ID No. 4).

[0103] B. Construction of prokaryotic expression vector and enzyme digestion verification

[0104] The pET-32a(+) empty plasmid ( Figure 1 ) The E. coli DH5α was transformed by heat shock method. The product obtained after transformation was applied to LB solid medium containing 50mg / LAmp and inverted at 37℃. After about 12h, a single colony was picked and cultured in LB culture medium containing Amp. When the culture medium was obviously turbid, the pET-32a(+) vector universal primer was used to detect the bacterial solution. The bacterial solution with obvious correct bands was further expanded, the plasmid was extracted, and the vector was prepared for enzyme digestion.

[0105] The T-carrier plasmid DNA with the correct insertion of the LhSorPR10-1 gene fragment identified by sequencing was used as a template. According to the LhSorPR10-1 target fragment sequence and the restriction site of the pET32a(+) expression vector, HindⅢ and BamHI were selected for the design of homologous recombination primers. LhSorPR10-1-BamHI-F and LhSorPR10-1-HindⅢ-R with restriction sites were used as primers, and PCR amplification was performed with TransStart FastPfu DNA polymerase; the vector was constructed using a homologous recombination kit. The bacterial solution with the correct band was sent for testing, and the bacterial solution with the correct sequencing result was selected for plasmid extraction and PCR verification. Figure 2 shown.

[0106] C. Transformation of recombinant plasmid into prokaryotic expression strain

[0107] The recombinant plasmid was transferred into E. coli BL21 (DE3) Chemically Competent Cell by heat shock method, and the transformed bacterial solution was subjected to PCR electrophoresis detection, and a single and bright band was obtained. Figure 3 As shown, Escherichia coli containing the recombinant plasmid pET32a(+)-LhSorPR10-1 was successfully obtained.

[0108] 2. Inducible expression of recombinant protein

[0109] The recombinant plasmid pET32a(+)-LhSorPR10-1 and the pET32a(+) empty vector were transformed into the prokaryotic expression engineering strain BL21(DE3)Chemically Competent Cell (refer to the BL21(DE3)Chemically Competent Cell manual), and single clones were picked for detection. The positive clones were expanded, plasmids were extracted, and PCR verification was performed ( Figure 3 The constructed prokaryotic expression engineering bacterial solution was induced with 1.0 mM IPTG inducer, and the bacterial solution was collected to prepare the electrophoresis sample, and the expression of the recombinant protein was detected by 15% SDS-PAGE electrophoresis ( Figure 4 ). The results are as follows: pET32a(+)-LhsorPR10-1 recombinant protein is significantly expressed between 35kDa and 45kDa, while the pET32a(+) empty vector as a control has no protein expression at the same position under the same induction conditions. Through the analysis of protein physicochemical properties, it is known that the molecular weight of the protein encoded by the LhsorPR10-1 gene is 16.8kDa, and the molecular weight of the previous tag (Trx, S, His) protein in the pET32a(+) prokaryotic expression vector is about 20kDa, so the theoretical molecular weight of the fusion protein should be about 35kDa. The experimental results are in line with expectations. There are no specific bands around 35kDa in the uninduced pET32a(+)-LhSorPR10-1 and the induced and uninduced pET32a(+)-BL21, which indicates that the target gene can express recombinant protein in the prokaryotic expression engineering strain BL21, that is, the lily LhSorPR10-1 protein is successfully expressed in Escherichia coli.

[0110] 3. Solubility analysis of recombinant protein

[0111] The induction expression conditions of the pET32a(+)-LhSorPR10-1 recombinant protein were optimized, and more suitable optimization conditions were obtained. Subsequently, the induction conditions of soluble proteins were analyzed based on the total protein induction optimization conditions. After optimizing the induction expression conditions of the recombinant protein soluble protein, it was finally determined that the conditions of 37°C, 180rpm, 1.0mM induction for 6h were the best soluble protein induction conditions for the pET32a(+) recombinant protein; and 28°C, 180rpm, 1.0mM induction for 6h were the best soluble protein induction conditions for the pET32a(+)-LhSorPR10-1 recombinant protein. The induced bacterial solution was subjected to repeated freeze-thaw treatment with liquid nitrogen, lysozyme, and ultrasonic disruption to break the cells, and the total protein, supernatant, and precipitate were collected. The solubility of the protein was analyzed by SDS-PAGE electrophoresis. Figure 5As shown, pET32a(+)-LhSorPR10-1 without induction by the inducer IPTG did not produce specific recombinant protein, but after induction by the inducer, the target protein of corresponding size was produced, and most of the protein was expressed in the supernatant, and only a small amount of recombinant protein existed in the form of inclusion bodies, indicating that the LhSorPR10-1 protein is a soluble protein in Escherichia coli.

[0112] 4. Purification of recombinant protein

[0113] After optimizing the induction expression conditions of the recombinant protein soluble protein, it was finally determined that the soluble protein of pET32a(+) was collected under the conditions of 37°C, 180rpm, 1.0mM induction for 8h, and the soluble protein of pET32a(+)-LhSorPR10-1 was collected under the conditions of 28°C, 180rpm, 1.0mM induction for 8h. The protein was purified using a Ni-NTA column, and each component was analyzed by electrophoresis. The protein purified by pET32a(+) empty vector was detected by electrophoresis. Figure 6 A; protein electrophoresis detection of pET32a(+)-LhSorPR10-1 purification is Figure 6 B: The size of the purified target protein band is about 35 kDa, which is consistent with the expected protein size and the size of the recombinant protein detected in the solubility analysis. Therefore, it can be preliminarily determined that all LhSorPR10-1 recombinant proteins can be purified, laying the foundation for the subsequent identification of the antibacterial activity and nuclease activity of LhSorPR10-1 protein.

[0114] 5. Analysis of antibacterial activity of recombinant protein

[0115] In order to analyze the antibacterial activity of LhSorPR10-1 protein against Botrytis cinerea, the filter paper method was used for in vitro antibacterial test. 40 μg, 60 μg, and 80 μg of purified protein solution were dropped on sterile filter paper of Botrytis cinerea and Botrytis cinerea culture medium, respectively. At the same time, equal amounts of ddH2O and Buffer were added as blank controls. After culturing in a constant temperature incubator at 28°C for 3 days, the growth of fungi was observed. The results are shown in Figure 7 As shown: the fungi near the filter paper in the control group grew normally and there was no inhibitory effect, while the two fungi near the filter paper with the LhSorPR10-1 recombinant protein added did not grow normally, and the growth of the two pathogens was inhibited to varying degrees. Figure 8 As shown in Figure 2, compared with the control, in three repeated experiments, the average growth inhibition areas of 460 μg and 80 μg of recombinant LhsorPR10-1 protein on Botrytis cinerea were 81.27 and 24.88 mm, respectively. 2; There was no significant inhibitory effect on Botrytis cinerea. The above results showed that the LhSorPR10-1 recombinant protein had a specific inhibitory effect on the growth of Botrytis cinerea.

[0116] 6. Analysis of nuclease activity of recombinant protein

[0117] The conserved "P-loop" domain contained in the PR10 protein is closely related to nuclease activity and is believed to be related to plant defense responses. Therefore, the DNase and RNase activities of the LhSorPR10-1 protein were determined.

[0118] The RNase activity analysis of LhSorPR10-1 protein was performed according to the method of Guevara-Morato et al. Fig. 9 As shown in the figure, no RNA degradation occurred when HIS-tagged protein and boiled LhSorPR10-1 protein were used as negative controls; uncooked LhSorPR10-1 protein was used to treat lily leaf RNA, which showed obvious degradation, indicating that LhSorPR10-1 protein has RNase activity. Subsequently, the RNase activity of LhSorPR10-1 protein at different temperatures was tested, and it was found that RNA could be degraded below 40°C, but RNA was not degraded above 40°C, indicating that the RNase activity of LhSorPR10-1 protein would be affected by temperature.

[0119] The DNase activity analysis of LhSorPR10-1 protein was also performed according to the method of Guevara-Morato et al. 2+ It has a certain promoting effect on DNase activity. Therefore, in the presence of Mg 2+ The DNase activity of LhSorPR10-1 was analyzed under the presence of Fig.10 As shown, after adding Mg 2+ Under the conditions of , HIS-tagged protein and boiled LhSorPR10-1 protein were used as negative controls and it was found that DNA did not degrade. DNA treated with LhSorPR10-1 protein was almost completely degraded, indicating that LhSorPR10-1 protein has DNase activity. Subsequently, the DNase activity of LhSorPR10-1 was also measured at different temperatures, and it was found that LhSorPR10-1 protein could degrade DNA at any temperature, indicating that the DNase activity of LhSorPR10-1 protein is not affected by temperature.

[0120] Example 2 Analysis of post-translational modification sites of LhSorPR10-1 protein

[0121] 1. Prediction of post-translational modification sites of LhSorPR10-1 protein

[0122] The phosphorylation sites on the LhSorPR10-1 protein were predicted by the NetPhos-3.1 website. The protein phosphorylation sites mainly occurred on serine (S), threonine (T), and tyrosine (Y) residues. A total of 25 corresponding amino acid sites were predicted for the LhSorPR10-1 protein by the NetPhos-3.1 website, of which 16 were phosphorylation sites, namely serine at 9, 12, 40, 47, 50, 58, 63, 106, 118, and 136, tyrosine at 155, and threonine at 4, 82, 91, 116, and 144. The phosphorylation sites predicted for the LhsorPR10-1 protein were mainly on serine residues ( Fig.11 ).

[0123] The ubiquitination sites of LhsorPR10-1 were predicted using the GPS-SUMO webpage, and a total of two ubiquitination sites were predicted, as shown in Table 1.

[0124] Table 1 Ubiquitination prediction results

[0125]

[0126] 2. Mass spectrometry analysis of post-translational modification sites of LhSorPR10-1 protein

[0127] A. Data Quality Assessment

[0128] (1) Peptide length distribution

[0129] The mass spectrometry MS1 scanning range is usually 350-1500 m / z. After the peptides are ionized, most of the charges are +2 (+3, +4, etc.), and the average molecular weight of the amino acid residues in the protein is about 110 kDa. Therefore, most of the peptides detected by the mass spectrometry are located between 7 and 27 amino acids. The length distribution of the peptides detected in this experiment is as follows: Fig.12 As shown in A.

[0130] (2) Peptide number distribution

[0131] The commonly used research strategy in proteomics is a "bottom-up" approach, that is, the information of proteins comes from the peptide information directly detected by mass spectrometry. In theory, each protein contains multiple detectable enzyme-digested peptides, but due to many factors such as peptide length, relative abundance, and ionization efficiency, the number of peptides supporting each protein varies. The more protein data there is in large-scale proteomics data, the higher the relative abundance of the protein is usually, and there is a certain degree of positive correlation between the two. The peptide number distribution graph statistics the distribution of proteins containing different numbers of peptides. The higher the proportion of proteins with peptides, the more reliable the proteomics results are at the protein level. The distribution of the number of peptides detected in this experiment is as follows: Fig.12As shown in B.

[0132] (3) Distribution of missed cutting sites

[0133] During the pre-treatment of protein samples for mass spectrometry detection, the protein will be enzymatically treated with trypsin. Trypsin will specifically hydrolyze the C-terminal peptide bonds of arginine and lysine in proteins, and hydrolyze the complete protein into peptides of different lengths for mass spectrometry detection. In some cases, such as when there are adjacent arginine or lysine in the protein sequence, or when there are post-translational modifications on the arginine or lysine side chains that affect trypsin binding, some arginine or lysine C-terminal peptide bonds will not be hydrolyzed by trypsin. Under normal circumstances, most of the detected peptides do not have missed cleavage sites, and a small number of peptides will have 1 or 2 missed cleavage sites. If the proportion of peptides with missed cleavage sites is significantly too high, it may indicate that the enzymatic cleavage efficiency is low during the enzymatic hydrolysis process. The missed cleavage sites of the peptides detected in this experiment are as follows: Fig.12 As shown in C.

[0134] (4) Quality deviation distribution

[0135] After mass spectrometry detection, the measured size of the peptide is compared with its theoretical size to evaluate the mass accuracy of the mass spectrometer. The deviation between the measured size and the theoretical size is often displayed in ppm (parts per million), which is related to the performance of the mass spectrometer and the status of the instrument. When the instrument is in good condition, the mass deviation of the mass spectrometer using the Orbitrap mass analyzer is generally distributed between -10 and +10ppm, and the mass deviation of the mass spectrometer using the Q-TOF mass analyzer is generally distributed between -20 and +20ppm. The distribution of the mass deviation of the peptides detected in this experiment is as follows Fig.12 As shown in D.

[0136] (5) Common contaminating proteins

[0137] During the preparation and pretreatment of protein samples, the introduction of certain common contaminating proteins is often unavoidable. When obtaining mass spectrometry data and performing database searches, in addition to the target protein sequence library, a common contaminating protein sequence library will also be added. In subsequent analysis, the identification results that match the contaminating protein library will be excluded. If the proportion of contaminating proteins in the identification results is too high, it may be that improper operation during sample collection has introduced too much protein contamination, which will affect the detection of the target protein; it may also be that the protein to be detected in the sample itself is a contaminating protein, and the identified contaminating protein entries need to be retained for subsequent analysis. The distribution of the number of protein spectra identified in this experiment is as follows: Fig.12 As shown in E, the red marks are common contaminating proteins.

[0138] B. Basic data analysis

[0139] The purified LhsorPR10-1 recombinant protein samples were tested and a total of 9 proteins were identified ( Fig.13), and further remove non-target proteins.

[0140] 2. Protein post-translational modification sites

[0141] Mass spectrometry detected a total of 5 phosphorylation sites (including 2 serines, 2 threonines, and 1 tyrosine), 7 ubiquitination sites, and 10 acetylation sites. The results are shown in Tables 2 to 5.

[0142] Table 2 Results of phosphorylation site detection of LhSorPR10-1 recombinant protein (Phospho)

[0143] Accessions accession number Amin acid Phospho(STY)ProbabilitiesPhosphorylation Sites A0A2U7M4J6; A0A221S8D2 S HGKKS(0.937)LAY(0.064)QIIY(0.999)R A0A7U3S049; A0A7U3NRI2 S YGS(1)IPVVR A0A097PLG2 T LNQIFAT(1)VGDKK A0A221S8C2 T GKDT(0.118)IANIIT(0.764)S(0.118)IR A0A2U7M4J6; A0A221S8D2 Y HGKKS(0.937)LAY(0.064)QIIY(0.999)R

[0144] Table 3 Results of ubiquitination site detection of LhSorPR10-1 recombinant protein (GlyGly)

[0145]

[0146]

[0147] Table 4 Results of ubiquitination site detection of LhSorPR10-1 recombinant protein (LRGG (K))

[0148]

[0149] Table 5 LhSorPR10-1 recombinant protein acetylation site detection results (Acetyl)

[0150]

[0151] Example 3 Analysis of the disease resistance function of LhSorPR10-1 in lily

[0152] In order to further analyze the function of LhSorPR10-1 gene, overexpression and antisense expression vectors were constructed, which were transformed into embryonic callus of 'Sorbonne' lily by Agrobacterium infection. The disease resistance of transgenic lily plants was analyzed to clarify the function of LhSorPR10-1 in lily gray mold disease resistance.

[0153] The plant material used was 'Sorbonne' lily, and the lily bulbs used for fungal infection were imported from the Netherlands and purchased from Yunnan; they were stored in a freezer (4°C, 16h day / 8h night) with a relative humidity of 60-80%.

[0154] A: Construction of plant overexpression and antisense expression vectors of lily LhSorPR10-1

[0155] According to the ORF frame sequence of LhSorPR10-1 gene and the plant expression vector pVM01-GFP ( Fig.14 ) on the multiple cloning site analysis, select BamHI and SacI to design primers, the primer sequences are as follows:

[0156] LhSorPR10-1-BamHI-F: tgttgtttggtgttacttGGATCCatgcctttcactgtagtggtcg (SEQ ID No. 5);

[0157] LhSorPR10-1-SacI-R: ctcgcccttgctcaccatGAGCTCgacataggcatcagggttggca (SEQID No. 6);

[0158] LhSorPR10-1-SacI-F: ctcgcccttgctcaccatGAGCTCatgcctttcactgtagtggtcg (SEQID No. 7);

[0159] LhSorPR10-1-BamHI-R: tgttgtttggtgttacttGGATCCgacataggcatcagggttggca (SEQ ID No. 8);

[0160] pVM01-F: cttggatgatggcatatgcagcag (SEQ ID No. 9);

[0161] pVM01-2R: tcgccggacacgctgaacttgtg (SEQ ID No. 10).

[0162] The pVM01-GFP plasmid after amplification and extraction of plasmid was subjected to linearization restriction digestion of the vector, and the T-carrier plasmid of the LhSorPR10-1 gene fragment was used as a template, and primers with BamHI and SacI restriction sites were used to PCR amplify the target fragment. The restriction digestion product and the PCR cloning product were subjected to 1% agarose gel electrophoresis, and the required size fragments were recovered from the gel, that is, the pVM01-GFP linear vector fragment of about 11100bp and the target fragment of about 500bp were recovered. The linearized pVM01-GFP fragment was homologously recombined with the LhSorPR10-1 fragment with homology arms and restriction sites using a homologous recombination kit. The ligation product was transferred into Escherichia coli DH5α competent cells by heat shock method, and sent for detection after comparison of electrophoresis bands ( Fig.15 ). Extract the correct bacterial plasmid and perform double enzyme digestion verification ( Fig.16 ), and finally determined to obtain the gene recombinant plasmid, namely pVM01-LhSorPR10-1, and the antisense inhibition vector Anti-pVM01-LhSorPR10-1 positive recombinant plasmid.

[0163] B: Genetic transformation of lily 'Sorbonne'

[0164] Using the Agrobacterium transformation method, the recombinant plasmid pVM01-LhSorPR10-1 and the antisense inhibition Anti-pVM01-LhSorPR10-1 positive recombinant plasmid were respectively transferred into EHA105 Agrobacterium for bacterial liquid detection. Fig.17 The embryonic callus of lily was used as raw material for Agrobacterium infection, and positive lily plants were obtained after pre-culture, co-culture and screening culture.

[0165] The CTAB method was used to extract the leaf DNA of the lily in the previous step, and PCR amplification was performed using specific primers designed upstream of the gene and downstream of the vector. Agarose gel electrophoresis detection proved that the LhSorPR10-1 gene had been transferred into the lily.

[0166] The RNA of the transgenic lily lines that successfully passed DNA detection was extracted using the Trizol method, and reverse transcription was performed to obtain cDNA. According to the kit instructions, real-time fluorescence quantitative PCR was performed on the LhSorPR10-1 gene transgenic lily lines to analyze the expression differences of the transgenic lines. The primer sequences are as follows:

[0167] qLhSorPR10-1-F: atcagtccacttcaagatcgagcc (SEQ ID No. 11);

[0168] qLhSorPR10-1-R: cgcaagaagtatggctggaccgcct (SEQ ID No. 12);

[0169] qLhSorActin (reference gene)-F: cacactggtgtcatggttgg (SEQ ID No. 13);

[0170] qLhSorActin (reference gene)-R: cacaataccgtgctcaattgg (SEQ ID No. 14);

[0171] qLhSorEF1 (reference gene)-F: gttgtggctgtggaggaagaagag (SEQ ID No. 15);

[0172] qLhSorEF1 (reference gene)-R: gacgcagaaccaaagagagtatccc (SEQ ID No. 16).

[0173] A total of 7 LhSorPR10-1 overexpressing strains were identified ( Fig.18 ) and 5 LhSorPR10-1 antisense expression lines ( Fig.19 ).

[0174] C: Identification of resistance of LhSorPR10-1 transgenic plants to Botrytis cinerea

[0175] To investigate the resistance of transgenic lilies overexpressing the LhSorPR10-1 gene to Botrytis cinerea, agar blocks containing Botrytis cinerea hyphae were inoculated on leaves of wild-type lilies and transgenic lilies. Fig. 20 As shown in the figure, 96 hours after inoculation, the leaves of wild-type lilies showed severe lesions and yellowing symptoms. In contrast, the leaves of the LhSorPR10-1 overexpressing strain only showed slight lesions, and the leaves of the lilies expressing LhSorPR10-1 antisense showed more serious lesions, indicating that LhSorPR10-1 has a certain defensive effect against the invasion of Botrytis cinerea. Image J was used to measure and count the lesion area of ​​the corresponding leaves, as shown in the figure below. Fig.21 shown.

[0176] The amount of ROS accumulation was roughly determined by leaf tissue staining, in which NBT and DAB chemical staining were used to analyze hydrogen peroxide (H2O2) and superoxide anion (O 2- ) accumulation. Fig. 20 Compared with the transgenic lilies, the wild-type lilies showed large dark brown patches after DAB staining, indicating H2O2 accumulation, and large dark blue patches after NBT staining, indicating O 2- On the contrary, the leaves of antisense-suppressed transgenic lilies showed more severe brown and blue colors than those of the wild type, which indirectly indicated the role of LhSorPR10-1 in lily resistance to gray mold.

[0177] D: Physiological index determination of LhSorPR10-1 transgenic plants

[0178] When plants are infected by pathogens, the reactive oxygen species (ROS) burst in response to biotic stress has a protective effect, however, excessive ROS can cause serious damage, and plants strictly regulate the production and detoxification of ROS. It is speculated that LhSorPR10-1 improves plant disease resistance by enhancing the activity of ROS scavenging enzymes and regulating ROS content. Malondialdehyde (MDA) is closely related to plant disease resistance. As a strong oxidant, it can destroy the structure and physiological integrity of cell membranes, and its content is directly related to the degree of membrane lipid peroxidation; the content of proline (Pro) in plant cells can reflect the strength of plant stress resistance; when plants are under stress, in addition to the accumulation of reactive oxygen species, there is also an increase in the content of lignin or lignin-like phenolic compounds, so lignin metabolism has a certain correlation with plant stress. Therefore, the contents of MDA, Pro, lignin, and antioxidant enzymes SOD, POD, and CAT in transgenic and wild-type lilies were determined.

[0179] The malondialdehyde (MDA) content in leaves of transgenic and wild-type lily seedlings was determined. Fig. 22 ). Under the infection of Botrytis cinerea, the MDA content in the leaves of transgenic and wild-type lilies showed a linear upward trend, and the content of the antisense suppression transgenic strain was the highest; when not inoculated with bacteria, the MDA content in the leaves of the overexpression transgenic strain and the antisense suppression transgenic strain was basically the same as that of the wild-type. These results indicate that the degree of plasma membrane damage in the antisense suppression transgenic plants and wild-type plants is more serious than that in the overexpression transgenic plants, and the degree of plasma membrane damage in the antisense expression transgenic plants is higher than that in the wild-type plants.

[0180] The contents of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) in leaves of transgenic and wild-type lily seedlings were determined. Fig.23 ). As shown in the figure, the activities of three antioxidant enzymes, SOD, POD, and CAT, in the transgenic plants overexpressing LhSorPR10-1 under the treatment of Botrytis cinerea were 2.31, 2.22, and 2.36 times higher than those in the wild type, respectively; while the activities of three antioxidant enzymes, SOD, POD, and CAT in the transgenic plants expressing antisense LhSorPR10-1 were 1.05, 0.94, and 0.98 times higher than those in the wild type, respectively. The three antioxidant enzyme activities of transgenic plants were always higher than those of WT plants. The above results show that overexpression of LhSorPR10-1 significantly reduced the degree of membrane lipid peroxidation and increased the activity of its antioxidant enzymes in 'Sorbonne' lily; while the antisense expression of LhSorPR10-1 also reduced the degree of membrane lipid peroxidation and increased the activity of its antioxidant enzymes, but the degree of reduction was lower than that of the wild type, indicating that the LhSorPR10-1 gene plays a very important role in the resistance of lily to gray mold.

[0181] The proline (Pro) content in leaves of transgenic and wild-type lily seedlings was determined ( Fig.24 ). After treatment, the proline content of the overexpressed transgenic strain was significantly higher than that of the wild-type and antisense inhibition transgenic strains, while the proline content of the wild-type and antisense inhibition transgenic strains was basically the same. The above experimental results further prove that the LhSorPR10-1 gene can enhance the antibacterial ability of the overexpressed lily strains.

[0182] The lignin content of leaves of transgenic and wild-type lily seedlings was determined ( Fig.25 ). Compared with the untreated wild-type lily strain and the transgenic strain overexpressing the LhSorPR10-1 gene, the lily strain overexpressing the LhSorPR10-1 gene had a higher lignin content; with the accumulation of Botrytis cinerea infection, the lignin content increased, and the lily plants overexpressing the LhSorPR10-1 gene had a greater increase in lignin content than the wild type. It can be inferred that the lily overexpressing the LhSorPR10-1 gene is more resistant to gray mold than the wild type.

[0183] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific implementation methods described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. Application of lily pathogenesis-related protein LhSorPR10-1, characterized in that: Its amino acid sequence is shown in SEQ ID No. 2; the application is at least one of the following: a. Application in improving plant disease resistance; b. Application in improving the antioxidant enzyme level, lignin content and / or proline content of diseased plants; c. Application in reducing the level of plasma membrane damage in diseased plants; d. Application in the preparation of plant gray mold inhibitors.

2. The application according to claim 1, characterized in that: The coding gene of the lily pathogenesis-related protein LhSorPR10-1 has a nucleotide sequence as shown in SEQ ID No.1; Alternatively, in a, b, c or d, the plant is lily.

3. The application according to claim 1, characterized in that: One of the following characteristics: In a, the disease resistance is resistance to gray mold; In b or c, the disease is infection with gray mold; In b, the antioxidant enzyme is SOD, POD and / or CAT.

4. The application according to claim 3, characterized in that: The pathogen of gray mold is Botrytis cinerea and / or Botrytis cinerea.

5. A method for using lily pathogenesis-related protein LhSorPR10-1, characterized in that: The amino acid sequence of the lily pathogenesis-related protein LhSorPR10-1 is shown in SEQ ID No. 2; the method of applying the lily pathogenesis-related protein LhSorPR10-1 is to increase the expression level of the lily pathogenesis-related protein LhSorPR10-1 in the plant; the purpose of the method is at least one of the following: e. Improve plant disease resistance; f. Increase the antioxidant enzyme level, lignin content and / or proline content of infected plants; g. Reduce the level of plasma membrane damage in infected plants.

6. The method according to claim 5, characterized in that: One of the following characteristics: In e, the disease resistance is resistance to gray mold; In f or g, the infection is infection with gray mold; In e, f or g, the plant is a lily.

7. The method according to claim 6, characterized in that: The pathogen of gray mold is Botrytis cinerea and / or Botrytis cinerea.

8. The method according to claim 5, characterized in that: The method for increasing the expression level of LhSorPR10-1 in plants comprises: transferring the coding gene of LhSorPR10-1 into the plants, so that the plants express LhSorPR10-1 protein; Preferably, the gene encoding the lily pathogenesis-related protein LhSorPR10-1 has a nucleotide sequence as shown in SEQ ID No.

1.

9. A plant gray mold inhibitor, characterized in that: Its main components are lily pathogenesis-related protein LhSorPR10-1, engineered bacteria expressing lily pathogenesis-related protein LhSorPR10-1, fermentation liquid of engineered bacteria expressing lily pathogenesis-related protein LhSorPR10-1 and / or secretions of engineered bacteria expressing lily pathogenesis-related protein LhSorPR10-1; the amino acid sequence of lily pathogenesis-related protein LhSorPR10-1 is shown in SEQ ID No.

2.

10. The plant gray mold inhibitor according to claim 9, characterized in that: The pathogen of gray mold is Botrytis cinerea and / or Botrytis cinerea; preferably, the plant is lily.

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