Coding gene of benzyl benzoate 2-hydroxylase 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 for regulating and controlling the endogenous salicylic acid content and disease resistance of rice by using an SA synthetic gene OSD3 participating in rice PAL dependence and application of the gene, and the nucleotide sequence of the OSD3 is SEQ ID NO: 1; the nucleotide sequence of a coding protein region is SEQ ID NO: 2; the amino acid sequence of the coded protein is SEQ ID NO: 3; according to the invention, an OSD3 gene is knocked out through CRISPR / Cas9 under the background of Zhonghua 11, a mutant osd3 of which the SA of a rice leaf is reduced to 20% of that of a wild type is obtained, and an OSD3 overexpression plant is created by constructing an OSD3-pMDC43 overexpression vector.

Description

Technical Field

[0001] The present invention belongs to but is not limited to the field of plant genetic engineering technology, and particularly relates to a gene encoding benzyl benzoate 2-hydroxylase, which participates in rice PAL-dependent salicylic acid synthesis and regulates the endogenous salicylic acid content and disease resistance of rice, and its application. 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. Furthermore, it has been reported that benzoic acid 2-hydroxylase (BA2H), a 160 kDa cytochrome P450 monooxygenase, catalyzes the conversion of benzoic acid (BA) to SA in tobacco. Barley inoculated with Pseudomonas syringae pv. syringae exhibits an increase in endogenous SA levels during infection. However, in rice, SA levels did not increase following infection with P. syringae, the rice blast pathogen Magnaporthe grisea, or Rhizoctonia solani. Salicylate hydroxylases (NahG), which can block SA accumulation in vivo, significantly increased in NahG transgenic Arabidopsis plants following infection with the pathogen Pto DC3000, suggesting that SA plays an important role in disease resistance in Arabidopsis. Rice plants expressing NahG show a significant increase in ROS levels after invasion by the blast fungus Pyricularia oryzae, indicating that reduced SA contributes to increased susceptibility to the fungus. Mutation of OsS5h increases SA content in rice and enhances resistance to bacterial blight. Overexpression of OsS5h reduces endogenous SA in rice and renders 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 suggest 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 rice yields.

[0006] Existing technologies still have significant deficiencies in understanding the PAL-dependent SA biosynthesis pathway in rice. Currently, only a few key enzymes, such as AIM1, have been clearly located within this pathway, while the remaining functional members involved in this pathway have yet to be systematically identified and functionally analyzed. Although studies reported as early as three decades ago that tobacco contains a type of cytochrome P450 monooxygenase BA2H activity that catalyzes the hydroxylation of benzoic acid to produce SA, the specific gene encoding this enzyme has not yet been successfully cloned. Therefore, the enzymatic composition of the PAL-dependent SA biosynthesis pathway remains incomplete, limiting our in-depth understanding of this metabolic network. Summary of the Invention

[0007] In response to the problems existing in the prior art, the present invention provides a gene involved in rice PAL-dependent salicylic acid synthesis gene OSD3, which regulates the endogenous salicylic acid content and disease resistance of rice, and its application.

[0008] The present invention is achieved by providing a gene involved in rice PAL-dependent salicylic acid synthesis, the gene being OSD3, and the nucleotide sequence of the gene being SEQ ID NO: 1.

[0009] Furthermore, the gene involved in rice PAL-dependent salicylic acid synthesis further comprises a DNA sequence having a homology of more than 90% with the DNA sequence shown in SEQ ID NO: 1;

[0010] The gene involved in rice PAL-dependent salicylic acid synthesis further includes one or more base-changed alleles or gene derivatives produced by base substitution, deletion, or addition;

[0011] The gene involved in rice PAL-dependent salicylic acid synthesis further includes: a DNA molecule that can hybridize with the DNA sequence shown in SEQ ID NO: 1 under stringent conditions.

[0012] Furthermore, the protein encoded by the gene involved in rice PAL-dependent salicylic acid synthesis has an amino acid sequence of SEQ ID NO: 3.

[0013] Furthermore, the protein encoded by the gene involved in the synthesis of SA in rice leaves and regulating the tolerance of rice to bacterial blight pathogen is characterized in that the nucleotide sequence of the encoded protein is SEQ ID NO: 2.

[0014] Furthermore, the protein encoded by the gene involved in rice PAL-dependent salicylic acid synthesis is characterized by:

[0015] 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;

[0016] 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;

[0017] The encoded protein further includes a fusion protein formed by linking the protein shown in SEQ ID NO: 3 with other tag proteins.

[0018] Another object of the present invention is to provide a method for catalyzing the production of benzyl salicylate (BS) using benzyl benzoate (BB) as a substrate using the gene involved in rice PAL-dependent salicylic acid synthesis.

[0019] Another object of the present invention is to provide a plant genetic transformation vector constructed using the gene involved in rice PAL-dependent SA synthesis, wherein the plant genetic transformation vector comprises an up-regulated expression vector; the up-regulated expression vector comprises 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 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;

[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 to provide a recombinant bacterium, plant callus and cell line expressed by the plant genetic transformation vector.

[0024] Another object of the present invention is to provide a method for regulating the resistance of rice leaves to bacterial blight bacteria. Knocking out the OSD3 gene leads to a decrease in SA of rice leaves, making them sensitive to bacterial blight bacteria. Overexpressing the OSD3 gene increases the resistance of rice to bacterial blight bacteria.

[0025] 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:

[0026] The present invention provides a rice SA synthesis gene OSD3 and its application, which describes that rice OSD3 can regulate rice SA synthesis ( Figure 1-Figure 3 ); It uses BB as substrate to catalyze the production of BS ( Figure 4 ); Create its over-expression material to increase rice disease resistance ( Figure 5 ). BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Identification of the gene OSD3 involved in the rice SA biosynthesis pathway: A, Location of the Cas9-induced mutagenesis target motif (red arrow) within the coding region of the OSD3 gene (orange box); B, Sanger sequencing peaks of the osd3 mutant; C, Amino acid sequence information of the wild-type and osd3 mutants; D, Phenotypes of wild-type and osd3 mutant plants at heading. Scale bar = 10 cm; E, SA and SAG content in leaves of wild-type and osd3 mutant plants at 14 days old; F, Lesion phenotype and lesion length statistics of wild-type and osd3 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 < 0.01, ***p < 0.001. Data are expressed as mean ± SD, n = 4 (E) and n = 20 (F) biologically independent samples.

[0028] Figure 2The expression pattern of OSD3: A, OSD3 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, OSD3 gene expression in leaves of Zhonghua 11 plants inoculated with Xoo at 0, 12, 24, 48, and 72 hours. OSD3 gene expression levels after Xoo inoculation were normalized to those of the corresponding control group. Statistical analysis was performed using a two-tailed Student's t-test; *p < 0.05, **p < 0.01. Data are expressed as mean ± SD, n = 3 biologically independent samples (A and B).

[0029] Figure 3 Subcellular localization of OSD3 in rice protoplasts. mCherry-HDEL was used as an endoplasmic reticulum localization marker. Scale bar, 10 μm.

[0030] Figure 4 Identification of OSD3 enzymatic activity products: (A) Purification of recombinant Flag-OSD3 protein from yeast strain WAT11. Lane 1, molecular weight marker; lanes 2 and 3, yeast microsomes containing either empty vector (EV) or Flag-OSD3, respectively. Recombinant protein expression was verified by immunofluorescence. (B) In vitro MRM (multiple reaction monitoring) spectra of BA and SA in the OSD3 product using BA as a substrate. The absence of SA in the OSD3 product indicates that the Flag-OSD3 recombinant protein cannot catalyze the production of SA using BA as a substrate. (C) Biochemical reactions catalyzed by OSD3 in vitro. (D) Comparison of spectra of the OSD3 in vitro enzymatic activity product and a BS standard. (E) DAD chromatogram of the BB product catalyzed by OSD3 and a BS standard. The retention time of the enzyme product is the same as that of the BS standard. (F) Structure and putative fragments of the OSD3 product. (G) The MS fragmentation pattern of the product of BB produced by OSD3 is the same as that of the BS standard. (H) Effect of pH on the enzymatic activity of Flag-OSD3. (I) Effect of temperature on the enzymatic activity of Flag-OSD3. (J) Reactions of Flag-OSD3 to various substrates, including methyl benzoate, ethyl benzoate, phenyl benzoate, phenylethyl benzoate, benzyl benzoate, and benzoic acid. Data are expressed as mean ± SD, n = 3 biologically independent samples (H–J).

[0031] Figure 5Overexpression of the OSD3 gene enhances rice disease resistance: A, Expression levels of the OSD3-pMDC43 / ZH11 overexpressing material at the tillering stage in the field. B, Field-grown plants of Zhonghua 11 and OSD3-pMDC43 / ZH11 inoculated with the Xoo pathogen at the tillering stage. Scale bar, 3 cm. C, Lesion lengths on leaves of Zhonghua 11 and three OSD3-overexpressing lines 14 days after inoculation with the Xoo pathogen. Statistical analysis was performed using a two-sided Student's t-test. ***p < 0.001, NS, not significant. Data are expressed as mean ± SD, n = 3 (A) and n = 20 (C) independent biological replicates. DETAILED DESCRIPTION

[0032] 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.

[0033] An embodiment of the present invention provides a gene involved in rice PAL-dependent salicylic acid synthesis, the gene is OSD3, and the nucleotide sequence of the gene is SEQ ID NO: 1.

[0034] The gene involved in rice PAL-dependent salicylic acid synthesis further includes a DNA sequence having a homology of more than 90% with the DNA sequence shown in SEQ ID NO: 1;

[0035] The gene involved in rice PAL-dependent salicylic acid synthesis further includes one or more base-changed alleles or gene derivatives produced by base substitution, deletion, or addition;

[0036] The gene involved in rice PAL-dependent salicylic acid synthesis further includes: a DNA molecule that can hybridize with the DNA sequence shown in SEQ ID NO: 1 under stringent conditions.

[0037] The protein encoded by the gene involved in rice PAL-dependent salicylic acid synthesis has an amino acid sequence of SEQ ID NO: 3.

[0038] The protein encoded by the gene that participates in the synthesis of SA in rice leaves and regulates the tolerance of rice to bacterial blight pathogen is characterized in that the nucleotide sequence of the encoded protein is SEQ ID NO: 2.

[0039] The protein encoded by the gene involved in rice PAL-dependent salicylic acid synthesis is characterized by:

[0040] 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;

[0041] 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;

[0042] The encoded protein further includes a fusion protein formed by linking the protein shown in SEQ ID NO: 3 with other tag proteins.

[0043] The embodiment of the present invention provides a method for using the gene involved in rice PAL-dependent salicylic acid synthesis to catalyze the production of benzyl salicylate using benzyl benzoate as a substrate.

[0044] The embodiment of the present invention provides a plant genetic transformation vector constructed using the gene involved in rice PAL-dependent salicylic acid synthesis, wherein the plant genetic transformation vector comprises an up-regulated expression vector; the up-regulated expression vector comprises a fusion expression vector constructed with a recombinant promoter or an organ-specific promoter;

[0045] 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.

[0046] 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;

[0047] 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.

[0048] The embodiments of the present invention provide a recombinant bacterium, plant callus tissue and cell line expressed by the plant genetic transformation vector.

[0049] The embodiment of the present invention provides a method for regulating the resistance of rice leaves to bacterial blight bacteria. Knocking out the OSD3 gene leads to a decrease in SA of rice leaves, making them sensitive to bacterial blight bacteria. Overexpressing the OSD3 gene increases the resistance of rice to bacterial blight bacteria.

[0050] Experiments with bacterial blight pathogens showed that osd3 mutants were more susceptible to the pathogen, and that overexpressing OSD3 exhibited even greater resistance. An OSD3-pESC-URA vector was constructed and its in vitro enzymatic activity verified, demonstrating that OSD3 catalyzes the direct conversion of BB to BS. Given that OSD3 functional loss significantly decreases rice SA content and increases sensitivity to bacterial blight pathogens, while OSD3 overexpression confers enhanced resistance to the pathogen, the function of OSD3 in rice SA biosynthesis was elucidated, and its application in improving crop disease resistance was explored.

[0051] Example 1: Identification of the gene OSD3 involved in the rice SA biosynthesis pathway

[0052] To elucidate the PAL pathway of SA biosynthesis, we used CRISPR / Cas9 technology to knock out the Os09g0441400 gene and validate its function. Os09g0441400 is predicted to be a cytochrome P450 71A1 gene and was named OSD3 (Oryza Sativa SA-Deficient gene 3). The gene is 2477 bp long, contains an intron, and encodes a 514-amino acid protein. The CRISPR / Cas9 technology was used to knock out the gene, and three homozygous strains were obtained. Among them, the osd3-1 strain had a T insertion at the 75th position from the ATG, resulting in a frameshift mutation at the 25th amino acid position and a premature termination at the 29th amino acid position in the protein it encoded. The osd3-2 strain had a 30-base deletion at the 50th position from the ATG, resulting in a 10-amino acid deletion in the protein it encoded. The osd3-3 strain had an A insertion at the 75th position from the ATG, resulting in a frameshift mutation at the 25th amino acid position and a premature termination at the 29th amino acid position in the protein it encoded. Figure 1 A–C). There is no significant difference between the osd3 mutant and the 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 OSD3 gene plays a crucial role in the synthesis of SA in rice. Subsequently, wild-type and osd3 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 osd3 mutant was significantly increased compared to the wild-type, with increases of 59.4%, 44.3%, and 30.5% in three independent lines, respectively, compared to the wild-type. Figure 1 F). The above results indicate that OSD3 is involved in the synthesis of rice SA. The deletion of the OSD3 gene leads to a significant decrease in rice SA content and makes rice more sensitive to pathogens.

[0053] Example 2: OSD3 gene expression and induced expression

[0054] The transcriptional expression pattern and expression regulation of genes are crucial to their biological functions. To study the transcriptional expression pattern of the OSD3 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 OSD3 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 OSD3 gene by Xoo. The results showed that 24 hours after inoculation, OSD3 was induced by Xoo and its expression increased by 13.9 times ( Figure 2 B). The above results indicate that SA synthesized by OSD3 plays an important role in rice disease resistance.

[0055] Example 3: Subcellular localization of OSD3 protein

[0056] The subcellular localization of a gene is often closely related to its biological function. To investigate the subcellular localization of OSD3, an OSD3 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. The GFP-OSD3 fusion protein colocalized with the endoplasmic reticulum marker protein mCHerry-HDEL, indicating that OSD3 is localized to the endoplasmic reticulum (ER) (Figure 3).

[0057] Example 4: OSD3 catalyzes the production of BS using BB as a substrate instead of SA using BA as a substrate

[0058] To verify the biological function of OSD3, the OSD3-pESC-URA vector was constructed and the Flag-OSD3 fusion protein was purified using the yeast WAT11 strain. The purified Flag-OSD3 fusion protein was approximately 58.1 kDa ( Figure 4 A). Reports indicate that tobacco contains a 160 kDa cytochrome P450 monooxygenase, BA2H, which catalyzes the conversion of BA to SA. To verify whether OSD3 is the rice BA2H, enzyme activity was performed using BA as a substrate, and the product was detected by LC-MS. The results showed that OSD3 had no peak at the retention time of the SA standard ( Figure 4 B), which indicates that OSD3 cannot generate SA using BA as substrate. Therefore, we speculate that OSD3 can generate BS using BB as substrate ( Figure 4 C). The product was detected and analyzed using LC-MS, and the spectrum and retention time of the product were consistent with those of BB ( Figure 4 D, E), based on its molecular formula, the possible fragmentation mode was speculated and compared with the MS fragments of BB standard. The results showed that the MS fragments of the two were consistent ( Figure 4 F, G), indicating that OSD3 catalyzes the production of BS using BB as a substrate. Based on its biochemical function, we named it benzyl benzoate 2-hydroxylase (BB2H). pH and temperature experiments showed that the optimal pH for OSD3 catalysis is 7.0 ( Figure 4 H), the optimum temperature is 30℃ ( Figure 4 I). The substrate specificity of OSD3 was tested under its optimal conditions using BB, methyl benzoate, ethyl benzoate, phenyl benzoate, phenylethylbenzoate and BA as substrates. The results showed that OSD3 could only catalyze BB and had no catalytic activity towards other substrates, indicating that OSD3 has strong substrate specificity ( Figure 4 J). The above results show that OSD3 can react with BB as substrate to generate BS, with its optimal pH at 7.0, optimal temperature at 30°C, and optimal substrate being BB, showing strong substrate specificity.

[0059] Example 5: OSD3 overexpression in rice plants increases resistance to Xoo

[0060] The OSD3 gene was overexpressed in the background of Zhonghua 11 using the 35S promoter, and three overexpression lines were screened. Their expression levels were 10.8, 3.6, and 5.3 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 28.0%, 33.6%, and 28.4%, respectively. Figure 5 B, C), indicating that overexpression of OSD3 can increase rice resistance to Xoo.

[0061] Examples 1-5 above demonstrate that OSD3 is a gene involved in PAL-dependent SA biosynthesis. OSD3 catalyzes the conversion of BB to BS but not BA to SA. The OSD3 gene is expressed in all plant tissues, its expression is induced by Xoo, and it is localized in the endoplasmic reticulum. Overexpression of OSD3 in rice can increase resistance to Xoo.

[0062] SEQ ID NO: 1:

[0063]

[0064] SEQ ID NO:2:

[0065]

[0066] SEQ ID NO: 3:

[0067] MELMLPPWASFVGVVLATVLFLKAVLGRSRRVYNLPPGPKPWPVIGNLNLVGTLPHRSIHNLSKKYGPLMYLRFGSFPVVVGSSVEMAKFFLKTHDVVFTDRPKTAAGKHTTYNYSDITWSPYGAYWR QARKMCLTELFSAKRLESYEYIRGEEVRALLRDLHGAAGGVVVLKDYLSTVSLNVITRMVLGKKYLDKDAGGSVTTPEEFKWMLDELFLLLNGVLNIGDSIPWLDWLDLQGYIKRMKKLGKMFDRFLEHV VDEHNERRRREGESFVAKDMVDVLLQFADNPNLEVKLKREGVKAFTQDLIAGGTESSAVTVEWALSELLKKPEVFAKATEELDRVVGRGRWVTEKDVPSLTYVDAIVKETMRLHPVAPMLVPRLSREDT SVDGYDIPAGTRVLVSVWTIGRDPKLWDAPEEFMPERFIGNKIDVKGQDFELLPFGSGRRMCPGYSLGLKVIQLSLANLLHGFAWRLPDGVTREQLSMEEIFGLSTPRKFPLEAVVEPKLPAHLYAAA*

[0068] 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 PAL-dependent salicylic acid synthesis in rice, characterized by: The gene is OSD3, and the nucleotide sequence of the gene is SEQ ID NO:

1.

2. The gene involved in rice PAL-dependent salicylic acid synthesis according to claim 1, wherein: The gene involved in rice PAL-dependent salicylic acid synthesis further includes a DNA sequence having a homology of more than 90% with the DNA sequence shown in SEQ ID NO: 1; The gene involved in rice PAL-dependent salicylic acid synthesis further includes one or more base-changed alleles or gene derivatives produced by base substitution, deletion, or addition; The gene involved in rice PAL-dependent salicylic acid synthesis 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 rice PAL-dependent salicylic acid synthesis 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 rice PAL-dependent salicylic acid synthesis 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 with BB2H or related metabolic enzyme activity, which can catalyze the key intermediate reaction step in the PAL-dependent SA biosynthesis pathway - catalyzing BB to produce BS, thereby promoting the enhancement of the PAL-dependent salicylic acid synthesis metabolic flow in rice.

7. A plant genetic transformation vector for constructing a gene involved in rice PAL-dependent salicylic acid synthesis according to 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 7, 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 expressing 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 by: Knocking out the OSD3 gene leads to a decrease in SA in rice leaves and sensitivity to bacterial blight pathogen. Overexpressing the OSD3 gene increases the resistance of rice to bacterial blight pathogen.

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