Coding gene of benzyl salicylate hydrolase participating in rice PAL-dependent salicylic acid synthesis route and application of coding gene

Abstract

The invention belongs to the technical field of plant genetic engineering, and discloses a gene OSD4 participating in a synthetic route of PAL-dependent salicylic acid (SA) of rice and application of the gene OSD4. A mutant osd4 is obtained by knocking out OSD4 through CRISPR / Cas9, the SA content of leaves of the mutant osd4 is reduced to 15% of that of a wild type, and the mutant osd4 is more sensitive to xanthomonas oryzae pv. And constructing an OSD4 overexpression vector to obtain a transgenic material with enhanced resistance. Hormone determination and inoculation experiments show that OSD4 regulates SA synthesis and participates in disease-resistant reaction. The in-vitro enzyme activity is verified by constructing an OSD4-pMAL carrier, and it is proved that OSD4 can catalyze BS to directly generate SA. The gene can be used for disease resistance improvement of crops such as rice.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and in particular relates to a gene encoding benzyl salicylate hydrolase involved in a rice PAL-dependent salicylic acid synthesis pathway and an application thereof. Background Art

[0002] Salicylic acid (SA) is an important plant hormone that participates in plant growth and development, as well as stress resistance, directly or through crosstalk with other hormones. SA is one of the many phenolic compounds synthesized by plants that contain hydroxyl groups or their derivatives. It participates in multiple life processes, including seed germination, cellular respiration, cell growth, stomatal opening and closing, flowering, and aging. Furthermore, SA is a key signal regulating heat production and disease resistance.

[0003] SA (Salt-Acid) synthesis in plants primarily occurs through two secondary metabolic pathways, originating from shikimate: the isochorismate synthase (ICS) pathway and the phenylalanine ammonia lyase (PAL) pathway. The SA signaling pathway plays a key regulatory role in plant disease defense responses. In rice, ICS1 is primarily involved in chlorophyllin quinone biosynthesis and not in SA biosynthesis. In contrast, the biochemical role of the PAL pathway in rice SA biosynthesis remains to be elucidated. However, functional deletion of AIM1 (ABNORMALINFLORESCENCE MERISTEM 1), a gene involved in the PAL-dependent SA biosynthesis pathway, reduced endogenous SA content in rice to approximately 10% of that in the wild type, indicating that the PAL pathway is the primary pathway for SA biosynthesis in rice. Barley inoculated with Pseudomonas syringae pv. syringae exhibited an increase in endogenous SA during infection, whereas rice did not experience an increase in SA levels following infection with P. syringae, the rice blast pathogen (Magnaporthe grisea), or Rhizoctonia solani. Salicylate hydroxylases (NahG), which block SA accumulation in vivo, significantly increased in NahG-transgenic Arabidopsis plants following infection with the pathogen Pto DC3000, suggesting a key role for SA in disease resistance. Rice expressing NahG also exhibited a significant increase in ROS levels following infection with Magnaporthe oryzae, indicating that reduced SA contributes to increased susceptibility to the pathogen. Mutation of OsS5h leads to increased SA content in rice and enhanced resistance to bacterial blight. Overexpression of OsS5h reduces endogenous SA in rice and makes it susceptible to bacterial blight, indicating that SA content is positively correlated with resistance to bacterial blight. Recent studies suggest that SA may also mediate defense signaling in wheat. Taken together, these results indicate that, at least in some monocotyledons, SA serves as a signal for defense responses, and that endogenous SA content is positively correlated with plant disease resistance.

[0004] Rice, a staple food crop for more than half the world's population, is a model monocot plant with high basal SA levels. Rice production is frequently impacted by various field diseases, the most devastating of which is bacterial leaf blight caused by Xanthomonas oryzae. Bacterial leaf blight can affect rice growth, development, or reproduction, resulting in severe yield losses of up to 50%. It is estimated that with population growth, global food production will need to increase by at least 50% by 2050 to meet global demand. Therefore, developing new high-yield and stable crop varieties is crucial for ensuring food security.

[0005] Rice SA cannot accumulate in large quantities after pathogen invasion, resulting in the plant being unable to effectively utilize SA to resist the invasion of external pathogens. In plant disease resistance improvement, any improvement in disease resistance is often accompanied by changes in growth and development and a decrease in yield. Therefore, it is crucial to improve rice resistance to pathogens while ensuring stable yields.

[0006] The identification and cloning of genes encoding key catalytic enzymes in the PAL-dependent SA biosynthesis pathway remains a key technical bottleneck in plant immune signaling research. The lack of these functional genes severely hinders the molecular understanding of the mechanisms of SA-mediated systemic acquired resistance (SAR) in plants, further hindering the development and application of strategies to precisely regulate SA biosynthesis through genetic engineering to enhance crop disease resistance. Therefore, the in-depth identification and functional validation of the core enzyme genes involved in PAL-dependent SA biosynthesis have become a key breakthrough for improving crop disease resistance and building disease-resistant germplasm resources. Summary of the Invention

[0007] In response to the problems existing in the prior art, the present invention provides a gene involved in the rice PAL-dependent salicylic acid A synthesis pathway and its application.

[0008] To address the above technical issues, the present invention provides a gene, OSD4, that regulates SA synthesis in rice leaves and resistance to bacterial blight, and its applications. Using CRISPR / Cas9 technology, the present invention screened a rice mutant with leaf SA reduced to less than 10% of that of the wild type. Functional studies have demonstrated that the gene is involved in the PAL-dependent salicylic acid synthesis pathway in rice, and that overexpression can enhance disease resistance in rice.

[0009] The present invention is achieved by a gene involved in the rice PAL-dependent salicylic acid synthesis pathway:

[0010] A salicylic acid synthesis gene, the gene is OSD4, and the nucleotide sequence is SEQ ID NO: 1.

[0011] Furthermore, the salicylic acid synthesis gene further comprises a DNA sequence having a homology of more than 90% with the DNA sequence shown in SEQ ID NO: 1.

[0012] Furthermore, the salicylic acid synthesis gene further includes alleles or gene derivatives with one or more base changes produced by base substitution, deletion, or addition.

[0013] Furthermore, the salicylic acid synthesis gene further comprises: a DNA molecule that can hybridize with the DNA sequence shown in SEQ ID NO: 1 under stringent conditions.

[0014] Another object of the present invention is to use the protein encoded by the rice salicylic acid synthesis gene, wherein the nucleotide sequence of the encoded protein is SEQ ID NO: 2.

[0015] Furthermore, the amino acid sequence of the encoded protein is SEQ ID NO: 3;

[0016] The encoded protein further comprises an amino acid sequence having a homology of more than 90% with the amino acid sequence shown in SEQ ID NO: 3;

[0017] The encoded protein further includes proteins and protein analogs having one or more amino acid changes produced by amino acid substitution, deletion, or addition based on the amino acid sequence shown in SEQ ID NO: 3;

[0018] The encoded protein further includes a fusion protein formed by linking the protein shown in SEQ ID NO: 3 with other tag proteins.

[0019] Another object of the present invention is a plant genetic transformation vector constructed using the salicylic acid synthesis gene, wherein the plant genetic transformation vector includes an expression vector for up-regulating OSD4; the expression vector for up-regulating OSD4 includes a fusion expression vector constructed with a recombinant promoter or an organ-specific promoter;

[0020] The plant genetic transformation vector further comprises: a DNA sequence as shown in SEQ ID NO: 1, or a DNA sequence having a homology of more than 90% with the DNA sequence as shown in SEQ ID NO: 1, or an allele or gene derivative with one or more base changes produced by base substitution, deletion, or addition of the nucleotide sequence as shown in SEQ ID NO: 1, or a DNA molecule capable of hybridizing with the DNA sequence as shown in SEQ ID NO: 1 under stringent conditions.

[0021] Furthermore, the plant genetic transformation vector further includes an up-regulation or down-regulation OSD4 expression vector, which down-regulates the expression of the gene shown in SEQ ID NO: 3 by CRISPR / Cas9 technology, T-DNA insertion technology, EMS mutagenesis, RNA interference technology, or gene silencing technology;

[0022] The plant genetic transformation vector up-regulates or down-regulates the expression level or activity of the protein shown in SEQ ID NO: 3 through a related protein regulator.

[0023] Another object of the present invention is a recombinant bacterium, plant callus and cell line using the plant genetic transformation vector for expression.

[0024] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0025] In rice, the PAL-dependent SA biosynthesis pathway has long been considered the primary secondary metabolic pathway in response to pathogenic stress. This pathway uses phenylalanine as its initial substrate and, through a series of enzymatic conversions, generates SA, which in turn mediates immune processes such as systemic acquired resistance. Although studies have revealed that benzoic acid (BA) may participate in this pathway as an intermediate and generate SA through hydroxylation, the gene encoding this key enzyme has yet to be successfully cloned and functionally verified, limiting the molecular mechanism of this pathway and its targeted application in genetic improvement. Current research has largely focused on in vitro inference at the enzyme activity level or dynamic analysis of upstream metabolic fluxes, lacking systematic identification of downstream catalytic factors, a core technical bottleneck restricting the targeted regulation of the PAL-dependent SA biosynthesis pathway.

[0026] This study, published in the journal Nature Communications, cloned and functionally characterized a key novel gene, OSD4, involved in rice PAL-dependent SA biosynthesis (SEQ ID NO: 1). Enzyme activity assays and substrate specificity experiments revealed that OSD4 specifically recognizes and catalyzes the conversion of benzyl salicylate (BS) to free SA, revealing a previously unidentified enzymatic link in the PAL pathway. This study, for the first time, identified BS as a bona fide substrate in the rice SA biosynthesis pathway and successfully incorporated it into enzymological models, establishing a BS-derived biosynthesis pathway centered on OSD4. This approach fills a critical gap in conventional models regarding substrate recognition and conversion mechanisms.

[0027] At the molecular genetic level, experiments have demonstrated that OSD4-overexpressing transgenic rice lines significantly increases free SA content in rice plants, leading to enhanced systemic resistance to bacterial blight (Xanthomonas oryzae pv. oryzae, Xoo). This confirms that the OSD4-dominated metabolic pathway plays an independent and effective defensive role in the plant immune system. This discovery, distinct from the traditional SA biosynthesis pathway, provides a new genetic target for constructing disease-resistant germplasm lines with high SA accumulation.

[0028] The cloning of OSD4 and the elucidation of its biochemical and genetic functions not only expand the regulatory landscape of the rice SA synthesis network but also propose a new SA synthesis model based on BS intermediates, challenging the traditional synthesis hypothesis that free benzoic acid is the direct precursor. At the technical implementation level, this invention provides new enzyme resources and metabolic interfaces that can be used to construct efficient SA synthesis modules in synthetic biology platforms. This achievement has significant innovative value in both scientific and practical applications, providing an important theoretical basis and practical path for disease-resistant breeding and metabolic engineering.

[0029] The present invention provides a key gene OSD4 for rice SA synthesis and its application in crop disease prevention and control, systematically revealing its functional mechanism in regulating endogenous SA levels, catalyzing key steps in the PAL-dependent SA synthesis process, and catalyzing BB to BS (see Figure 1-Figure 5 ), and verified its disease resistance enhancement effect under pathogen stress by creating its overexpression material (see Figure 5 ), providing innovative and applicable solutions for plant immune metabolism regulation and crop genetic improvement. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is an identification diagram of the gene OSD4 involved in the rice SA biosynthesis pathway provided by an embodiment of the present invention.

[0031] A, Location of the Cas9-induced mutagenesis target motif (red arrow) within the coding region of the OSD4 gene (orange box); B, Sanger sequencing peaks of the osd4 mutant; C, Amino acid sequence information of wild-type and osd4 mutant plants; D, Phenotypes of wild-type and osd4 mutant plants at heading. Scale bar, 10 cm; E, SA and SAG content in leaves of 14-day-old wild-type and osd4 mutant plants; F, Lesion phenotype and lesion length statistics of wild-type and osd4 mutant plants 14 days after inoculation with Xoo. Scale bar, 3 cm. Statistical analysis was performed using a two-tailed Student's t-test. ***p < 0.001. Data are expressed as mean ± SD, n = 4 (E) and n = 20 (F) biologically independent samples.

[0032] Figure 2 1 is an expression pattern diagram of OSD4 provided in an embodiment of the present invention.

[0033] A, OSD4 gene expression in roots, stems, leaves, and panicles of 70-day-old Zhonghua 11 plants. Gene expression levels were normalized to those of the internal reference gene OsUBQ5. B, OSD4 gene expression in leaves of Zhonghua 11 plants inoculated with Xoo at 0, 12, 24, 48, and 72 hours. Gene expression levels in Xoo-inoculated plants were normalized to those in the corresponding control group. Statistical analysis was performed using a two-tailed Student's t-test. **p < 0.01. Data are expressed as mean ± SD, n = 3 biologically independent samples (A and B).

[0034] Figure 3 This is a subcellular localization map of OSD4 provided in an embodiment of the present invention.

[0035] Subcellular localization of OSD4 in rice protoplasts. Chloroplast autofluorescence was used to determine the cytoplasmic localization of the GFP-OSD4 fusion protein. Scale bar, 10 μm.

[0036] Figure 4 This is an identification diagram of the enzyme activity product of OSD4 provided in an embodiment of the present invention.

[0037] (A) Biochemical reactions catalyzed by OSD4 in vitro. (B) Purification of recombinant protein MBP-OSD4 from Escherichia coli BL21 DE3 strain. Lane 1, molecular weight marker; lanes 2 and 3, crude extracts of E. coli containing MBP-OSD4, uninduced or induced with IPTG, respectively; insoluble fractions (lane 4) and soluble fractions (lane 5) of E. coli sonicated with IPTG-treated MBP-OSD4; lane 6, purified recombinant protein MBP-OSD4. Arrows point to MBP-OSD4 protein. Proteins were visualized by Coomassie Blue R-250 staining; (C) Comparison of spectra of the OSD4 in vitro enzymatic reaction product and SA standard; (D) DAD chromatograms of the OSD4-catalyzed product and SA standard. The retention time of the enzyme product is the same as that of the SA standard. (E) Structure and putative fragments of the OSD4 product. (F) The MS fragmentation pattern of the OSD4-catalyzed product is the same as that of the SA standard. (G) Effect of pH on the enzymatic activity of the recombinant protein MBP-OSD4. (H) Effect of temperature on the enzymatic activity of the recombinant protein MBP-OSD4. (I) Kinetic curve of MBP-OSD4 on BS. The enzymatic activity curve was fitted with the Michaelis-Menten equation. (J) Kinetic analysis of the recombinant protein MBP-OSD4 on different substrates, including BS, phenylethyl salicylate, phenylethyl benzoate, methyl salicylate, ethyl salicylate, phenylethyl phenylacetate, methyl benzoate, ethyl benzoate, and benzyl benzoate. Data are expressed as mean ± SD, n = 3 biologically independent samples (G–J).

[0038] Figure 5This is a diagram showing that overexpression of the OSD4 gene can enhance rice disease resistance, as provided by an embodiment of the present invention.

[0039] A, Expression levels of OSD4-pMDC43 / ZH11 overexpressing materials at the tillering stage in the field. B, Field-grown plants of Zhonghua11 and OSD4-pMDC43 / ZH11 inoculated with Xoo at the tillering stage. Scale bar, 3 cm. C, Lesion lengths of leaves of Zhonghua11 and three OSD4-overexpressing lines 14 days after inoculation with Xoo pathogen. Statistical analysis was performed using a two-sided Student's t-test, ***p < 0.001. Data are expressed as mean ± SD, n = 3 (A) and n = 20 (C) independent biological replicates. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0041] The present invention provides a gene, OSD4, that regulates SA synthesis in rice leaves and resistance to bacterial blight, and its applications. Using CRISPR / Cas9 technology, the present invention screened a rice mutant with leaf SA reduced to less than 10% of the wild type. Functional studies have demonstrated that the gene is involved in the PAL-dependent salicylic acid synthesis pathway in rice, and that overexpression can enhance disease resistance in rice.

[0042] The present invention is achieved by providing a salicylic acid synthesis gene, the gene is OSD4, and the nucleotide sequence is SEQ ID NO: 1.

[0043] The salicylic acid synthesis gene provided by the embodiment of the present invention further includes a DNA sequence having a homology of more than 90% with the DNA sequence shown in SEQ ID NO: 1.

[0044] The salicylic acid synthesis gene provided by the embodiment of the present invention further includes alleles or gene derivatives with one or more base changes produced by base substitution, deletion, or addition.

[0045] The salicylic acid synthesis gene provided by the embodiment of the present invention further includes: a DNA molecule that can hybridize with the DNA sequence shown in SEQ ID NO: 1 under stringent conditions.

[0046] An embodiment of the present invention provides a protein encoded by the rice salicylic acid synthesis gene, wherein the nucleotide sequence of the encoded protein is SEQ ID NO: 2.

[0047] The amino acid sequence of the protein encoded by the embodiment of the present invention is SEQ ID NO: 3;

[0048] The encoded protein further comprises an amino acid sequence having a homology of more than 90% with the amino acid sequence shown in SEQ ID NO: 3;

[0049] The encoded protein further includes proteins and protein analogs having one or more amino acid changes produced by amino acid substitution, deletion, or addition based on the amino acid sequence shown in SEQ ID NO: 3;

[0050] The encoded protein further includes a fusion protein formed by linking the protein shown in SEQ ID NO: 3 with other tag proteins.

[0051] The embodiment of the present invention provides a plant genetic transformation vector constructed using the salicylic acid synthesis gene, wherein the plant genetic transformation vector includes an expression vector for up-regulating OSD4; the expression vector for up-regulating OSD4 includes a fusion expression vector constructed by a recombinant promoter or an organ-specific promoter;

[0052] The plant genetic transformation vector further comprises: a DNA sequence as shown in SEQ ID NO: 1, or a DNA sequence having a homology of more than 90% with the DNA sequence as shown in SEQ ID NO: 1, or an allele or gene derivative with one or more base changes produced by base substitution, deletion, or addition of the nucleotide sequence as shown in SEQ ID NO: 1, or a DNA molecule capable of hybridizing with the DNA sequence as shown in SEQ ID NO: 1 under stringent conditions.

[0053] The plant genetic transformation vector provided by the embodiment of the present invention further includes an up-regulation or down-regulation OSD4 expression vector, which down-regulates the expression of the gene shown in SEQ ID NO: 3 by CRISPR / Cas9 technology, T-DNA insertion technology, EMS mutagenesis, RNA interference technology, or gene silencing technology;

[0054] The plant genetic transformation vector up-regulates or down-regulates the expression level or activity of the protein shown in SEQ ID NO: 3 through a related protein regulator.

[0055] The embodiments of the present invention provide a recombinant bacterium, plant callus tissue and cell line using the plant genetic transformation vector for expression.

[0056] The present invention is specifically implemented:

[0057] Example 1: Identification of the gene OSD4 involved in the rice SA biosynthesis pathway

[0058] To elucidate the PAL pathway of SA biosynthesis, we used CRISPR / Cas9 technology to knock out the Os05g0410200 gene and verify its function. Os05g0410200 was predicted to be a carboxylesterase and was named OSD4 (Oryza Sativa SA-Deficient gene 4). The gene is 1089 bp long, has no introns, and encodes a protein composed of 362 amino acids. Using CRISPR / Cas9 technology to knock it out, three homozygous strains were obtained. The osd4-1 strain had a 55-base deletion at position 208 from the ATG, resulting in an 18-amino acid deletion and a frameshift mutation in its gene. The osd4-2 strain had a T insertion at position 207 from the ATG, resulting in a frameshift mutation at amino acid 69 in its gene. The osd4-3 strain had a C insertion at position 207 from the ATG, resulting in a frameshift mutation at amino acid 69 in its gene. Figure 1 A–C). There is no significant difference between the osd4 mutant and WT during growth ( Figure 1 D), the determination of endogenous SA and SAG contents in its 14-day-old leaves showed that both SA and SAG contents decreased to less than 20% of the wild type ( Figure 1 E), indicating that the OSD4 gene plays a crucial role in the synthesis of SA in rice. Subsequently, wild-type and osd4 mutants were inoculated with Xoo in the field during the tillering stage. Statistical analysis of disease incidence 14 days after inoculation revealed that the lesion length of the osd4 mutant was significantly increased compared to the wild-type, with increases of 55.4%, 47.1%, and 54.2% in three independent lines, respectively, compared to the wild-type. Figure 1 F). The above results indicate that OSD4 is involved in the synthesis of rice SA. The deletion of OSD4 gene leads to a significant decrease in rice SA content and makes rice more sensitive to pathogens.

[0059] Example 2: OSD4 gene expression and induced expression

[0060] The transcriptional expression pattern and expression regulation of genes are crucial to their biological functions. To study the transcriptional expression pattern of the OSD4 gene, qRT-PCR was used to detect the transcriptional levels in roots, stems, leaves, and panicles of wild-type ZH11 at the heading stage. The results showed that the OSD4 gene was expressed in all tissues of rice ( Figure 2 A) Rice is more sensitive to Xoo after SA deficiency ( Figure 1 F), therefore, ZH11 was sampled within 72 hours after inoculation to detect the induction of OSD4 gene by Xoo. The results showed that 24 hours after inoculation, OSD4 was induced by Xoo and its expression increased by 2.4 times ( Figure 2B). The above results indicate that SA plays an important role in rice disease resistance.

[0061] Example 3: Subcellular localization of OSD4 protein

[0062] The subcellular localization of a gene is often closely related to its biological function. To investigate the subcellular localization of the OSD4 protein, an OSD4 protein vector containing an N-terminal GFP fusion protein driven by the 35S promoter was constructed. Rice protoplasts were transformed and their subcellular localization was observed. Chloroplast autofluorescence was used as a control. The results showed that OSD4 was localized in the cytoplasm (Figure 3).

[0063] Example 4: OSD4 catalyzes benzyl salicylate to produce salicylic acid

[0064] The biochemical function of OSD4 is speculated to be to catalyze BS to produce SA and benzyl alcohol (BAlc) ( Figure 4 A) To verify the biological function of OSD4, the pMAL-OSD4 vector was constructed and the MBP-OSD4 fusion protein was purified using the E. coli Rosetta2 (DE3) strain. The purified MBP-OSD4 fusion protein was approximately 81.8 kDa ( Figure 4 B). The product was detected and analyzed using LC-MS, and the spectrum and retention time of the product were consistent with those of SA ( Figure 4 C, D), based on its molecular formula, the possible fragmentation mode was speculated and compared with the standard SA by MS fragmentation. The results showed that the MS fragmentation of the two was consistent ( Figure 4 E, F) indicate that OSD4 uses BS as a substrate to generate SA and BAlc. Based on its biochemical function, we named it benzyl salicylate hydrolase (BSH). pH and temperature experiments showed that the optimal catalytic activity of OSD4 is at pH 8.0 and at a temperature of 10-50°C ( Figure 4 G, H). These results indicate that OSD4 can catalyze BS to produce SA. Enzyme kinetic analysis of OSD4 using different concentrations of BS showed that OSD4 obeys Michaelis-Menten kinetics, with a maximum reaction rate V max 15.11 nmol mg -1 min -1 , Michaelis constant K m is 324.7, and R2 is 0.9749 ( Figure 4I, J). The specificity of OSD4 was analyzed under its optimal conditions using BS, phenethyl salicylate, phenyl benzoate, methyl salicylate, ethyl salicylate, phenylethyl benzoate, benzyl benzoate, methyl benzoate, and ethylbenzoate as substrates. The results showed that the catalytic activities of OSD4 towards phenethyl salicylate, phenyl benzoate, methyl salicylate, ethyl salicylate, phenylethyl benzoate, and benzyl benzoate were 72.63%, 77.16%, 0.54%, 1.45%, 32.04%, and 16.10% of those of BS, respectively, while OSD4 had no catalytic activity towards methyl benzoate and ethyl benzoate ( Figure 4 J) The above results indicate that OSD4 can catalyze the production of SA and BAlc using BS as a substrate, with an optimal pH of 8.0 and an optimal temperature of 10-50°C. OSD4 obeys Michaelis-Menten kinetics, and the optimal substrate is BS.

[0065] Example 5: OSD4 overexpression in rice plants increases resistance to Xoo

[0066] The OSD4 gene was overexpressed in the background of Zhonghua 11 using the 35S promoter, and three overexpression lines were screened. Their expression levels were 65.6, 39.7, and 16.1 times that of Zhonghua 11, respectively. Figure 5 A). Xoo inoculation experiments were conducted during the tillering stage. 14 days later, the disease progression was observed and statistically analyzed. The results showed that compared with the wild type, the lesion lengths of the three overexpression lines were reduced by 31.0%, 35.0%, and 35.7%, respectively. Figure 5 B, C), indicating that overexpression of OSD4 can increase rice resistance to Xoo.

[0067] According to the above Examples 1-5, OSD4 is a gene involved in the biosynthesis of PAL-dependent SA, expressed in all tissues of the plant, induced by Xoo, and located in the cytoplasm. Overexpression of OSD4 can increase the resistance of rice to Xoo.

[0068] SEQ ID NO: 1:

[0069]

[0070] SEQ ID NO:2:

[0071]

[0072] SEQ ID NO: 3:

[0073] MAIATESQQAQVQAAATDAVAAAAVGRKVVDEVSGWLRVFDDGTVDRTWTGPPEVLPLMQPVPAYAEPRDGHTLHDLPGEPNLRVYLPEVALAERRLPVVVQLHGGGFCISHPSWLMYHHFYARLACALPAVVVAVELPLAPERRLPAHIDTGVDGLRRLRSIALSDAAALGDPAAELLRT AADFSRVFLIGDSSGGNLVHHVGARVGEDGADSWAPLRVAGGIPLHPGFVHATRSKSELEPRPDSVFFTLDMLDKFLAMALPEGATKDHPYTCPMGPNAPPLESVPLPPLLVAVAEHDLIRDTNLEYCDALRTAGKDVEVLVNRGMSHSFYLNKYAVDMDPATGERTRELVDAIKSFVDRH*

[0074] The above description is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with this technical field within the technical scope disclosed by the present invention and within the spirit and principles of the present invention should be covered by the scope of protection of the present invention.

Claims

1. A gene involved in the PAL-dependent salicylic acid biosynthesis pathway in rice, characterized in that: The nucleotide sequence of the gene is SEQ ID NO:

1.

2. The gene involved in the rice PAL-dependent salicylic acid biosynthesis pathway according to claim 1, characterized in that: The gene involved in the rice PAL-dependent salicylic acid synthesis pathway further includes a DNA sequence having a homology of more than 90% with the DNA sequence shown in SEQ ID NO: 1; The genes involved in the rice PAL-dependent salicylic acid synthesis pathway further include alleles or gene derivatives with one or more base changes produced by base substitution, deletion, or addition; The gene involved in the rice PAL-dependent salicylic acid synthesis pathway further includes: a DNA molecule that can hybridize with the DNA sequence shown in SEQ ID NO: 1 under stringent conditions.

3. The protein encoded by the gene involved in the rice PAL-dependent salicylic acid biosynthesis pathway according to claim 1 or 2, characterized in that: The amino acid sequence of the protein is SEQ ID NO:

3.

4. The protein encoded by the gene involved in SA synthesis in rice leaves and regulating rice tolerance to bacterial blight according to claim 3, characterized in that: The nucleotide sequence of the encoded protein is SEQ ID NO:

2.

5. The protein encoded by the gene involved in the rice PAL-dependent salicylic acid synthesis pathway according to claim 3, characterized in that: The encoded protein further comprises an amino acid sequence having a homology of more than 90% with the amino acid sequence shown in SEQ ID NO: 3; The encoded protein further includes proteins and protein analogs having one or more amino acid changes produced by amino acid substitution, deletion, or addition based on the amino acid sequence shown in SEQ ID NO: 3; The encoded protein further includes a fusion protein formed by linking the protein shown in SEQ ID NO: 3 with other tag proteins.

6. The gene according to any one of claims 1 or 2, characterized in that The gene encodes a protein having the enzymatic activity of benzyl salicylate hydrolase or a related metabolic enzyme, which can catalyze the key intermediate reaction step in the PAL-dependent SA biosynthesis pathway - hydrolyzing benzyl salicylate to directly generate salicylic acid, thereby promoting the enhancement of the PAL-dependent salicylic acid synthesis metabolic flow in rice.

7. A plant genetic transformation vector constructed using the gene involved in the rice PAL-dependent salicylic acid biosynthesis pathway as claimed in claim 1, characterized in that: The plant genetic transformation vector includes an up-regulated expression vector; the up-regulated expression vector includes a fusion expression vector constructed by a recombinant promoter or an organ-specific promoter; The plant genetic transformation vector further comprises: a DNA sequence as shown in SEQ ID NO: 1, or a DNA sequence having a homology of more than 90% with the DNA sequence as shown in SEQ ID NO: 1, or an allele or gene derivative with one or more base changes produced by base substitution, deletion, or addition of the nucleotide sequence as shown in SEQ ID NO: 1, or a DNA molecule capable of hybridizing with the DNA sequence as shown in SEQ ID NO: 1 under stringent conditions.

8. The plant genetic transformation vector according to claim 6, wherein The plant genetic transformation vector further includes a down-regulating gene expression vector, which down-regulates the expression of the gene shown in SEQ ID NO: 3 by CRISPR / Cas9 technology, T-DNA insertion technology, EMS mutagenesis, RNA interference technology, or gene silencing technology; The plant genetic transformation vector up-regulates or down-regulates the expression level or activity of the protein shown in SEQ ID NO: 3 through a related protein regulator.

9. A recombinant bacterium, plant callus and cell line expressed using the plant genetic transformation vector according to claim 7 or 8.

10. A method for regulating resistance of rice leaves to bacterial blight bacteria, characterized in that: Knocking out the OSD4 gene leads to a decrease in SA in rice leaves and sensitivity to bacterial blight pathogen. Overexpressing the OSD4 gene increases the resistance of rice to bacterial blight pathogen.

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